genetic and molecular basis of inherited visual …
TRANSCRIPT
GENETIC AND MOLECULAR BASIS OF INHERITED
VISUAL DISORDERS
Ph.D THESIS
DR. SHAKEEL AHMED SHEIKH
MBBS
MOLECULAR BIOLOGY & HUMAN GENETICS
LIAQUAT UNIVERSITY OF MEDICAL & HEALTH SCIENCES
JAMSHORO, PAKISTAN
(2017)
GENETIC AND MOLECULAR BASIS OF INHERITED
VISUAL DISORDERS
A THESIS SUBMITTED TO THE
LIAQUAT UNIVERSITY OF MEDICAL & HEALTH SCIENCES
JAMSHORO, PAKISTAN IN FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY IN
MOLECULAR BIOLOGY AND HUMAN GENETICS
By
DR. SHAKEEL AHMED SHEIKH
MBBS
SUPERVISOR
Dr. Ali Muhammad Waryah
PhD
CO-SUPERVISORS
Dr. Zubair M. Ahmed
PhD
Dr. Ashok Kumar Narsani
FCPS
(2017)
DEDICATED TO:
MY LOVING MOTHER
i
TABLE OF CONTENTS
………………………………………………………………………… Page
List of Tables ……………………………………………………… viii
List of Figures ……………………………………………………… ix
List of Appendices…………………………………………………… xii
List of Abbreviations and Symbols…………………………………. xiii
Acknowledgments……………………………………………………. xv
Abstract ……………………………………………………………… xvii
INTRODUCTION 01
CHAPTER 1
LITERATURE REVIEW
06
SECTION I
INHERITED VISUAL DISORDERS
07
SECTION II EYE STRUCTURES RELATED TO INHERITED VISUAL
DISORDERS
12
1.1 Anatomy of Eye………………………………………………………. 13
1.1.1 Embryology of Eye …………………………………………………. 13
1.1.2 Orbit …………………………………………………………………. 14
1.1.3 The Eyeball ------------------------------------------------------------------ 14
1.1.4 Conjunctiva -------------------------------------------------------------------- 15
1.1.5 Sclera --------------------------------------------------------------------------- 15
1.1.6 Cornea -------------------------------------------------------------------------- 16
1.1.7 Uvea ---------------------------------------------------------------------------- 17
1.1.8 Iris ------------------------------------------------------------------------------- 17
1.1.9 Ciliary Body -------------------------------------------------------------------
1.1.10 Choroid ------------------------------------------------------------------------- 18
ii
1.1.11 Lens ----------------------------------------------------------------------------- 18
1.1.12 Aqueous Humor and its Outflow Pathway -------------------------------- 19
1.1.13 Anterior Chamber Angle ----------------------------------------------------- 20
1.1.14 Intra-Ocular Pressure --------------------------------------------------------- 21
1.1.15 Vitreous ------------------------------------------------------------------------ 21
1.1.16 Retina --------------------------------------------------------------------------- 21
1.1.17 Optic Disc ---------------------------------------------------------------------- 23
SECTION III GLAUCOMA, STARGARDT DISEASE, CONGENITAL
CATARACT
25
1.2 Glaucoma ---------------------------------------------------------------------- 26
1.2.1 Prevalence ---------------------------------------------------------------------- 26
1.2.2 Classification of Glaucoma -------------------------------------------------
A. Primary Open-Angle Glaucoma
B. Primary Angle-Closure Glaucoma
C. Normal Tension Glaucoma
D. Secondary Glaucoma
E. Primary Congenital Glaucoma
i. Pathophysiology of Primary congenital glaucoma
ii. Genetics of Primary Congenital Glaucoma
a) GlC3A Locus
b) GLC3B Locus
c) GLC3C Locus
d) GLC3D Locus
iii. Role of Myocilin in PCG
28
28
31
32
32
33
34
36
36
44
44
44
46
1.2.3. Diagnosis of Glaucoma ---------------------------------------------------- 46
1.2.4 Management of Glaucoma --------------------------------------------------- 47
1.3 Stargardt Disease ----------------------------------------------------------- 48
1.3.1 Variants of Stargardt Disease:------------------------------------------------
A. Stargardt Disease-1 (STGD-1)
B. Stargardt Disease-3 (STGD-3)
C. Stargardt Disease-4 (STGD-4)
50
51
53
55
1.3.2. Treatment of Stargardt Disease ---------------------------------------------- 55
1.4 Congenital Cataract --------------------------------------------------------- 56
iii
1.4.1 Prevalence ---------------------------------------------------------------------- 56
1.4.2 Classification ------------------------------------------------------------------ 57
1.4.3
Genetics of Congenital Cataract --------------------------------------------
1.4.3.1 Role of various Genes in Cataract Development ----------------
61
63
1.4.4 Role of Unfolded Protein Response (UPR) in Congenital Cataract---- 67
1.4.5 Age-Related Cataract --------------------------------------------------------- 68
1.4.6 Treatment Options for Cataract---------------------------------------------- 68
SECTION IV OPHTHALMOLOGICAL EXAMINATION
70
1.5 Ocular Examination ----------------------------------------------------------- 71
1.5.1 Visual Acuity------------------------------------------------------------------- 71
1.5.2 Corneal Diameter--------------------------------------------------------------- 73
1.5.3 Corneal Edema and Haab’s Striae------------------------------------------- 73
1.5.4 Corneal Opacity---------------------------------------------------------------- 74
1.5.5 Tonometry----------------------------------------------------------------------- 74
1.5.6 Gonioscopy---------------------------------------------------------------------- 75
1.5.7 Ophthalmoscopy (Fundoscopy)---------------------------------------------- 77
1.5.8 Cup to Disc Ratio -------------------------------------------------------------- 79
1.5.9 Optical Coherence Tomography--------------------------------------------- 79
SECTION V
WHOLE EXOME SEQUENCING
80
CHAPTER 2
MATERIALS AND METHODS
84
2.1 Methodology ------------------------------------------------------------------
2.1.1 Place of Study
2.1.2. Inclusion Criteria
2.1.3 Exclusion Criteria
85
85
85
85
2.1.4 Field Work -------------------------------------------------------------------
2.1.4.1. Identification and Enrollment of Families
2.1.4.2. History Recording
2.1.4.3.General & Systemic Examination
86
86
87
88
iv
2.1.4.4. Clinical Assessment of Inherited Visual Disorders
i. Visual Acuity Test
ii. Measurement of IOP
iii. Measurement of Corneal Diameter
iv. Fundoscopy
v. Optical Coherence Tomography
88
88
88
88
89
89
2.1.5. Laboratory Work ……………………………………………………
A. DNA Extraction
B. Quantification of DNA
C. Preparation of Working Dilutions of Extracted DNA
D. STR Markers Used for Genotyping
E. Typing STR Markers by PCR
F. Quantification of PCR Products for Genotyping
G. Designing DNA Plate Map for Genotyping
H. Sample Preparation for Genotyping using ABI PRISM 3130
Genetic Analyzer
I. Haplotype Analysis
J. DNA Sequencing
K. Designing Sequencing Primers
L. Preparation of Stock Solutions of Sequencing Primers
M. Preparation of Working Dilutions of Sequencing Primers
N. Optimization of PCR Conditions for Sequencing Primers
O. Amplification of DNA Samples using Sequencing Primers
P. Confirmation of Amplification through Agarose Gel
Electrophoresis
Q. Purification of Amplified PCR Products by Ethanol
Precipitation
R. Sequencing PCR
S. Precipitation of Sequencing PCR Products
T. Preparing Plate for Sanger Sequencing at University of
Maryland
U. PCR Purification by ExoSAP
V. Precipitation of Sequencing PCR Products
W. Analysis of DNA Sequences
89
89
91
92
93
94
96
96
96
97
97
98
98
98
98
99
100
100
100
101
101
102
103
104
2.1.6 Whole Exome Sequencing of Selected Families at University of
Maryland ………………………………………………………………
105
CHAPTER-3
RESULTS
108
SECTION-I GENETIC CHARACTERIZATION OF FAMILIAL PCG
110
3.1 Homozygosity mapping of common PCG Loci---------------------------- 111
3.1.1 Reported CYP1B1 Mutations-------------------------------------------------
A. p.R390H
i. PCG-02
ii. PCG-03
iii. PCG-04
111
111
111
112
113
v
iv. PCG-15
v. PCG-16
vi. PCG-17
vii. PCG-19
B. p. E229K (PCG-07)
C. p.P437L (PCG-10)
D. p.R290fs*37 (PCG-13)
E. p.A115P (PCG-06)
114
115
116
117
119
121
121
122
3.1.1.1 Clinical Features of Patients with Reported CYP1B1 Mutations -------- 123
3.1.2 Novel Mutations in CYP1B1 ------------------------------------------------
A. p.G36D(PCG-08)
B. p.G67-A70del (PCG-09)
129
129
132
3.2 Unlinked PCG Families ------------------------------------------------------
3.2.1 PCG-11
3.2.2 PCG-12
3.2.3 PCG-14
3.2.4 PCG-20
135
135
135
136
136
SECTION-II MOLECULAR CHARACTERIZATION OF STARGARDT
DISEASE
138
3.3 Linkage Analysis of Stargardt Disease ------------------------------------- 139
3.3.1 Whole Exome Sequencing Revealed a Novel Gene for Stargardt
Disease---------------------------------------------------------------------------
139
3.3.1.1 LUSG-03 ----------------------------------------------------------------------- 139
3.3.1.2 LUSG-04 ----------------------------------------------------------------------- 142
3.3.2 Unlinked Stargardt Disease Families ---------------------------------------
3.3.2.1- LUSG-02
3.3.2.2- LUSG-07
3.3.2.3-LUSG-08
147
147
148
149
SECTION-III GENETICS STUDY OF CONGENITAL CATARACT
150
3.4 Linkage Analysis of Congenital Cataract----------------------------------- 151
3.4.1 LUCC-15 -----------------------------------------------------------------------
3.4.1.1- Clinical Features of affected patients:
151
153
3.4.2 Unlinked Congenital Cataract Families ------------------------------------
3.4.2.1- LUCC-01
3.4.2.2- LUCC-02
3.4.2.3- LUCC-04
3.4.2.4- LUCC-13
155
155
156
157
157
vi
CHAPTER-04
DISCUSSION
159
SECTION-I GENETIC CHARACTERIZATION OF FAMILIAL PCG
161
4.1 Genetic Characterization of Familial PCG---------------------------------- 162
SECTION-II MOLECULAR CHARACTERIZATION OF STARGARDT
DISEASE
168
4.2 ARL3- A Novel Gene for Stargardt Disease ------------------------------- 169
SECTION-III GENETIC STUDY OF CONGENITAL CATARACT
172
4.3 INPP5K- A Novel Gene for Congenital Cataract ------------------------- 173
CONCLUSION ---------------------------------------------------
176
APPENDICES----------------------------------------------------- 177
REFERENCES --------------------------------------------------- 187
vii
LIST OF TABLES
Table No. Particulars Page No.
1.1 The Classification of human xenobiotics metabolizing forms
of P450s 39
1.2 Various types of mutations identified in CYP1B1 43
1.3 Loci and their corresponding genes for syndromic and non-
syndromic Congenital Cataracts 62
2.1 Reported Loci for Primary Congenital Glaucoma(PCG) 93
2.2 Reported Loci/Gene for Stargardt disease 94
2.3 Common Genes/Loci for Congenital Cataract Screened in
LUCC-15 94
2.4 The components of PCR reaction mixture for amplification
of STR markers 95
2.5 Sequencing Primers used for CYP1B1 gene amplification 99
2.6 Reaction Mixture for amplification of PCR Fragments 99
2.7 The Components of master mix for Sequencing PCR 100
2.8 Variants Sequenced for Segregation in LUSG-03 and LUSG-
04 107
2.9 Sequencing Primers of ARL3 107
2.10 Variants Sequenced for Segregation in LUCC-15 107
2.11 Sequencing Primers of INPP5K 107
3.1 Clinical Features of Affected Individuals of Families with
Reported CYP1B1 Mutations 126
3.2 Clinical Features of all individuals homozygous for p.A115P
in PCG-06. 128
3.3 Clinical features of affected individuals in CYP1B1 linked
families with Novel mutations 134
3.4 Protein Prediction of ARL3 (Arg99Ile) by Various
Bioinformatics Tools 146
3.5 Clinical Features of Affected Individuals of LUCC-15 154
3.6 Protein Prediction of INPP5K (p.Ile50Thr) by Various
Bioinformatics Tools 155
viii
LIST OF FIGURES
Fig. No. Particulars Page
No.
1.1 Human Eye Ball 14
1.2 Human Eye Showing Cornea and Limbus 15
1.3 Layers of Cornea 16
1.4 A-Zones of lens showing Y-sutures, B- Magnified view of
lens showing subcapsular epithelial termination 19
1.5 Trabecular meshwork outflow pathway of aqueous humor. 20
1.6 Anterior Chamber Angle 21
1.7 Layers of retina. OS-Outer segment; IS-Inner segment. 23
1.8 Normal optic disc.
24
1.9 Diagram depicting aqueous outflow pathway in A) Normal
Eye, B) POAG and C) PACG. 30
1.10 Structure of human CYP1B1 gene and mRNA transcript 40
1.11 Metabolic pathway of CYP1B1 42
1.12 The Human Crystalline lens 59
1.13 The Snellen Chart 72
1.14 Goldmann Applanation Tonometer 75
1.15 The Normal Irido-corneal angle 77
1.16 Irido-corneal angle in Open-angle and Angle-Closure
Glaucoma 77
1.17 Fundus in a normal person and a glaucomatous patient 78
2.1 Diagrammatic Representation of PCR Program MultiCemb
54oC 95
2.2 Diagrammatic Representation of PCR Program Touchdown
64-54oC 95
2.3 Diagrammatic Representation of ExoSAP PCR Incubation 102
2.4 Variants Filtration Scheme for Whole Exome Sequencing 106
3.1 PCG-02 Pedigree with Haplotype 112
3.2 PCG-03 Pedigree with Haplotype 113
3.3 PCG-04 Pedigree 114
ix
3.4 PCG-15 Pedigree with Haplotype 115
3.5 Photographs showing affected individuals of PCG-15 115
3.6 Pedigree of PCG16 116
3.7 Photographs of affected children of PCG-16 116
3.8 PCG-17 Pedigree with Haplotype 117
3.9 PCG-19 Pedigree with Haplotype 118
3.10 Photographs of affected children of PCG-19 118
3.11 Chromatogram of p.R390H in CYP1B1 in PCG families 119
3.12 Haplotype analysis of PCG-07 120
3.13 Ocular photographs of affected individuals of PCG-07 120
3.14 Pedigree of PCG-10 121
3.15 Haplotype analysis of PCG-13 122
3.16 Pedigree of PCG-06 123
3.17 Photograph Photographs of affected individuals and normal
homozygous of PCG-06 128
3.18 Pedigree of PCG-08 family 129
3.19 Chromatograms of Normal, mutant and carrier of PCG-08 130
3.20 Photograph of patient IV:3 of PCG-08 130
3.21 Multiple Sequence alignment of p.G36D in various species 131
3.22 HOPE protein prediction of p.G36D 131
3.23 Pedigree of PCG-09 132
3.24 Chromatograms of PCG-09 133
3.25 Multiple sequence alignment of CYP1B1 proteins from
various species. 133
3.26 Pedigree of PCG-11 135
3.27 Pedigree of PCG-12 136
3.28 Pedigree of PCG-14 136
3.29 Pedigree of PCG-20 137
3.30 Pedigree of LUSG-03 140
3.31 Chromatograms of affected and carrier in LUSG-03 140
x
3.32 Fundus photographs of affected patients of LUSG-03 141
3.33 Optical Coherence Tomography showing retinal thickness of
affected patients of LUSG-03 142
3.34 Pedigree of LUSG-04 143
3.35 Chromatogams of heterozygous and homozygous individuals
of LUSG-04 143
3.36 Fundus photographs of affected patients of LUSG-04 144
3.37 Optical Coherence Tomography showing retinal thickness of
affected patients of LUSG-04 145
3.38 Multiple sequence alignment of ARL3 gene in various species 146
3.39 Diagrammatic depiction of ARL3 gene 147
3.40 ARL3 protein domain 147
3.41 ARL3 protein modelling obtained through Phyre-2 online tool 147
3.42 Pedigree of LUSG-02 148
3.43 Pedigree of LUSG-02 148
3.44 Pedigree of LUSG-08 149
3.45 Pedigree of LUCC-15 152
3.46 Chromatograms of wild-type, carrier and affected individuals
of LUCC-15 152
3.47 Multiple Sequence Alignment of INPP5K gene in various
species 153
3.48 Photographs of affected children of LUCC-15 154
3.49 Diagrammatic depiction of INPP5K gene 155
3.50 INPP5K protein with its domain 155
3.51 Pedigree of LUCC-01 156
3.52 Pedigree of LUCC-02 156
3.53 Pedigree of LUCC-04 157
3.54 Pedigree of LUCC-13 158
xi
LIST OF APPENDICES
Appendix Particulars Page No.
I Consent Form for Participation in Clinical Research 178
II Proforma for Identificaiton of Patients with Glaucoma 180
III Ophthalmological Examination &Assessment Proforma 181
IV DNA Extraction Sheet 183
V Optical Density, DNA Quantification & DNA Working
Dilution Preparation Sheet 184
VI List of Softwares/Website Accessed 185
VII List of Publications 186
xii
LIST OF ABBREVIATIONS AND SYMBOLS
A260 Absorption at 260 nanometer wavelength
A280 Absorption at 280 nanometre
ABCA4 ATP-binding cassette transporter
ABI Applied Biosystems
AC angle Anterior chamber angle
AMD Age-related macular degeneration
ASR Allele Size Range
Bp Base pair
CADD Combined Annotation Dependent Depletion
CF Counting fingers
cM Centi Morgan
C-Terminal Carboxy terminal
CYP1B1 Cytochrome P450, subfamily 1, polypeptide 1
dH2O Distilled Water
DNA Deoxyribonucleic Acid
dNTPs Deoxy Nucleoside Triphosphates
EDTA Ethylene Diamine Tetra acetic Acid
ELOVL4 Elongation of very long chain fatty acids like 4 gene
ExAC The Exome Aggregation Consortium
GWAS Genome wide association Studies
HM Hand movements
IOP Intra-ocular Pressure
JOAG Juvenile open-angle glaucoma
Kb Kilo base
kDa Kilo Dalton
LOD Likelihood of Odds
Mb Mega base
mg Milli gram
Ml Millilitre
mM Milli molar
mmHg Millimetres of Mercury
mRNA Messanger Ribonucleic Acid
MYOC Myocilin
Ng Nano gram
OD Optical Density
ONH Optic Nerve Head
p Short arm of chromosoma
PACG Primary Angle Closure Glaucoma
PCG Primary Congenital Glaucoma
PCR Polymerase Chain Reaction
PL Perception of light
pM Pico Mole
POAG Primary Open-angle glaucoma
q Long arm of chromosome
xiii
RGC Retinal ganglion cells
RPE Retinal pigment epithelium
RPM Revolutions per minute
SDS Sodium Dodecyl Sulphate
STGD Stargardt Disease
STRs Short Tandem Repeats
Taq Thermus Aquaticus
TE Buffer Tris EDTA Buffer
TNE buffer Tris Sodium EDTA Buffer
UCSC University of California, Santa Cruz
UV Ultra violet
VA Visual acuity
WDR36 WD repeat-containing protein 36
mg Micro gram
μM Micro mole
xiv
ACKNOWLEDGEMENTS
I offer most humble gratitude to Almighty Allah, the Most Beneficent,
the Most Merciful. He is the Creator of the universe and has bestowed upon the man
knowledge and wisdom to search for secrets and explore the natural phenomena to
use it for the benefit of human mankind. I earnestly bow before His compassionate
endowment.
The person to whom I am indebted the most is my Supervisor, Dr. Ali
Muhammad Waryah, Incharge, Molecular Biology and Genetics Department, Liaquat
University of Medical and Health Sciences, Jamshoro. I must say that this thesis owes
its existence to my great supervisor. With great patience and dedication, he has guided
me at every step in this entire journey. Without his kind support and encouragement,
the journey of my research work would have never ended up.
I am greatly thankful to Prof.Dr.Zubair M.Ahmed while working under
his supervision at his laboratory (Dr.Ahmed’s Lab) at department of
Otorhinolaryngology, School of Medicine, University of Maryland, Baltimore, USA.
It was really great working with him and learning new techniques related to genetics
and molecular biology. He has been great to show his confidence in me and infused
new passion for learning and becoming familiar with latest advancements in the field.
He is not only a great mentor but also a great human being who provided every
possible support whenever I needed in my difficult time during my stay in USA. He
will remain a source of inspiration for me throughout my life.
I am also indebted to my Co-supervisor, Dr. Ashok Kumar Narsani,
Professor, Department of Ophthalmology, Liaquat University of Medical & Health
Sciences, from whom I have received consistent warm support and advices. He has
been very kind and instrumental during clinical evaluation of the patients under study.
The completion of this thesis would not have been possible without
consistent support of my family, whereas, special thanks goes to my affectionate
father and my loving (late) mother for their encouragements and love. I am also
thankful to my eldest brother Prof.Dr.Saghir Ahmed Sheikh for his continuous
support and encouragement throughout my research work.
I have really been enjoying my Research work and I am thankful to all
my colleagues, faculty members, staff members from whom I got valuable services
over the years. I would say special thanks to Mr.Yar Muhammad Waryah and
Ms.Hina Shaikh, Ph.D Scholars at Molecular Biology & Genetics Department, for
their kind help in my PhD research work. I am also thankful to my friends Dr.Rizwan
Yousif, Dr.Sairah Yousif, Asaad Usmani, Dr.Muhammad Yaqoob Shahani and
Dr.Abdul Sattar Khan for being with me in all odds and encouraged me throughout,
while doing field work outside the laboratory and doing research work in the
laboratory.
I am grateful to Dr.Azam Memon, Dr.Amber, Mr. Ali Raza Rao,
Mr.Irfan Tufail, Mr.Muhammad Ali and other staff members of MBGD for being
very co-operative and polite towards me.
xv
In the last I apologize to anybody if he/she feels underrepresented or even has gone
overlooked.
Dr.Shakeel Ahmed Sheikh
xvi
ABSTRACT
The inherited visual disorders are leading cause of blindness all over
the world. In Pakistan, where consanguinity is common, high prevalence of
genetically transmitted visual disorders is a serious health problem from socio-
economic aspect. The present study was aimed to investigate the genetic and
molecular basis of various inherited visual disorders in Pakistani population. For this
purpose, we enrolled Twenty seven families suffering from Primary Congenital
Glaucoma, Stargardt disease and Congenital Cataract from different cities of Sindh
province. Blood samples were obtained from affected as well as normal individuals
from all enrolled families and detailed medical history and ophthalmological
examination were carried out.
All families were first subjected to genotyping to known/reported loci
or genes for Primary Congenital Glaucoma (PCG), Stargardt disease and Congenital
Cataract. Whole Exome Sequencing (WES) was done for the families found not
linked to any known locus/gene and candidate variants were subjected to direct
Sanger sequencing for segregation with the disease phenotype.
Seventeen families with PCG were enrolled for present study. Thirteen
PCG families were found linked to CYP1B1 gene. Sequencing further revealed two
novel mutations in CYP1B1i.e. p.G36D and deletion of 12 bp (p.G67-A70del) in
PCG-08 and PCG-09 respectively. p.R390H was found in eight PCG affected families
whereas p.E229K and p.R290fs*37 (c.868_869insC) was found once in two families.
p.A115P was found in one family with four phenotypically normal homozygotes as
well most probably due to either non-penetrance or reduced penetrance of CYP1B1.
Four families remained unlinked to any reported locus or gene for PCG. Five
Stargardt disease affected families and five families with Congenital Cataract were
screened for linkage to known or common loci/genes. After excluding linkage to
reported genes, WES for two Stargardt disease families revealed a novel gene ARL3,
which has not been reported earlier. Likewise we carried out WES for a single
congenital cataract and it was found linked to INPP5K, a novel gene and has recently
been reported in association with syndromic form of congenital cataract in 2017.
xvii
In brief, the study reports CYP1B1 as most common mutated gene for
patients with PCG in our population. Two novel mutations, a missense and a deletion
in CYP1B1 were found, in addition to already reported mutations in other PCG
families whereas a novel gene (ARL3) was identified in association with Stargardt
disease. In Congenital Cataract, INPP5K (a novel gene when it was first identified in
November, 2016) was found to be segregated with disease phenotype. All these novel
findings are suggestive of genetic heterogeneity of Pakistani population for inherited
visual disorders and genetic factors responsible for corresponding phenotype. The
data may be beneficial for public awareness and genetic screening of our population
to improve the prognosis of corresponding genetic disorder by early diagnosis. In
addition, the findings of this thesis may contribute to already existing data on
inherited visual disorders especially when no significant work in this regard has been
carried out in people of Sindh province of Pakistan.
1
INTRODUCTION
Inherited visual disorders constitute an important group among genetic
disorders which occur due to structural changes in genes destined to normal
development and function of ocular structures (Cascella et al., 2015b). A study
identified the cataract followed by glaucoma as the leading causes of visual
impairment worldwide with retinal disorders constituting a significant proportion
(Pascolini and Mariotti, 2012). It has been estimated that around 90% of people with
visual disorders belong to developing countries (WHO, 2010). In countries like
Pakistan, where consanguinity is common, prevalence of genetically transmitted
visual disorders is high as there is a strong association between consanguinity and
autosomal recessive diseases (Adhi et al., 2009). According to a study, the prevalence
of blindness in Pakistan is 2.7% with 11,40,000 people being blind (Jadoon et al.,
2006). Another study identified the causes of visual impairment in Pakistan with
Cataract being the first and glaucoma as the 4th leading cause (Dineen et al., 2007).
The present study was aimed to explore the genetic and molecular
basis of inherited visual disorders in Pakistani population in Sindh province. Various
families with primary congenital glaucoma (PCG), Stargardt disease and Congenital
Cataract were enrolled from different areas of the province. All families were visited
to record detailed medical history and blood samples were obtained after taking
informed written consent. DNA was extracted from blood samples and linkage
analysis tool was used to link the families to reported loci for PCG, Stargardt disease
and Congenital Cataract.
Glaucoma is a group of ocular disorders characterized by degeneration
of retinal ganglionic cells (RGC) leading to optic nerve atrophy and irreversible
blindness. It is one of the leading causes of irreversible blindness in the world. It is
usually associated with elevated intraocular pressure (IOP) which leads to
degeneration of retinal ganglionic cells and atrophy of optic nerve. Patients may be
asymptomatic during early course of disease but there is gradual loss of vision that
usually ends with irreversible blindness(Gauthier and Liu, 2017). On the basis of
etiology, glaucoma can be classified into primary and secondary glaucoma but it is
2
more elaborative to classify it on the basis of anatomy of anterior chamber’s angle
into open-angle glaucoma and closed-angle glaucoma (Bordeianu, 2014). Primary
open-angle glaucoma is the most common form of glaucoma and is usually
manifested during adult life. It is genetically heterogeneous and is associated with
complex mode of inheritance. (Reis et al., 2016). Closed-angle glaucoma is
characterized by narrow anterior chamber angle which causes obstruction to aqueous
outflow causing elevation in IOP (Ni Ni et al., 2014). Based on age of onset,
glaucoma can be classified into congenital glaucoma, juvenile-onset glaucoma and
adult-onset glaucoma. Primary congenital glaucoma (PCG) is the most common type
of glaucoma occurring during infancy and childhood with onset usually in first two
years of life. It accounts for 1-5% of all infantile and childhood glaucoma cases and is
transmitted in an autosomal recessive way. It usually manifests clinically as increased
IOP with protruding eye ball called bupthalmos, corneal edema, photophobia and
excessive lacrimation (Cascella et al., 2015b). The prevalence of PCG varies in
different parts of world with 1:10000 in Western populations to as high as 1:1200 in
populations with high consanguinity (Suri et al., 2015). In Pakistan, it is the fourth
leading cause of reported blindness whereas British Infantile and Childhood
Glaucoma Study has reported that PCG is nine times more common in Pakistani
children than in Caucasians (Bashir et al., 2014). Four chromosomal loci have been
identified in association with PCG i.e. GLC3A at 2p21, GLC3B at 1p36, GLC3C at
14q24.3 and GLC3D at 14q24.2-14q24.3. However, only two genes have been
identified with relation to PCG i.e. CYP1B1 on GLC3A and LTBP2 on GLC3D. In
spite of recent advancements in the field of molecular biology and genetics and
extensive research on genetics of glaucoma, very little information is available
regarding pathogenesis of PCG due to mutations in these genes (Firasat, 2016).
Of all four reported loci and two genes, CYP1B1 has been reported as
most common mutated gene for PCG and its prevalence in consanguineous and inbred
populations ranges between 70-100% (Abu-Amero et al., 2011). We enrolled 17 PCG
affected families and identified CYP1B1 mutations in 13 families (76%) segregating
with the disease phenotype. This is the highest prevalence of CYP1B1 mutations
reported from any population as previously highest CYP1B1 prevalence (70%) was
reported from Iran (Chitsazian et al., 2007). Linkage analysis studies in PCG families
identified p.R390H as predominant mutation in eight families whereas E229K and
3
c.868-869insC was found once in two families. Sanger sequencing further revealed
two novel mutations i.e. p.G36D in PCG-08 and a deletion of 12 bp i.e. p.G67-A70del
(c.198_209del12) in PCG-09. PCG-06 revealed p.A115P in all three affected
individuals but four phenotypically normal individuals were also found to be
homozygous for the same change which could be due to non-penetrance or reduced
penetrance. It was concluded that these phenotypically normal individuals
homozygous for the change will be followed up periodically for possible development
of late-onset glaucoma for next 10-20 years.
Stargardt disease is a type of retinal degenerative disorders which
constitutes an important cause of irreversible blindness worldwide. Almost all retinal
disorders are associated with damage to outer layers in retina such as photoreceptors
and retinal pigment epithelium (RPE) (Wiley et al., 2016). Its prevalence is
approximately 1:10,000 individuals and accounts for nearly 7% of all retinal
disorders. Clinically it usually manifests during second decade which makes it as one
of the most common cause of juvenile macular degeneration. It is heterogeneous both
clinically and genetically and may be transmitted either in autosomal dominant or
recessive mode. On the basis of gene involved, it is classified into three variants i.e.
STGD-1 is associated with mutations in ABCA4 (ATP-Binding Cassette, Subfamily
A, Member 4) gene at 1p22.1, STGD-3 with ELOVL4 (Elongation of very long chain
fatty acids like 4) at 6q14.1 and STGD-4 with mutations in PROM1 (Prominin 1)
gene at 4p15.32 (Tran et al., 2016). The usual symptoms in all three variants are loss
of central vision, paracentral scotomas, gradual adaptation to darkness and
photaversion. On fundoscopy, macular atrophy and yellow-white flecks which
represent lipofuscin accumulation within RPE can be seen (Strauss et al., 2016). The
mutations in genes for Stargardt disease result in impaired trafficking of visual cycle
metabolites across photoreceptors and RPE leading to degeneration of photoreceptors
and RPE by apoptotic cell death and subsequent visual loss (Wiley et al., 2014).
After confirming the diagnosis, five families with Stargardt disease
were first subjected to linkage analysis for three reported genes i.e. ABCA4, ELOVL4
and PROM-1. After excluding the linkage to these mentioned genes, Whole Exome
Sequencing was performed for two families (LUSG-03 and LUSG-04) and candidate
genes were sequenced for segregation. ARL3 was found to be segregated with disease
4
phenotype in both families. This is a novel genes and has not been reported earlier in
association with Stargardt disease.
Congenial cataract is the leading cause of treatable blindness in
childhood and is multifactorial in origin. It is defined as opacification of human eye
lens and occurs as an isolated ophthalmological abnormality in non-syndromic form
or co-exists with other ocular or systemic abnormalities as syndromic cataract. It may
be inherited as autosomal recessive, autosomal dominant or X-linked pattern (Francis
et al., 2000). Congenital cataract accounts for one third of all infantile blindness cases
with lens being affected in more than 60% of the cases (Chen et al., 2011). Its
prevalence ranges from 0.6-6/10,000 in developed countries to 5-15/10,000 in
developing countries (Chen C, 2015). Based on phenotype, it is classified into Non-
syndromic Congenital Cataract and Syndromic Congenital Cataract (Ma et al., 2016)
and on the basis of age of onset into Congenital Cataract and Senile Cataract (Shiels
and Hejtmancik, 2016). Congenital cataract is genetically and clinically
heterogeneous in nature. Till date, more than 40 loci and 26 genes have been found in
association with congenital cataract (Chen et al., 2011). Inspite of recent
advancements in surgical management of cataract, it is still a leading cause of low
vision and blindness in children (Shiels and Hejtmancik, 2016). After excluding the
linkage to common loci/genes for five congenital cataract families, Whole Exome
Sequencing was carried out for one family (i.e.LUCC-15). Sequencing done for all
possible pathogenic variants based on highest CADD score and protein prediction
which revealed segregation of INPP5K (Inositol polyphosphate-5-phosphatase K)
with disease phenotype in all individuals of the family. This gene has been recently
reported for syndromic congenital cataract in families from Pakistan and Bangladesh
(Osborn et al., 2017) & (Wiessner et al., 2017).
In short, this study demonstrates CYP1B1 as most common mutated
gene for PCG like other studies. Additionally, the identification of two novel
mutations in CYP1B1 and a novel gene i.e. ARL3 in Stargardt disease indicates
genetic heterogeneity of Pakistani population for genetic visual disorders. The results
of this thesis are novel and are valuable addition to the already existing data available
for PCG, Stargardt disease and Congenital Cataract. The data may be used for
understanding the pathophysiological phenomena involved in the etiology of these
5
disorders. In addition, the data could be used for creating public awareness about
these disorders among affected families by providing genetic counselling whereas the
identification of reported or novel mutations could be used for future genetic
screening in diagnosis of inherited visual disorders in our population to improve the
prognosis by early diagnosis.
6
CHAPTER-1
LITERATURE REVIEW
7
SECTION-I
INHERITED VISUAL DISORDERS
8
Human race has been blessed with five senses that make it possible to
create a protective environment suitable for its nurture. Sense of sight is most
important among those five senses as nearly 75% of information related to outside
world is dependent upon the ability of eyes to transform it in the form of image. Its
deterioration in anyway however leads to an impaired quality of life. This situation
has become more life threatening in developing countries due to lack of sufficient
health facilities. Due to such immense importance of eye as an organ, any damage or
malformation to any part of it may result in various clinical phenotypes that may be
associated with a range of visual loss to irreversible blindness. Although such
disorders may result due to certain environmental factors including infectious agents,
aging process but a large number of these disorders occur due structural changes in
genes responsible for normal development and functioning of ocular
structure(Cascella et al., 2015a).
According to the International Classification of Diseases, there are
4 levels of visual function (WHO, 2010):
1. Normal vision
2. Moderate visual impairment
3. Severe visual impairment
4. Blindness.
Moderate visual impairment together with severe visual impairment
has been grouped in category of “low vision”: low vision taken together with
blindness represents all visual impairment (WHO, 2010). Blindness is defined as
visual acuity of less than 20/200 on a Snellen’s eye chart or a visual field of less than
20 degrees (Gupta and Chen, 2016).
It is stated by World Health Organization (WHO) that approximately
284 million people worldwide suffer from some sort of visual impairment including
39 million blind people (Eballe et al., 2011) & (WHO, 2010).
The prevalence of visual disorders leading to blindness is not equally
distributed across the globe. In France it is estimated to be around 0.2%, in United
States it ranges between 0.2-0.4%, in Eastern Europe it is 0.7% whereas in Africa it is
9
1.4% , in Cameron it is approximately 1% (Eballe et al., 2011). In Pakistan, the
prevalence of blindness is approximately 2.7% (Jadoon et al., 2006).
A survey was conducted in Cameroon to identify the leading causes of
blindness. According to the survey Cataract was the major cause with a frequency of
50.1% and Glaucoma was the second leading cause of blindness with a frequency of
19.7%. However one must not forget the irreversible nature of blindness associated
with glaucoma due to late diagnosis and damage to optic nerve leading to its atrophy.
The other causes in the series included age-related macular degeneration, retinitis
pigmentosa. Interestingly there was no gender discrimination among affected
individuals(Eballe et al., 2011).
Pascolini et al determined the worldwide prevalence of visual
impairment and blindness in six WHO regions for three different age groups i.e. 0 to
14 years, 15 to 49 years and older than 50 years for both genders. They concluded that
glaucoma is the second leading cause of blindness worldwide whereas posterior
segment or retinal diseases constitute a significant proportion of visual impairment
etiology. They are of opinion that retinal diseases would share a major burden of
visual loss with the growth of ageing population. Their study further pointed out the
fact that glaucoma, retinal diseases and age-related macular degeneration together are
responsible for a greater proportion of visual impairment and blindness than the
infective ophthalmological causes (Pascolini and Mariotti, 2012).
It is estimated that around 90% of people with visual disorders live in
developing countries and they constitute 20% of global population. Due to increasing
elderly people in many countries, it is expected that the number of visually impaired
affected would increase due to ageing process and chronic eye diseases. In pediatric
group, around 19 million children are visually impaired of which 1.4 million children
are irreversibly blind who need rehabilitation services for personal and psychological
growth to prevent them becoming a social liability. Over the last twenty years the
number of visually impaired people has decreased but this is largely due to decrease
in infectious ophthalmological disorders causing irreversible blindness. This has been
made possible through various social and medical measures and improved
infrastructure in developed countries (WHO, 2010).
10
A Study has suggested that approximately 25% of childhood visual
disorders could be arrested and properly managed if proper screening facilities are
available. Screening school-age children for any visual impairment carries a great
significance in terms of early diagnosis of preventable blindness (Walker, 2009).
Some children may show signs of vision deterioration before attending to school, but
most of visual problem are diagnosed once children start attending their schools.
Many visual disorders may affect children which may be treated medically, surgicaly
while some remain untreatable by any means (G O Ovenseri-Ogbomo, 2010) (Abu
EK, 2015). It has been estimated that more than 1/4th of visual disorders can be
prevented if proper eye-screening programs exist at the level of schools. It is therefore
strongly recommended that all children should undergo a comprehensive visual
screening to improve their ocular health (Walker, 2009). Undetected visual disorders
leading to blindness in children are of concern as they directly affect the life of
patients and their families from socio-economic aspect as well (World Health
Organization [WHO], 1999). In relation to disability-adjusted life years (DALY),
childhood blindness is ranked as second leading cause of burden due to blindness
(Scheiman et al., 2002). It has been estimated by American Optometric Association
that 80% of a child’s learning is dependent on his visual acuity (Scheiman et al.,
2002), emphasizing the importance of early diagnosis and treatment of childhood
inherited visual disorders in order to either minimize or maintain the functional ability
of children intact (Casser, Carmiencke, Goss, Kneib, & Morrow, 2005; WHO, 1999).
Epidemiological data of visual disorders in children and other age groups is therefore
required for every region in the world to devise policies and allocate resources to
sufficiently provide ophthalmological health facilities to all.(G O Ovenseri-Ogbomo,
2010) (Abu EK, 2015).
The pathophysiology of many visual disorders leading to blindness is
still not clear. Furthermore it has remained an uphill task to identify individuals who
are at risk of developing visual disorders and blindness. Despite the availability of
modern treatment facilities both conservative and surgical, many people suffer from
visual disorders and progress to blindness (Semba et al., 2013).
The yearly health cost on visual impairment and blindness in 2010 has
been estimated to be around 3 trillion U.S.Dollars (Semba et al., 2013). The Vision
11
2020 initiative launched by WHO to improve the infrastructure for patients suffering
from visual disorders consists of three components i.e. elimination of preventable
blindness, human resource development and infrastructure development (Pizzarello et
al., 2004).
The increasing burden of inherited visual disorders in a country like
Pakistan with its specific cultural and religious background prompts the urgent need
of a system where such disorders could be reported at the earliest so that specific
rehabilitative efforts to prevent visual loss, educational services aimed at creating
awareness among people and support to the affected individuals could be initiated.
Inherited visual disorders remain a serious public health problem in
Pakistan due to high rate of consanguineous marriages. The diagnosis of inherited
visual disorders through molecular techniques may be helpful in identification of
carrier status and such information can be used to reduce the incidence of such
disorders in future. It is encouraging to note that inherited visual disorders have
acquired special attention by geneticists and molecular biologists for translational
gene therapy because of its appropriate anatomical structures, extent of disability
suffered by patients all over the world and earlier diagnosis owing to improved health
facilities especially in developed countries. These factors have led to enhancement of
increased number of research trials in order to find a cure for ocular disorders using
benefits of gene therapy. In this regard, identification of new loci, genes, mutations
together with modifier genes must be appreciated to correctly identify the role of
genes and environmental factors in pathophysiology of visual disorders(Cascella et
al., 2015b). It is important that all geographic areas must be genetically screened to
identify the varying nature of visual disorders for proper diagnosis and subsequent
management accordingly.
12
SECTION-II
EYE STRUCTURES RELATED TO
INHERITED VISUAL DISORDERS
13
1.1 Anatomy of Eye:
Eye is an organ of sight or vision. It functions to receive the external
light stimuli and encodes those stimuli through neurons of optic nerve to the visual
center located in occipital lobe in brain and enables a person to perceive the images
thus formed. A complete understanding of the structures of the eye is essential in
order to properly interpret the ocular disorders. A brief account of the anatomy of the
eye is described below to have a clear understanding of the inherited visual disorders
that follow afterwards (Riordan-Eva, 2011).
1.1.1 Embryology of the Eye:
Eye is derived from two of the three germinal layers i.e. surface
ectoderm and neural ectoderm (neural crest), and mesoderm. Endoderm does not
participate in the development of the eye. (Riordan-Eva, 2011)
1.1.1.1 Structures derived from Surface Ectoderm:
1. Lens
2. Lacrimal gland
3. Corneal epithelium
4. Conjunctiva and adnexal glands
5. Epidermis of eyelids
1.1.1.2 Structures derived from Neural Crest:
1. Corneal endothelium and keratinocytes
2. Trabecular meshwork
3. Stroma of iris and choroid
4. Ciliary muscle
5. Fibroblasts of sclera
6. The vitreous
7. Optic nerve meninges
1.1.1.3 Structures derived from Neural Ectoderm:
1. Optic cup and optic vesicle and therefore gives rise to retina and retinal
pigment epithelium (RPE)
14
2. Pigmented and non-pigmented layers of ciliary epithelium
3. Posterior epithelium
4. Dilator and sphincter muscles of iris
5. Optic nerve fibers and glia.
1.1.2 Orbit:
It is a pyramid formed by four walls and converging posteriorly. Its
structure can be compared with that of a pear having optic nerve as its stem. The
average volume of an adult orbit is around 30ml whereas eye ball occupies its 1/5th
space only. The remaining space is occupied by fat and muscle. Ophthalmic artery
(first branch of internal carotid artery inside cranium) supplies the orbit and its
structures (Riordan-Eva, 2011).
1.1.3 The Eyeball:
An average adult eyeball is roughly spherical in shape and its
anteroposterior diameter is approximately 24mm (Fg1.1) (Riordan-Eva, 2011).
Fig-1.1: Human Eye Ball. Adapted from: (Riordan-Eva, 2011)
15
1.1.4 Conjunctiva:
It is a thin and transparent mucous membrane covering the posterior
surface of the lids whereas anteriorly it covers the sclera. Its lining epithelium
contains 2-5 layers of stratified columnar epithelium. Conjunctival arteries, which are
derived from anterior ciliary and palpebral arteries, are responsible for its
nourishment.
1.1.5 Sclera:
It is a thin and dense outer fibrous protective layer of the globe and
consists of collagen. It is white in color and continues with cornea anteriorly (Fig-1.2)
and with dural sheath of the optic nerve posteriorly. A thin layer of fine elastic tissue
lines the anterior surface of sclera and is called episclera. Embedded inside episclera
are many blood vessels which provide nourishment to sclera. Many parallel and
interlacing collagen bundles (10-16 µm thick and 100-140 µm wide) form the
structure of sclera. Its structure is very much similar to that of cornea but unlike
cornea it is opaque due to irregular alignment of collagen fibres, very high content of
water and less proteoglycans.
Fig-1.2: Human Eye Showing Cornea and Limbus
16
1.1.6 Cornea:
It is the primary barrier of eye against infectious agents and is the first
structure present at the front of eye ball (Fig-1.2). It is avascular and is completely
transparent in nature. It constitutes the first refractive surface along with tear film for
the light rays entering the eye globe. The average diameter of cornea in adults is about
11.5 to 12.0 mm along horizontal axis whereas vertically it ranges between 10.5 to
11.0mm (DelMonte and Kim, 2011). The normal horizontal diameter in newborns
ranges between 9.5-10.5 mm whereas in infants it is between 10-11.5mm. Horizontal
corneal diameter greater than 12mm in newborns may be abnormal and should be
investigated for primary congenital glaucoma or bupthalmos (Chan JYY, 2015). It is
thicker at the periphery than at the center thus creating an aspherical optical surface. It
is inserted into sclera (Fig-1.2) at limbus (the junction of cornea and sclera). It
consists of five layers from front to backward (Fig-1.3):
1. Epithelium
2. Bowman’s layer
3. Stroma
4. Descement’s membrane
5. Endothelium
Fig-1.3: Layers of Cornea.
The corneal epithelium is stratified squamous non-keratinizing in
nature and consists of 5-6 layers of cells. During fetal life, it is derived from surface
ectoderm germ layer at around 5-6 weeks of gestation. The irregularities along the
17
epithelial surface are made smooth by a uniform film of tear and is important optically
as it provides more than 50% of the refractive power of human eye (DelMonte and
Kim, 2011).
Bowman’s layer is an avascular layer and is considered a modified
portion of the stroma. Approximately 90% of corneal thickness is due to corneal
stroma and is composed of collagen fibrils. Descement’s membrane is about 3 µm
thick at the time of birth but continues to grow in thickness throughout life and may
reach upto 10-12 µm in adults. The endothelium consists of single layer of cells and
their loss of function could lead to corneal edema.
Cornea derives its nourishment from vessels present in limbus, from
aqueous and tears. Superficial cornea is also nourished by oxygen present in the
atmosphere. The transparent nature of the cornea can be attributed to its uniformity in
structure, avascularity and deturgescence.
1.1.7 Uvea:
Uvea or uveal tract consists of three structures i.e. iris, ciliary body and
choroid anteriorly to posteriorly. It forms the middle vascular layer of eyeball whereas
anteriorly it is protected by cornea and sclera. Retina gets its blood supply from uvea.
1.1.8 Iris:
It is shallow cone-like structure with a central aperture called pupil. Iris
is situated just in front of the lens and separates the anterior chamber from posterior
chamber. Both these chambers contain aqueous humor which passes freely through
pupil. Iris controls the amount of light entering the eye by adjusting the size of the
pupil through a balance between sympathetic and parasympathetic activity.
1.1.9 Ciliary Body:
It is triangular in shape extending from anterior end of choroid to the
root of iris. It is composed of three layers of fibres i.e. longitudinal, radial and
circular. Their function is to adjust the tension on the lens capsule in order to focus
the near and distant objects accordingly. The longitudinal fibres are inserted into the
18
trabecular meshwork and may influence its pore size. The blood supply is provide
through major circle of iris.
1.1.10 Choroid:
It is posterior segment of uvea and is situated between retina and
sclera. It harbors choroidal blood vessels in three layers i.e. large, medium and small.
Posteriorly choroid is attached to optic nerve whereas on its anterior side it is attached
with ciliary body. Its main function is to provide nourishment to outer portion of
retina.
1.1.11 Lens:
Lens is a transparent, clear and avascular tissue which consists of
epithelial and fiber cells (Semba et al., 2013)). It is biconvex in shape and completely
colorless and transparent structure. It is about 4mm thick with a diameter of 9mm.
Lens is located behind iris, being suspended by zonule, connecting it with the ciliary
body. Zonule is a suspensory ligament consisting of fibrils which originate from
ciliary body and are inserted into the equator of the lens.
It consists of 65% water, about 35% proteins and trace of some
minerals with potassium being present in the highest amount. Ascorbate and
glutathione are also present in the lens both in reduced and oxidized states. Lens
separates the posterior chamber containing aqueous anteriorly from the vitreous
located posteriorly. The lens capsule consists of a semipermeable membrane that
could allow the movement of water and electrolytes. Anteriorly, a sub-capsular
epithelium is present. Aging results in production of sub-epithelial lamellar fibers that
making lens bigger in size with reduced elasticity. The nucleus and cortex are formed
by long concentric lamellae. The joining of these lamellae form Y-shaped sutures
which can be viewed with a slit-lamp. The lens does not contain any pain fibres,
blood vessels or nerves.
19
Fig-1.4: A-Zones of lens showing Y-sutures, B- Magnified view of lens showing subcapsular
epithelial termination. Adapted from (Riordan-Eva, 2011)
1.1.12 Aqueous Humor and its Outflow Pathway:
It is a clear liquid formed by ciliary body and is present both in anterior
and posterior chambers of eyeball. Total volume of aqueous humor is around 250 µL
and its average rate of synthesis is approximately 2.5 µL/min. There is diurnal
variation in its rate of production. The composition of aqueous is comparable with
plasma but aqueous contains more quantity of ascorbic acid, pyruvic acid and lactic
acid and low amounts of protein, urea and glucose so that its osmotic pressure is
somewhat higher than that of plasma.
Aqueous is an ultrafiltrate of plasma formed by ciliary body, and
modified in its composition due to barrier function and secretory processes of ciliary
epithelium (Fig-1.5). From posterior chamber it enters into anterior chamber through
pupil and finally drains through trabecular meshwork located in the anterior chamber
angle. The trabecular meshwork is composed of collagen fibers and elastic tissue. The
collagen fibers and elastic tissue are covered by trabecular cells and together they
form a filter with a gradual decrease in their pore size as Schlemm canal is
approached. Contraction of ciliary muscle leads to increase in the size of pores and
hence increased aqueous drainage. Approximately 30 collector channels and 12
aqueous veins are present in endothelial lining of Schlemm’s Canal which conduct
this drained fluid to venous system. Some aqueous also escapes into suprachoroidal
20
space through the bundles of ciliary muscle and then ultimately into the veins of
ciliary body, choroid and sclera.
Fig-1.5: Trabecular meshwork outflow pathway of aqueous humor.
Adapted from (Goel et al., 2010)
1.1.13 Anterior Chamber Angle:
Anterior chamber (AC) angle is located at the junction formed by
cornea and the root of iris. The main structures forming anterior chamber angle are
Schwalbe’s line, trabecular meshwork and scleral spur (Fig-1.5).
Schwalbe’s line is a terminal extension of corneal endothelium. The
trabecular meshwork forms a filter for drainage of aqueous humor. Trabecular
meshwork also contains the longitudinal fibers of ciliary muscle. Scleral spur can be
considered an extension of sclera between the ciliary body and Schlemm’s canal.
21
Fig-1.6: Anterior Chamber Angle. Adapted from (Riordan-Eva, 2011)
1.1.14 Intra-Ocular Pressure (IOP):
The intra-ocular pressure is the pressure difference across cornea. It is
generated due to flow of aqueous humor against resistance and is responsible for
maintaining the shape and refractory properties essential for proper vision. It is
measured by corneal applanation or indentation. It is the sum of pressure in anterior
chamber and the pressure external to eye which is exactly equal to the atmospheric
pressure (Jonas et al., 2015). The circulating aqueous humor provides nourishment to
transparent structure of the eye, cornea and lens. Aqueous humor also provides a
refractive index of 1.33, thus contributing to optical system of the eye (Kaufman,
2003).
1.1.15 Vitreous:
It is a clear and avascular structure, gelatinous in nature and makes up
the 2/3rd of the volume of the eye. It occupies the space between retina and lens. It is
approximately 99% water and remaining 1% is composed of collagen and hyaluronate
which gives the vitreous its gel-like consistency (Riordan-Eva, 2011).
1.1.16 Retina:
It is thin sheet made up of many layers and derived from neural tissue.
It lines the inner walls of posterior two thirds of eye globe. Anteriorly it extends from
ora serrata (about 6.5 mm behind Schwalbe’s line). Retina is 0.1mm thick at its point
of origin from ora serrata but it is 0.56 thick on its posterior pole. The center of
22
posterior retina contains macula (5.5-6.0 mm in diameter) and is defined as an area of
retina which contains more than one-cell ganglion layer. In contrast, fovea which is
1.5 mm in diameter is defined histologically by thinning of outer nuclear layer and
absence of other parenchymal layers (Riordan-Eva, 2011).
Retina is composed of 10 layers starting from inner aspect in following order (Fig-
1.7):
1. Internal limiting membrane (ILM)
2. Nerve fiber layer
3. Ganglion cell layer (GCL)
4. Inner plexiform layer (IPL)
5. Inner nuclear layer (INL)
6. Outer plexiform layer (OPL)
7. Outer nuclear layer (ONL)
8. External limiting membrane
9. Photoreceptor layer of rods and cones
10. Retinal pigment epithelium (RPE)
Broadly, all these above layers can be categorized into two layers i.e.
the neural layer and pigment cell layer. The neural layer containing neurons in
alternating layers, is responsible for processing light and carrying sensory information
to visual center in the brain. The pigment layer called retinal pigment epithelium is
important for maintenance of photoreceptors (rods and cones). Outer segments of
photoreceptor cells contain a stack of discs with visual pigments (rhodopsin)
necessary for process of phototransduction (process of converting light stimulus into
action potential). The discs within the outer segments of photoreceptor cells are
phagocytosed by RPE as they age and are replaced by newer discs formed and
transported from inner segments (Loh et al., 2009).
23
Fig-1.7- Layers of retina. OS-Outer segment; IS-Inner segment.
Adapted from (Boye et al., 2016)
1.1.17 Optic Disc:
Optic disc or more precisely optic nerve head is located posteriorly in
the eye globe on its nasal side. It is the entry point for three important structures i.e.
axons of retinal ganglion cells, central retinal artery and central retinal vein (Jonas et
al., 2015). Examination of optic disc is an important component of ophthalmological
evaluation to diagnose and follow the progression of glaucoma in any patient. The
optic disc has two components, the optic cup and neuro-retinal rim. The cup is devoid
of axons and represents the central depression whereas neuro-retinal rim is a band of
tissue between the cup and disc margin. Cup to disc ratio or simply CDR is a measure
of the size of cup to that of disc (Fig-1.8). It is a useful measure to assess the damage
incurred due to glaucoma. In glaucoma patients, size of cup progressively becomes
larger due to loss of retinal ganglion cells (RGC) and their axons greatly because of
increased intraocular pressure. Usually CDR up to 0.3 is considered normal whereas
in glaucoma patients one may find total cupping of the disc which corresponds to
CDR of 1.0 (Tatham et al., 2013)
24
Fig-1.8: Normal optic disc. Adapted from (Bourne, 2012)
25
SECTION-III
GLAUCOMA, STARGARDT DISEASE
&
CONGENITAL CATARACT
26
1.2 GLAUCOMA
Glaucoma is a group of optic neuropathies associated with both
structural and functional defects in eye. It is the second leading cause of blindness
worldwide and most common cause of preventable blindness (Butt NH, 2016). There
is progressive loss of retinal ganglion cells (RGC) present in the inner retinal layers
and their axons within the optic nerve head (ONH)(Werkmeister et al., 2013). The
structural damage almost invariably leads to functional damage such as decrease in
visual acuity and ultimately irreversible blindness (Marvasti et al., 2013). The
structural damage due to glaucoma includes retinal nerve fiber layer defects, thinning
of nuero-retinal rim and excavation of optic nerve head termed as cupping of optic
disc. Among several identified risk factors for glaucoma such as elevated intra-ocular
pressure (IOP), family history of glaucoma, increasing age, gender, and systemic
hypertension etc, elevated intra-ocular pressure alone is the most important
modifiable risk factor. Reducing the elevated IOP remains the corner stone of any
form of treatment such as medical, laser or surgical (Butt NH, 2016).
1.2.1 Prevalence:
Glaucoma is the second leading cause of irreversible blindness in the
world (Kumbar et al., 2015). It has been estimated that more than 12 million people
have already become blind due to glaucoma all over the world. Surveys in various
populations have suggested its prevalence from 0.5-0.7% (Suri et al., 2015). It has
further been estimated that more than 58 million people would have been suffering
from some type of glaucoma by the year 2020 and nearly 10% of these would be
affected bilaterally. Most of the people would have suffered from some visual loss at
the time of diagnosis that could have serious impact on their quality of life. The
irreversible blindness is the ultimate outcome if the disease remains unchecked due to
its silent nature (Tatham et al., 2013). A survey conducted in Nigeria in 2015 finds its
prevalence at 5.02% with one in every five persons with glaucoma was blind (Kyari et
al., 2015). According to a recently published study, in Nigeria more than one million
are blind with glaucoma accounting for approximately 16% of total cases (Abdull
MM, 2016). According to a National Health Survey which was conducted in 2003 in
Pakistan, the incidence of blindness is approximately 2.7% with Glaucoma as the
fourth leading cause of irreversible blindness in Pakistan. British Infantile and
27
Childhood Glaucoma Study has reported the incidence of Primary Congenital
Glaucoma nine times higher in Pakistani children than Caucasians (Bashir et al.,
2014).
An American study has estimated that by 2020, approximately 80
million people will be suffering from glaucoma and more than 11 million would be
bilaterally blind due to some type of glaucoma. It has further been reported that more
than 2 millions of American above 40 years of age are suffering from glaucoma and
nearly 50% of these cases are undiagnosed or have not received any treatment. In
some ethnic groups in United States, glaucoma is the most common cause of
blindness even more than diabetic retinopathy, age-related macular degeneration or
even cataracts. For example, in American Blacks and Hispanics, blindness due to
glaucoma accounts for more than 25% of all cases and this is highest proportion of
blind people in any community due to glaucoma in USA. Even those patients who are
not blind due to glaucoma usually suffer from some functional limitation such as
driving or other activities that may require intact visual acuity. The estimated annual
expenditure on Glaucoma patients within USA accounts for one of the high spending
accounts(Gupta and Chen, 2016).
The risk factors for glaucoma include positive family history of
glaucoma, age, race and ethnicity, and diabetes mellitus. Individuals having a positive
family history of glaucoma have increased chance of developing it as compared to
normal individuals. The incidence of glaucoma increases with increasing age and is
more common in ethnic groups e.g. angle-closure glaucoma is more prevalent in
people of Chinese ethnicity(Gupta and Chen, 2016).
Primary Congenital Glaucoma (PCG) induced blindness has been
estimated to be responsible for 18% children in institutions for blind and 5% pediatric
blindness all over the world. The main factors contributing to this high degree of
blindness in children are delayed presentation due to decreased awareness among
masses and few options as far as the management of the disease is concerned (Moore
et al., 2013). PCG is the most severe form of glaucoma that usually manifests in first
three years of life and is characterized by increased intra-ocular pressure, corneal
edema, excessive tearing (lacrimation), photophobia, enlargement of ocular globe
(Bupthalmos), corneal opacification and optic nerve damage (Suri et al., 2015).
28
1.2.2 Classification of Glaucoma:
Several efforts have been made to classify glaucoma but only two
systems of classification have gained popularity. First of these named Donder’s
classification is based on clinically available data and does not include into account
the anterior chamber’s angle (the angle between iris and posterior trabecular
meshwork). The second classification known as Gonioscopic Classification is more
elaborative as it classifies glaucoma on basis of view of anterior chamber angle as
follows (Bordeianu, 2014):
1. Primary open angle glaucoma (POAG)
2. Primary angle closure glaucoma (PACG)
3. Secondary Glaucoma
4. Primary Congenital Glaucoma (PCG)
In secondary glaucoma, where the cause of glaucoma is known, it is usually present
along with other manifestations in the form of a syndrome. The word primary
indicates that the cause is unknown and it is further sub classified into three types on
the basis of anatomy of anterior chamber angle (Suri et al., 2015).
A) Primary Open Angle Glaucoma:
Primary open angle glaucoma (POAG, OMIM 137760) is the most
common type of glaucoma. Of all the sub-types of glaucoma, the primary open-angle
glaucoma has posed difficulties from its diagnosis to treatment. It is a chronic disease,
insidious in onset and usually progresses over the years without any manifestation.
When a patient presents with any detectable visual field defects, more than one third
of nerve fibers in optic nerve have already been damaged and more than 90% of the
axons are lost (Kumbar et al., 2015).
POAG is most common type of glaucoma in Western and some other
populations with considerable variations both in terms of severity and phenotype (Suri
et al., 2015). In USA & Europe, POAG is seven times more prevalent than primary
angle closure glaucoma (PACG) (Gupta and Chen, 2016). It has been revealed
through gonioscopy that the anterior chamber angle is open like as in normal
individuals creating no obstruction to drainage of aqueous humor (Fig-1.9). Even then
most of the patients affected with POAG have increased intra-ocular pressure possibly
due to yet some unknown anomalies in trabecular meshwork which could obstruct the
29
aqueous outflow. POAG patients with high IOP are diagnosed as high tension POAG
patients. Other POAG patients, where IOP is within normal limits, are termed as
Normotensive Glaucoma patients. On the contrary, yet there is another class of
patients where IOP remains elevated but there is no associated optic nerve damage.
Such patients are classified as having Ocular hypertension. In classical POAG,
symptoms usually appear after 40 years of age. But in some patients with POAG,
symptoms may appear in childhood or before 40 years of age. These patients are
categorized into a sub-class of POAG termed as Juvenile Open-angle Glaucoma.
Symptoms in JOAG patients are usually more severe than in patients with POAG
(Suri et al., 2015).
30
Fig-1.9: Diagram depicting aqueous outflow pathway in A) Normal Eye, B) POAG and C)
PACG. Adapted from (Gupta and Chen, 2016)
POAG and PACG may progress to chronic form associated with
irreversible blindness if not checked within time. They are usually associated with
asymmetrical loss of peripheral vision which is not felt by the patient due to
compensatory effect of the other eye. That’s the reason that POAG is often detected
31
as an incidental finding during routine ophthalmological examination due to its
insidious and silent pathological nature (Gupta and Chen, 2016).
Several loci (Thirty three) have been shown to be associated with
POAG from GLC1A to GLC1O, but only four causative genes have been identified so
far. These genes are MYOC (GLC1A, OMIM 601652), OPTN (GLC1E, OMIM
602432), WDR36 (GLC1G, OMIM 609669) and NTF4 (GLC1O, OMIM 613100).
MYOC codes for myocilin, OPTN for optineurin, WDR36 for WD repeat containing
protein 36 and NTF4 for neurotrophin-4. Their functions in relation to eye are still
unknown and they all together account for less than 10% of all POAG patients.
MYOC was the first gene to be identified in POAG patients and it has also been
associated with sporadic cases of POAG and patients with Juvenile-onset glaucoma.
Myocilin is bipartite protein having a myosin-like NH2 terminal domain and an
olfactomedin homology COOH terminal domain. Most MYOC associated mutations
affect olfactomedin domain. CYP1B1 has also been implicated in POAG patients
independently although digenic expressivity of both MYOC and CYP1B1 in POAG
patients has been suggested by some researchers(Suri et al., 2015).
B) Primary Angle Closure Glaucoma:
The characteristic feature of closed angle glaucoma is obstruction to
flow of aqueous humor through angle in the anterior chamber located between iris and
cornea known as irido-corneal angle (Fig-1.9). The main factor that plays an
important role in the pathogenesis is the width of the angle. A narrow angle causes
obstruction to the flow of aqueous humor and leads to increase in intra-ocular
pressure (IOP) that ultimately causes angle closure glaucoma (Ni Ni et al., 2014).
When patients with PACG are viewed on gonioscopy, they have closed
or very narrow anterior chamber angle. Acute PACG is usually characterized by
sudden rise in IOP accompanied by severe orbital pain, redness of eyes, appearance of
halos and blurred vision. In some patients of PACG, gonioscopy reveals closed angle
but there is no associated optic nerve damage or atrophy. Such patients are termed as
primary angle closure suspects (PACS). PACG is comparatively more prevalent in
Far East Asia than in Caucasian and African populations. It has been reported from
some Far East Asian regions such as China, India and Singapore that bilateral
32
blindness due to PACG is more common than due to POAG or PCG. Only two
countries (Mongolia and Myanmar) in the World have reported the highest prevalence
of PACG than any other type of glaucoma in their regions(Suri et al., 2015).
C) Normal Tension Glaucoma:
Normal tension glaucoma (NTG) is a special type of glaucoma and is
an optic neuropathy having multifactorial etiology. Although intra-ocular pressure
remains below the upper normal limit yet it manifests with loss of visual field owing
to death of retinal ganglion cells (RGCs). The pathophysiology of normal tension
glaucoma is not clearly understood but it has been suggested that factors associated
with ocular blood flow play an important role in its development and progression.
These findings have further been supported by newly developed ocular imaging
techniques such as fluorescent angiography, color Doppler imaging, magnetic
resonance imaging and laser speckle flowgraphy. These imaging techniques have
demonstrated impairment in ocular vascular auto regulation mechanism. The exact
pathway of how these factors lead to visual loss in normal tension glaucoma is not
clearly known, but it has been hypothesized that free radicals induced oxidative stress,
vascular spasm, and vascular endothelial dysfunction might be responsible for the
visual loss associated with it. Several vascular systemic disorders are thought to be
associated with normal tension glaucoma including Alzheimer’s disease, migraine,
hypotension and primary vascular dysregulation. Important risk factors associated
with normal tension glaucoma include race (more common in Japan), gender
(frequently seen in women) and low blood pressure (Fan et al., 2015).
D) Secondary Glaucoma:
Secondary types of glaucoma are usually accompanied by some other
clinical manifestations as well, although elevated intra-ocular pressure is a universal
finding in all cases. There are many types of secondary glaucoma such as
Pseudoexfoliation (PEX) syndrome which is characterized by deposition of PEX type
material in anterior chamber of eye. Other types of secondary glaucoma include
neovascular glaucoma manifested by formation of new blood vessels in eye,
pigmentary glaucoma showing pigmentary granules entering aqueous humor, uveitic
glaucoma characterized by inflammation of uvea and traumatic glaucoma that occurs
due to any kind of injury to eye. Secondary glaucomas also include drug induced
33
glaucoma or glaucoma occurring as a result of long-standing uncontrolled diabetes
mellitus (Suri et al., 2015).
E) Primary Congenital Glaucoma:
Primary congenital glaucoma (PCG) is a very rare sub-type of
glaucoma but it is most common type of glaucoma occurring in infancy and
childhood. PCG is responsible for 1-5% of all cases of glaucoma and is inherited in an
autosomal recessive pattern. Clinically, PCG manifests as increased intra-ocular
pressure more than 21 mmHg, protruding eye ball (Buphthalmos), corneal edema (or
corneal opacification in later stages), photophobia and excessive lacrimation.
Fundoscopic examination reveals optic cupping causing decreased CD (cup to disc)
ratio (Cascella et al., 2015b). Cloudy cornea and bupthalmos are the most common
manifestations which are present in more than 40% of patients at the time of
presentation. Other signs and symptoms may include lack of eye contact, facial
birthmarks, pupillary abnormalities and nystagmus, but they are found to be present in
less than 3% of all patients(Chan JYY, 2015).
PCG can further be classified into three sub-types depending upon the age of onset as
follows:
1. Neonatal, when PCG occurs either at birth or during first month of life
2. Infantile, when diagnosed from first month till two years after birth.
3. Late onset, when PCG is diagnosed after two years of birth.
PCG usually affects both eyes in 70-80 % of cases (Zagora et al., 2015).
Prevalence of PCG in Western populations has been estimated at
1:10,000 where as in inbred populations with high consanguinity, it has been
estimated at around 1:1200 to 1:3300 (Suri et al., 2015). In North America and other
Western developed countries it ranges from 1 in 10,000 - 68,000 live births. A general
ophthalmologist working in a non-specialist center usually comes across a case of
congenital glaucoma once every five years (Moore et al., 2013).
Although the data from developing countries is limited and
unorganized yet in some developing countries, it is much more prevalent
(approximately 10 times more common) in certain ethnic and religious groups where
34
consanguinity is common owing to its exclusive autosomal recessive nature of
inheritance. In regions of sub-Saharan Africa, the condition is responsible for 0.4% to
4% of all new cases of glaucoma. The highest prevalence has been reported from
Saudi Arabia and Slovakian Gypsies where 1 in every 2500 and 1 in every 1250
individuals are affected with primary congenital glaucoma respectively (Moore et al.,
2013). The prevalence in Chinese population ranges from 1 in 5000 to 1 in 25000.
Although PCG is transmitted in an autosomal recessive pattern, but it
has been shown that it is familial in nature in 10-40% of cases with variable
penetrance (Sarfarazi and Stoilov, 2000). Its prevalence is highest in populations
having increased rate of consanguinity(Suri et al., 2015). Consanguinity has been
attributed as the main reason for familial cases. However high rate of concordance
among monozygotic twin and discordance among dizygotic twins has also been
reported (Sarfarazi and Stoilov, 2000). Few authors have doubted the autosomal
recessive mode as the only mode of inheritance in patients with primary congenital
glaucoma (Jay, 1978) & (Demenias, 1979). Their observations were based on unequal
distribution of the disease in terms of gender in affected individuals. They demanded
an explanation over presence of the disease twice among boys as compared to girls
and also to why lower numbers of siblings were affected than expected. Furthermore
many families with primary congenital glaucoma were reported showing the presence
of the disease in successive generations, a feature consistent with autosomal dominant
mode of inheritance. All above observations and discrepancies, however can be
explained through the phenomenon of genetic heterogeneity (Sarfarazi and Stoilov,
2000).
i) Pathophysiology of Primary Congenital Glaucoma:
It was Hippocrates who first documented the abnormal enlargement of
eyeballs in infants 400 BC. Afterwards in 18th century, Berger proposed the elevated
intra-ocular pressure as the cause of eye globe enlargement. Then in 1869, von Muralt
established bupthalmos as one of the types of glaucoma (Chan JYY, 2015). According
to Westerlund, Grelios in 1836 first reported the endemic occurrence of the disease in
Jewish population of Algiers (Westerlund, 1947) & (Sarfarazi and Stoilov, 2000). In
1842, Junkgen suggested the autosomal recessive mode of inheritance for patients
35
affected with primary congenital glaucoma. He came across a family with seven
affected offsprings with two normal siblings and parents. These two studies
established the congenital glaucoma as an inherited disorder (Sarfarazi and Stoilov,
2000).
Many risk factors have been associated with glaucoma such as age,
ethnicity, and family history, but the most important risk factor for its development
and progression is the elevated intra-ocular pressure (Thomas Yorio, 2008). Increased
intra-ocular pressure in PCG occurs due to a phenomenon known as
trabeculodysgenesis. According to this process, there is an obstruction to drainage of
aqueous humor due to enlarged bundles of trabecular meshwork together with
defective development of iris, ciliary body and other structures forming anterior
chamber angle leading to increased IOP and enlargement of eyeball (Cascella et al.,
2015b).
The main abnormality that has been detected almost in all patients with
PCG is defective development of anterior chamber angle, which is considered in
terms of evolution a modified ocular structure. Like many other structures such as
skeletons and nerves of craniofacial and cervical region, eye is also derived from
neural crest cells. This shows that molecular defects responsible for pathogenesis of
PCG may have some links with early stages of differentiation of neural crest cells.
Various authors have suggested different views about the possible pathogenesis of
PCG. Barkan was of the view that outflow of aqueous humor at anterior chamber
angle is blocked by presence of endothelial membrane (Barkan, 1954). Maumenee
suggested that posterior displacement of scleral spur together with abnormal insertion
of ciliary muscle fibers cause blockade to drainage of aqueous humor at anterior
chamber (Maumenee, 1958). Anderson attributed to unusual exposure of trabecular
meshwork due to failure of maturation of iris and anterior ciliary body. Earlier, many
authors held atrophy of structures responsible for deepening of anterior chamber
angle. Inspite of all these studies the actual pathogenesis of PCG is still speculative
and elusive as it is impossible to trace back the pathogenesis to exact triggering
molecular defect (Sarfarazi and Stoilov, 2000). It has been suggested that optic nerve
atrophy in glaucoma may be due to misbalance among cerebrospinal fluid pressure,
intro-ocular pressure and systemic blood pressure (Jonas et al., 2015).
36
The role of proteins encoded by the known genes in the pathogenesis
of primary congenital glaucoma is still not clear. Bouhenni RA et al (2010) tried to
identify the role of proteins with PCG associated genes by comparing the composition
of aqueous humor of PCG affected patients with that of patients undergoing cataract
surgery using global proteomics approach. They concluded that in patients with PCG,
levels of three proteins were considerably higher namely Apolipoprotein A-IV
(APOA-IV), Albumin and Antithrombin compared to the controls. They also found
significant lower quantities of Transthyretin, Prostaglandin-H2 D-isomerase, Opticin
and interphotoreceptor Retinoid binding protein in aqueous humor of PCG patients.
As these proteins have been found to be associated with Alzheimer’s disease owing to
their role in binding and transport of retinoic acid, it was suggested that availability of
retinoic acid in anterior chamber could be affected by the same pathologic changes
(Bouhenni et al., 2011).
ii) Genetics of Primary Congenital Glaucoma:
Human genome organization has established a specific nomenclature
for glaucoma associated loci and genes. According to this nomenclature, GLC
represents all genes related to glaucoma whereas 1, 2, 3 stand for primary open-angle
glaucoma, primary angle-closure glaucoma and primary congenital glaucoma
respectively. A, B, C and D indicate loci and genes that have been reported for each
type of glaucoma. Till date, four loci have been mapped in relation with PCG i.e.
GLC3A, GLC3B, GLC3C and GLC3D (Cascella et al., 2015b).
a) GLC3A Locus and Role of CYP1B1 in PCG:
This is the first locus that was identified in association with primary
congenital glaucoma by Sarfarazi et al in 1995 using both candidate regional and
general positional mapping strategies in a study that included PCG patients (Sarfarazi
et al., 1995). Later in 2000, it was Sarfarazi again who mapped the gene (i.e.
CYP1B1) associated with this locus for PCG (Sarfarazi and Stoilov, 2000).
Prior to identification of CYP1B1 locus association with PCG, large
number of mutations and chromosomal abnormalities had been identified in patients
with primary congenital glaucoma but the exact location of the locus remained
37
unknown. It was in 1995 when first locus (GLC3A; OMIM 231300) was reported by
Mansoor Sarfarazi and his co-researchers using a combination of various strategies
such as candidate chromosomal region, candidate gene markers and a general
positional mapping. They recruited 86 families from Turkey and Canada harboring
patients affected with primary congenital glaucoma without any other associated
abnormality. This group of 86 families consisted of 119 affected individuals (with 72
males and 47 females) and their 182 close relatives who were normal. From this
group of identified families, they initially screened 17 families comprising 113
individuals of whom 40 were affected with primary congenital glaucoma and
published their findings. They screened the genome with a total of 126 STR micro
satellite markers and obtained positive LOD scores with two markers (D2S406 and
D2S405) closely associated with 2p13-p23 region of short arm of chromosome 2. Use
of additional 14 STR markers closely linked to this region of chromosome 2 revealed
a strong evidence of linkage in 11 families whereas 6 families did not show any
linkage to this region. This region was found to be flanked by two STR markers
(D2S367 and D2S119) which previously had been localized to 2p21 region of
chromosome 2. Construction of haplotypes in all families further confirmed that
individuals in 6 unlinked families inherited different chromosomes from their parents
excluding their linkage to chromosome 2. Sarfarazi et al further confirmed their
findings using various bioinformatics tools and assigned the GLC3A gene symbol
found linked to chromosome 2. Furthermore, the finding of GLC3A locus to 2p21 for
primary congenital glaucoma was not an isolated event as four other disorders had
already been mapped to the same region of chromosome 2. Four disorders associated
with 2p21 include spastic paraplegia (autosomal dominant) (Hazan et al., 1994),
Cystinuria (Pras et al., 1994) (Calonge et al., 1994),Holoprosencephaly type 2
(Muenke et al., 1994) and hereditary non-polyposis colorectal cancer (Green et al.,
1994), (Wijnen et al., 1995), (Sarfarazi et al., 1995).
After the identification of the first locus associated with PCG, an active
search was initiated by researchers to identify the disease causing genes within that
locus and their possible defective proteins. In 1997, the first gene (CYP1B1) was
identified by Sarfarazi et al which was directly involved in the pathogenesis of
primary congenital glaucoma. Although this gene had been placed previously within
2p21 region of chromosome 2 using in-situ hybridization, Sarfarazi used high
38
resolution mapping to identify its location within GLC3A critical region. On
sequencing of 11 families found linked to GLC3A in their previous study (described
above;Sarfarazi et al 1995), they were able to identify 3 different mutations
segregating with disease phenotype in five families and were not present in normal
individuals of same ethnic groups. In the remaining families linked to GLC3A locus,
they assumed to carry mutations in promoter or control regions of the gene. In all
these families, the mutant alleles co-segregated with phenotypes in an autosomal
recessive mode and both sexes were equally affected. In these families, the observed
penetrance was 100%, though a case of reduced penetrance has been previously
reported in a study from Saudi Arabid. This could be due to a modifying effect of a
locus which is not genetically linked to CYP1B1 gene (Sarfarazi and Stoilov, 2000).
The identification of association of CYP1B1 gene with pathogenesis of
primary congenital glaucoma was quite unexpected as members of CYP1B1 gene
family are mostly involved in metabolism of xenobiotics. However when researchers
carefully analyzed the available data and old hypotheses regarding pathogenesis of
PCG, they arrived at a logical conclusion as enzymes of cytochrome P450 metabolism
are mostly involved in oxidative reactions which are vital for biosynthesis of some
hormones and compounds essential for intermediary metabolism. From this point of
view it became quite obvious that mutations affecting genes responsible for such
enzymes could lead to recessive phenotypes as in normal heterozygous individuals the
normal allele exerts its compensatory effect (Sarfarazi and Stoilov, 2000).
Previously cytochrome P450 CYP1 gene family was considered a
single subfamily with two well established members CYP1A1 and CYP1A2. However
a third member of the subfamily was identified in 1994 through cloning from human
keratinocyte cell line. This third member of the sub-family CYP1B showed only 40%
similarity with other two members (i.e CYP1A1 and CYP1A2) and was therefore
included by P450 nomenclature committee (Table-1.1) in a new CYP1B subfamily. It
currently contains only one member i.e. CYP1B1 (GenBank Accession nos. U56438
and U03688) and this fact has further been supported by DNA hybridization studies
(Murray et al., 2001).
39
Table 1.1: The Classification of human xenobiotics metabolizing forms of P450s. Adapted
from (Murray, 2000)
P450
Family
P450 Sub-
family
Individual
P450
Substrate(s)
CYP1 CYP1A CYP1A1 Polycyclic aromatic hydrocarbons
CYP1A2 Heterocyclic amines, flutamide
CYP1B CYP1B1 Polycyclic aromatic hydrocarbons,
heterocyclic amines, estradiol
CYP2 CYP2A CYP2A6 Aflatoxin
CYP2A7 ?
CYP2A13
CYP2B CYP2B6 Aflatoxin, cyclophosphamide
CYP2C CYP2C8 Benzopyrene, paclitaxel
CYP2C9 Paclitaxel
CYP2C18 ?
CYP2C19 ?
CYP2D CYP2D6 Methylnitrosaminopyridyl butanone
(NNK)
CYP2E CYP2E1 Notrosamines, ehanol, benzene
CYP2F CYP2F1 ?
CYP2J CYP2J2 ?
CYP3 CYP3A CYP3A4 Aflatoxin, Polycyclic aromiatic
hydrocarbons, Ifosphamide
CYP3A5 Paclitaxel, etoposide, vinca alkaloids,
tamoxifen.
CYP3A7
Cytochrome P450 1B1 belongs to CYP450 superfamily of heme-
binding mono-oxygenases which are involved in oxidative metabolism and
detoxification of many endogenous and exogenous compounds. The metabolic
reactions catalyzed by CYP1B1 include oxidative, peroxidative and reductive changes
into chemical structures of various small molecules. Cytochrome P450 is also
responsible for metabolism of environmental pollutants and chemicals, various
exogenous drugs, either detoxify them or converting them into more toxic metabolites
which could play role in carcinogenesis or pathogenesis of various disorders (Zhao et
al., 2015).
CYP1B1 is located on chromosome 2p22-21 and spans about 12
kilobases (kb) of human DNA whereas the other two members of the CYP1 family
are located on chromosome 15. Human CYP1B1 gene consists of three exons with
two intervening introns in contrast to other CYP1 members which are composed of
seven exons and six introns each (Murray et al., 2001). The exon number one of
40
CYP1B1 is composed of 371 bp, exon number two of 1044 bp and exon number three
consists of 3707 bp (Fig-1.10). The two introns are 390 and 3032 bp in length (Zhao
et al., 2015). The mRNA transcript of CYP1B1 consists of 5.2 kb. Its coding region of
CYP1B1 consists of exon two and three only with open reading frame starting at 5’ of
second exon in contrast to other P450s which all begin at exon 1. The gene product
has been estimated to contain 543 amino acids accounting for the largest known
human P450 both in terms of mRNA size and number of amino acids (Murray et al.,
2001).
Fig-1.10: Structure of human CYP1B1 gene and mRNA transcript. The first five N-terminal
and last five C-terminal amino acids are shown in the figure. Adapted from (Murray et al.,
2001).
In humans, CYP1B1 proteins are expressed in various adult and fetal
extrahepatic tissues including ocular tissues, brain, lungs, prostate, cervix, uterus,
kidneys, placenta, lymph nodes and skeletal muscles. CYP1B1 plays an important role
in normal development of eye structures in both humans and mice due to its
conserved expression. During fetal life CYP1B1 expression levels are higher than
during adult life pointing to its enormous role in normal development of ocular
tissues. It has been further estimated that CYP1B1 transcript level in human and mice
eyes is five times higher than transcript levels of other CYP450s emphasizing its
enormous role in normal development of eye (Zhao et al., 2015).
CYP1B1 is a member of cytochrome P450 superfamily and encodes an
enzymatic monooxygenase which is involved in the metabolism of large number of
endogenous and exogenous substances. The mutations in CYP1B1 have been
responsible for various types of glaucomas including PCG. It has been demonstrated
that CYP1B1 mutant protein exhibits decreased protein stability and thus enzymatic
41
activity. Several studies conducted in mice with CYP1B1 mutations have shown
abnormalities in trabecular meshwork together with irregular collagen distribution,
increased susceptibility to damage due to oxidative reactive species and decreased
secretion of periostin (Postn) by trabecular meshwork. These findings in mice have
been confirmed by studies conducted on trabecular meshwork isolated from human
patients affected with glaucoma. Furthermore, functional characterization of missense
mutations in CYP1B1 has shown decreased levels of retinoic acid which plays an
important role in the development of eye during fetal life (Reis et al., 2016).
Another study presented a molecular model that suggested the possible
mechanism by which mutations in CYP1B1 gene might interfere with normal
functioning of CYP1B1 protein. The hypothesis is based on membrane bound
cytochromes which share a similar molecular structure as CYP1B1. Membrane bound
cytochromes possess a transmembrane domain, an intervening hinge region and a
cytoplasmic domain. The proline rich hinge region permits flexibility between
membrane spanning domain and cytoplasmic domain. The membrane spanning
domain lies at amino (-NH2) terminal whereas cytoplasmic domain is located at
carboxy (-COOH) terminal. The carboxy-terminal ends are highly conserved among
different members of the cytochrome P450 superfamily. They contain a set of
conserved core structures (CCS) which are thought to be responsible for the heme-
binding ability of these molecules. Between the hinge and the CCS lies a less
conserved substrate-binding region. It has been reported that mutations in hinge
region interfere with proper folding and heme-binding properties of cytochrome P450
molecules. For missense mutations, a map was constructed against 3-D model of
CYP1B1 using homology modelling. The data obtained indicated that missense
mutations affect either highly conserved amino acid residues located in hinge region
or amino acids forming conserved core structure of cytochrome P450 protein
molecule leading to interference with basic properties such as proper folding, heme-
binding, substrate accommodation and interacting with any redox reactant. Another
group of mutations that has been reported, belong to frameshift type of mutations that
mostly lead to introduction of premature stop codons in CYP1B1 open reading frame.
Such type of mutations have drastic effect eliminating heme-binding region of
CYP1B1 molecule essential for its normal functioning. It has further been suggested
42
that frameshift type of mutations could interfere with normal RNA metabolism by
non-sense mediated mRNA decay mechanism (Sarfarazi and Stoilov, 2000).
Sarfarazi and Stoilov hypothesized the role of CYP1B1 in pathogenesis
of PCG by suggesting its role in the metabolism of an endogenous molecule such as a
steroid, a fatty acid or a prostanoid compound (Fig-1.11). Based on this hypothesis,
they elucidated the functioning CYP1B1 molecule and made following two
observations:
1. CYP1B1 produces an active compound which acts on some unknown
downstream target.
2. CYP1B1 deactivates other biologically active compounds.
Unfortunately, the identity of CYP1B1 substrate is still not clear
otherwise the biochemical cascade controlling the development of anterior chamber
angle could have been unearthed.
Fig-1.11: Metabolic pathway of CYP1B1. Adapted from (Sarfarazi and Stoilov, 2000)
Data from Iran and Japan has reported that PCG affects males more
than females which suggests some interaction of steroid hormones with CYP1B1 gene
expression or metabolism of its product. It has been shown that transcription of
CYP1B1 gene is induced by arylhydrocarbon receptor with estradiol acting as a
substrate for CYP1B1 protein and that the mutation in CYP1B1 gene affects
hydroxylation of estradiol. However data for PCG patients shows statistical
significance for male predominance only in patients without CYP1B1 mutations and
43
not in patients with CYP1B1 mutations. This suggested the role of other factors or
genes in PCG phenotypes in sex-dependent study (Suri et al., 2015).
So far (by July, 2016) more than 200 mutations have been reported in
CYP1B1 according to The Human Gene Mutation Database (HGMD). The
distribution of different types of mutations in CYP1B1 gene has been shown in the
following table.
Table 1.2: Various types of mutations identified in CYP1B1
(Source: The Human Gene Mutation Database)
S.# Mutation Type Number 1. Missense/Nonsense 163
2. Regulatory 04
3. Small Deletions 36
4. Small Insertions 12
5. Small Indels 06
6. Gross Deletions 06
7. Gross Insertions 03
8. Complex 01
Total 231
In inbred populations, most of CYP1B1 associated cases are caused by
one or few mutations whereas there is considerable diversity among mutations in
populations such as France and Japan. In 2005-06, screening of 104 glaucoma patients
for CYP1B1 revealed that most of the PCG cases are caused by mutations in CYP1B1
and that four mutations alone are responsible for majority of the cases of PCG. It was
also reported that most of the mutations in Iranian patients are identical to those found
in neighboring countries (Suri et al., 2015).
Mutations in CYP1B1 have also been reported for JOAG and POAG in
various studies. A study conducted in Iran reported the presence of CYP1B1
mutations in nearly 20% of patients with JOAG. Likewise CYP1B1 have also been
reported for late onset POAG patients though with a low frequency as compared to for
PCG (Suri et al., 2015). CYP1B1 has also been reported to act as a modifier locus for
MYOC in pathogenesis of POAG suggestive of digenic inheritance. It has been
suggested that presence of CYP1B1 mutation along with MYOC accelerates the
disease progression. Mutant MYOC leads to accumulation of a protein in cell
44
cytoplasm and endoplasmic reticulum which subsequently results in apoptotic cell
death. Hence the presence of mutant MYOC along with mutant CYP1B1 speeds up
this process of cell death in trabecular meshwork. From all these studies, it is quite
evident that CYP1B1 has a much larger role in pathogenesis of various sub-types of
glaucoma (Mookherjee et al., 2012).
b) GLC3B Locus:
When data for six families remained unlinked to CYP1B1 locus on
chromosome 2 (Sarfarazi et al., 1995) was further analyzed for two point linkage and
haplotype transmission, a chromosomal region was identified with the help of STR
markers showing positive LOD scores. Initial results failed to produce any
encouraging outcome however 17 STR markers flanking 1p36 region (from 1p36.2 to
1p36.1) on chromosome 1(D1S1635, D1S228, D1S507, D1S407, D1S1368) showed
segregation with disease phenotypes (positive LOD scores) in four families. All these
families showed an autosomal recessive mode of inheritance in all successive
generations. This locus was given the name GLC3B with OMIM # 600975(Akarsu et
al., 1996). GLC3B is located at 1p36.2–1p36.1 but still no gene has been assigned to
this locus (Cascella et al., 2015b).
c) GLC3C Locus:
The third locus (GLC3C, OMIM 613085) for primary congenital
glaucoma was identified by Stoilov, IR and Sarfarazi, M in 2002 and was mapped to
14q24.3–14q31.1, flanked by STR markers D14S61 and D14S1000 (Firasat et al.,
2008). Still no gene has been identified for this locus (Cascella et al., 2015b). The
GLC3C locus contains 40 genes and spans a region of 5.77Mb. LTBP2 is located
within 1.3 cM proximal to GLC3C locus (Chen et al., 2016).
d) GLC3D Locus:
In 2008, Firasat et al mapped one of thirteen families to Chromosome
14, although overlapping but adjacent to previously identified GLC3C locus. They
identified the critical region for this locus as 14q24.2-24.3 (Firasat et al., 2008).
Moreover it was also suggested by Noorie-Nejad M et al in 2009 that at least one
more PCG locus existed other than the three loci described above. Their findings were
45
based on absence of linkage to any of the three loci in nine Iranian families(Mehrnaz
Noorie-Nejad, 2009). They performed their analysis using high density microarray
chips and identified mutations in two families coding for LTBP2 (Latent
Transforming growth factor Beta binding Protein 2) gene (Suri et al., 2015). In 2009
Ali M et al identified LTBP2 as the second PCG-causative gene in four
consanguineous families from Pakistan and Gypsy ethnicity (Ali et al., 2009).
LTBP2 gene is located very close to GLC3C on chromosome 14q24.2-
14q24.3. In National Centre for Biotechnology Information (NCBI), LTBP gene has
been positioned within the locus GLC3D (OMIM 613086). LTBP2 (Latent
Transforming Growth Factor Beta binding Protein 2) protein is a member of
superfamily of proteins comprising fibrillins and latent transforming growth factor
beta binding proteins. It is expressed in elastic tissues and microfibrils containing
fibrillin-1 and is thought to modulate TGF-β activities. TGF-β belongs to a family of
cytokines involved in production of extracellular matrix and oxidative stress response.
The exact function of LTBP2 is still unknown, but it is thought to play a role in
various mechanisms following cell injury and inflammation such as tissue repair, cell
adhesion and some functions related to microfibrils and elastin fibers. It is thought
that LTBP2 binds latent TGF-β at the site of firillin-1 containing microfibrils and
modulates its response through covalent and non-covalent interactions. Tissue culture
studies have demonstrated the expression of LTBP2 in eyes especially trabecular
meshwork and ciliary process. Consequently any mutation in the gene may lead to
some defects in TM and subsequent hindrance in aqueous drainage through anterior
chamber angle leading to elevation in IOP. Likewise LTPB2 mutations have also
shown to affect TGB-β signaling pathways resulting in glaucoma (Suri et al., 2015).
Ali M et al studied the role of LTBP2 in anterior segment of eyes of
cow and mice. They concluded that mutations in LTBP2 increase the elasticity of
ciliary body structures with subsequent alteration in structural support for surrounding
tissues. The mutations could also affect the elasticity of Schlemm’s canal leading to
decrease in aqueous outflow. Furthermore, change in elasticity of scleral spur could
also affect the structural architecture of trabecular meshwork in the anterior chamber
of the eye further increasing the IOP (Ali et al., 2009).
46
Besides PCG, mutations in LTBP2 have also been reported for
megalocornea, microspherophakia, ectopia lentis (EL), Weil-Marchesani Syndrome
(WMS) and Marfan Syndrome (MFS). Glaucoma is sometimes found accompanied
by some of these conditions. Especially EL has been reported in a number of PCG
patients with LTBP2 mutations. Likewise, WMS and MFS are usually found in
association with EL or glaucoma or both. These findings suggested the involvement
of LTBP2 in various types of syndromic glaucomas. Additionally, LTBP2 has also
been implicated in some cases of POAG and PEX justifying its screening in patients
with these disorders. Several functional studies have also suggested the role of LTBP2
mutations affecting ECM in various forms of glaucomas. Furthermore, it has been
indicated that at least one more PCG locus exists in addition to already four known
loci (Suri et al., 2015).
iii) Role of Myocilin in PCG:
Myocilin (MYOC) gene, which is also known as Trabecular meshwork-
inducible glucocorticoid response (TIGR) gene, has been reported to be responsible
for primary open-angle glaucoma and Juvenile-onset open-angle glaucoma. The exact
mechanism by which mutant MYOC gene leads to development of glaucoma is still
not clear but it has been suggested that mutations in the gene leads to reduced
secretion of myocilin protein and synthesis of insoluble aggregates due to wild-
type/mutant heterooligomers. Some studies have reported the disease causing variants
of MYOC gene even in the presence or absence of CYP1B1 mutations. It is quite
possible that in those individuals CYP1B1 might be acting as modifier or two genes
may act through a common pathological pathway (Do et al., 2016).
Previously some studies implicated MYOC in pathogenesis of PCG
either independently or in combination with CYP1B1. In one Chinese study, mutation
in MYOC was found to be responsible for 2.6% of all cases of PCG included in the
study. In another study from Korea, mutation in MYOC was found in two of 85
unrelated PCG cases (Kim et al., 2011).
1.2.3 Diagnosis of Glaucoma:
As blindness associated with glaucoma is irreversible in nature, it is
quite important that it must be diagnosed at the beginning of its course so as to
47
minimize the visual loss. The diagnosis includes the recognition of characteristic
changes in optic nerve head and retinal nerve fiber layer (Tatham et al., 2014).
Measuring intra-ocular pressure as a screening tool for diagnosis of glaucoma is not
an authentic method as nearly 50% of POAG patients have normal intra-ocular
pressure when measured and individuals with elevated intra-ocular pressure do not
necessarily suffer from glaucoma. In addition, often there is diurnal variation in intra-
ocular pressure. Measurement of intra-ocular pressure or doing fundus imaging alone
has been reported to be specific in 90% and sensitive in less than 50% of cases only.
Furthermore, diagnosis of glaucoma is strongly related to age, race and family history
of the individual. Latest diagnostic tools that measure the thickness of nerve fibers of
optic nerve (such as OCT) are also associated with poor sensitivity and specificity.
Diagnosis through visual field testing carries higher sensitivity and specificity, but it
requires trained personnel and special ophthalmological equipment that is usually not
available in primary health care facilities. Accurate diagnosis of glaucoma requires
measuring intra-ocular pressure, visualizing fundus and testing visual fields. Such
examinations should be repeated at regular intervals to look for any tissue loss in optic
nerve head or to detect scotomas in visual fields that could be the early signs of
glaucoma. Ophthalmoscopy alone by a physician is inadequate and testing visual field
is essential for the highest accuracy. Although U.S.Preventive Services Task Force
(USPSTF) and American Academy of Family Physicians do not demand for screening
of glaucoma patients at Primary health care facility, but American Academy of
Ophthalmologists recommend regular eye examination for all individuals who are
above 40 years of age with more frequent examinations for persons who are at
increased risk of developing glaucoma (Gupta and Chen, 2016).
1.2.4 Management of Glaucoma:
Childhood blindness is usually an outcome of preventable or treatable
ophthalmological disorders. The World Health Organization has put a great emphasis
on strategies directed at preventing infancy or childhood blindness in its WHO 2020
Vision Program. It has been acknowledged by WHO that treatment of conditions
responsible for blindness during childhood is more difficult as compared to adults and
Primary Congenital glaucoma is one of leading causes of blindness in infants and
children. Low prevalence of PCG coupled with delayed diagnosis due to decreased
48
awareness put primary congenital glaucoma as one of the difficult conditions to
manage. Majority of affected children usually present in advanced stage of the disease
when treatment options are very limited. Although surgery is cornerstone of the
disease management but in advanced cases, even surgery has a limited success. In
western countries with improved medical facilities and awareness, children having
primary congenital glaucoma are usually diagnosed within months after birth. In
contrast, in developing third world countries where limited medical resources are
available combined with decreased awareness among communities, diagnosis is
usually delayed as far as up to 3 years on average when disease has progressed to
advanced stage. (Moore et al., 2013)
The main goal in patients with PCG is to reduce the intra-ocular
pressure (IOP).The medical treatment has a very limited role in controlling elevated
intra-ocular pressure in these patients. It has been estimated that the medical therapy
effectively reduces IOP in less than 10% of the patients. The medical therapy is
therefore only used as an adjunct to surgical treatment to reduce the corneal edema till
the commencement of surgery (Chan JYY, 2015).
Surgery (Trabeculectomy or Goniotomy) plays a key role in the
management of primary congenital glaucoma. It should be undertaken as early as
possible with the aim to maintain the visual acuity and preserving ocular structures
from damage that could be incurred due to increased intra-ocular pressure (Moore et
al., 2013). Various surgical options are available depending on the severity of corneal
diameter. Goniotomy or trabeculotomy are performed in mild PCG with corneal
diameter less than 13mm. Moderate PCG corresponding to corneal diameter between
13 to 16 mm requires drainage surgery or other options such as combined
trabeculotomy-trabeculectomy or glaucoma drainage implant. Cyclophotocoagulation
is the only option for patients with severe PCG having corneal diameter more than
16mm (Chan JYY, 2015). The role of more advanced techniques such as
cyclodestructive and non-penetrating surgeries is yet to be defined (Moore et al.,
2013).
1.3 Stargardt Disease
Retinal degenerative disorders constitute a very large and
heterogeneous group of ophthalmological disorders and are considered to be the
49
leading cause of irreversible blindness in the world. Important disorders in this group
include age-related macular degeneration (AMD), Retinitis pigmentosa, Stargardt
disease, Leber congential amaurosis and Best disease. All the above listed disorders
are common in a sense that they are associated with some damage to outer retinal
layers such as photoreceptors, retinal pigment epithelium (RPE) and choroidal
vessels. AMD is a complex disease that segregates due to interaction of multiple loci
and environmental factors whereas the other disorders follow Mendelian principles
occurring primarily due to either complete absence or some abnormality in their
respective protein (Wiley, 2015). There are millions of people who suffer from retinal
diseases all over the world. So far more than 238 genes have been identified in
association with various retinal disorders. Strategies for mutational screening for these
genes have made a tremendous progress during last 20 years and genetic technology
has been improving with each passing day. Inspite of all these advancements, the
pathophysiological mechanism associated with various retinal disorders is still not
clear. Of all retinal disorders, Stargardt disease needs a special attention as it is the
most common form of macular dystrophy during childhood (Tsipi et al., 2016).
Stargardt disease (or Stargardt macular dystrophy) was first identified
by a German ophthalmologist Karl Stargardt in 1909. It is usually inherited in
autosomal recessive manner in association with ABCA4 whereas autosomal dominant
cases have also been reported with mutations in ELOVL4 and PROM1. Patients
usually manifest with progressive loss of vision that may begin in first or second
decade of life although cases with early or late adult-onset have also been reported.
Patients with late onset of the disease usually have better prognosis. Fundoscopy
reveals macular atrophy and yellow-white lesions termed as flecks within retinal
pigment epithelium. Flecks result due to abnormal accumulation of lipofuscin, a
material composed of mainly lipids but proteins and various fluorescent compounds
originating from chromophore are also present. Such fleckts are present in entire
retina being more marked in region around macula (Strauss et al., 2016). The
prevalence of Stargardt disease is around 1 per 10,000 individuals and has been
reported to be responsible for nearly 7% of all type of retinal dystrophies. Stargardt
disease is both clinically and genetically heterogenous (Tran et al., 2016).
50
Stargardt disease is an inherited visual disorder that involves retina and
is characterized by deposition of phototransduction metabolites in retinal pigment
epithelium (RPE). RPE later undergoes atrophy with degeneration of photoreceptors
and choriocapillaries. It has been suggested that choroidal angiopathy plays a role in
the pathogenesis of Stargardt disease. A recently published study has shown that
choroid becomes irregular or S shaped in majority of patients with Stargardt disease
leading to inaccurate measurement of choroidal thickness due to focal or diffuse
defects in RPE. Furthermore, thickest point of choroid from under the foveal centre
was observed to be displaced in majority of the patients whereas thinning of the
choroid on nasal side was present in upto one third of patients with Stargardt disease.
Some patients also demonstrated loss of large choroidal vessel layer in comparison to
normal healthy eyes which may have some effect in progressive loss of visual acuity
in those patients (Adhi et al., 2015).
1.3.1 Variants of Stargardt Disease:
It is very difficult to classify various phenotypes associated with
Stargardt disease due to similarity in clinical features and fundus findings but some
researchers suggest that classic Stargardt disease should be restricted to those patients
with mutations in ABCA4 gene and Stargardt like or juvenile macular dystrophy
should be used for phenotypes associated with mutations in other genes like ELOVL4
and PROM1 (Zhang et al., 2014). Usually, Stargardt disease can be classified into
three variants on basis of gene involvement. STGD-1 is the most common variant of
Stargardt disease is responsible for nearly two third of all cases of this disease. It
involves mutations in ABCA4 (ATP-binding cassette transporter) retina specific gene
(chromosome 1p21-22; OMIM 601694) and is predominantly associated with
autosomal dominant mode of inheritance. The other two variants of Stargardt disease
are responsible for rest of the cases. STGD-3 (600110) is associated with mutations in
ELOVL4 (Elongation of very long chain fatty acids like 4 gene) gene, located on
chromosome 6 and is transmitted in autosomal dominant pattern. STGD-4 (603786)
occurs due to mutations in PROM1 (prominin1) gene on chromosome 4 (Tran et al.,
2016).
A) Stargardt Disease-1 (STGD-1):
51
Stargardt disease type-I (OMIM 248200) is the most common
phenotypic variant of this disease which is caused by mutation in ABCA4 (OMIM
601691) gene. The association of ABCA4 with Stargardt disease was first identified
in 1997 by Allikmets et al (Zhang et al., 2014). This gene encodes a protein named
Adenosine triphosphate-binding cassette transporter. When it is mutated, metabolism
of photoreceptors is affected in a way that A2E, a component of lipofuscin, is
gradually accumulated in photoreceptor cells and retinal pigment epithelium (RPE)
leading to injury within both. Although there is significant variability in the
phenotypic expression, affected patients manifest with early loss of central vision and
lesions within and around macula and lipofuscin flecks seen as small yellowish sub-
retinal flecks scattered throughout the retina when seen through fundoscopy
(Huckfeldt et al., 2016).
Stargardt disease is one of the leading causes of Juvenile to Adult-
onset macular dystrophies. Its prevalence varies from 1:8000 to 1:10,000 live births.
Clinically it manifests as gradual loss of central vision during childhood or
adolescence. There is variable degree of atrophy of retinal pigment epithelium (RPE)
around macula and peri-macular region. On fundoscopy, retina appears like a “beaten
metal” or “snail-slime” with dispersion of yellow-white flecks in and around fovea in
initial stages while in later stages fundoscopy may reveal retinal pigment epithelium
and chorio-retinal atrophy. On fluorescein angiography, typical dark choroid is
observed in most affected individuals due to deposition of lipofuscin like substance in
retinal pigment epithelium (Jiang et al., 2016).
So far, more than 800 mutations have been identified in ABCA4 gene.
Most of the identified mutations in ABCA4 are rare and unique and are extremely
heterogeneous. Several of these mutations are population specific, for example the
most common mutation in European population is p.G1961E and it has a highest
allele frequency of more than 20% whereas in Spain the most common mutation is
p.R1129L with an allele frequency of 22.4%. In Mexican population, p.A1773V and
p.G818E have been identified with a frequency of 17% and 15% respectively.
Recently it has been reported by a study that in African-American patients, pR2107H
was found to be the most common mutation with a frequency of 19.32% whereas the
same mutation was found with a very low frequency of 1.02% in patients of European
52
origin. It has also been suggested that some common variants are in fact founder
mutations such as p.R1129L in Spanish patients, 16 p.A1773V in Mexican patients,
and p.N965S in the Danish population.(Jiang et al., 2016)
Jiang F et al screened the ABCA4 gene in 96 patients affected with
STGD1 disease and found mutations in 84 of the patients with a frequency of 87.5%
whereas 9 patients were found to be heterozygous and no mutation was found in
ABCA4 gene in 3 patients. This amounts to be approximately 96.5% mutation
detection rate and according to author this high rate could be attributed to careful
clinical evaluation of those patients confirming those patients as typical stargardt
disease patients. More than 50% of these mutations were not reported previously from
any region indicating that these mutations might be specific to Chinese
population.(Jiang et al., 2016)
It has been hypothesized that ATP-binding cassette transporter coded
by ABCA4 gene is involved in the clearance of a metabolic byproduct of vitamin A
metabolism during visual cycle. Impaired clearance leads to abnormal accumulation
of lipofuscin in retinal pigment epithelial (RPE) cells and is toxic to the cells with
subsequent cell death. The accumulation of this lipofuscin pigment in retinal epithelial
cells is considered to be the hallmark of this disease. The usual symptoms of this
disease include central visual loss, inability to discriminate among colors, paracentral
scotomas, gradual dark adaptation and photoaversion. Diagnosis is mainly based on
identification of yellow flecks in retina caused by accumulation of lipofuscin pigment
and a dark-colored choroid on fluorescein angiography.
Another mechanism suggests the flippase action of ABCA4 protein
which is involved in the translocation of an intermediate metabolite of visual cycle
namely N-retinylidene-phosphatidylethanolamine (NR-PE) from intradiscal leaflet to
cytoplasmic leaflet of the photoreceptor cells. After being transported, NR-PE
undergoes reduction to Vitamin A and is then transported to RPE where it is
isomerized to 11 cis-retinal. Due to mutation in ABCA4, the protein loses its flippase
function leading to abnormal accumulation of bisretinoid N-retinylidene-
phosphatidylethanolamine (A2E). This product is insoluble and is toxic to both RPE
and photorecetors. Together with lipofuscin, these A2E gets progresseively deposited
53
in RPE and manifest clinically with gradually enlarging central scotomas (Wiley,
2015).
Mutations in ABCA4 are also found to be associated with some other
variants of retinal dystrophies such as autosomal recessive retinitis pigmentosa
(arRP), autosomal recessive cone-rod dystrophy (CORD) and age-related macular
degeneration collectively called as ABCA4-associated retinopathies (Tsipi et al.,
2016) & (Jiang et al., 2016).
It has been observed for more than a decade that some ABCA4
mutations are associated with more severe phenotypes where as some disease-causing
alleles will manifest clinically only when they are paired with some severe alleles and
not when they are paired with some milder allele and with the same allele. The
exclusion of latter cases from the data of affected individuals explains the difference
observed between the prevalence of disease-causing alleles seen in population
databases and reported cases of ABCA4 associated retinal dystrophies (Huckfeldt et
al., 2016). Autosomal dominant pattern of inheritance has also been reported for
Stargardt disease with mutations in ABCA4 gene (Zhang et al., 2014).
B) Stargardt Disease 3 (STGD-3):
Stargardt disease type-3 is another phenotype of juvenile-onset
macular dystrophy and is transmitted in an autosomal dominant manner by mutation
in ELOVL4 (Elongation of very long chain fatty acids-4). ELOVL4 codes for a
transmembrane protein which is involved in catalysis of initial rate limiting
condensation reaction in the biosynthesis of very long chain fatty acids. The
mutations result in alteration of coding sequence of C-terminal and delete predicted
dilysine endoplamic reticulum retention motif. Culture studies have shown that
mutant ELOVL4 are not able to synthesize very long chain polyunsaturated fatty acid
(VLC-PUFA) thereby inhibiting the activity of wild-type ELOVL4 (Martin-Paul
Agbaga, 2014).
The highest expression of ELOVL4 has been reported to occur in
retina whereas skin, brain and testis have also demonstrated its expression to some
54
extent. Very long chain fatty acids are compounds with more than 20 carbons in their
alkly chain and are involved in many biochemical processes such as sphingolipid
biosynthesis, inflammatory reactions, various immunological mechanisms, fetal
development and its growth. The elongation of fatty acids occurs in endoplasmic
reticulum and 2 carbons are added in each cycle of four steps via acyl-CoAs. The four
reactions of each cycle are condensation, reduction, dehydration and reduction. The
second, third and fourth steps are catalyzed by enzymes namely reductases 3-
ketoacyl-CoA reductase (KAR), 3-hydroxyacyl-CoA dehydratase (HACD) proteins
(HACD1–4) and trans-2,3-enoyl-CoA reductase(TER) respectively. First step (i.e.
condensation) of this four reaction cycle is a rate limiting reaction and is catalyzed by
one of seven elongases (ELOVL1-7). All seven elongases differ in their specificity for
substrates and it is reported that substrates for ELOVL4 are faty acyl CoAs with very
long chain lengths (>C26). Fatty acids of length >C26 are found only in some tissues
such as retina, skin, brain and sperms. It has been demonstrated in mutant
heterozygous knock-in mice that they exhibit the same STGD-3 like phenotype with
accumulation of lipofuscin pigment in retinal pigment epithelium and progressive
degeneration of photoreceptors. Furthermore, quantitative lipid analyses have shown
reduced levels of phosphatidylcholines fatty acids having lengths between C26-C32
in retina of these mutant heterozygous knock-in mice. In fact, mutations in ELOVL4
affect the entire machinery involved in elongation of fatty acids and hence result in
multiple types of damage in cellular process leading to STGD-3 pathology (Okuda A,
2010).
It has been demonstrated that very long chain polyunsaturated fatty
acids (VLC-PUFA) work in close association with rhodopsin and are involved in
photo transduction. Very long chain fatty acids are not present in blood and are
usually expressed in tissues involved in lipid metabolism such as retina, skin, brain
and testis. Patients with heterozygous ELOVL4 genotype exhibit STGD-3 like
phenotype with progressive loss of central vision, macular atrophy and degenerative
retinal pigment epithelium. Recently, patients with recessive homozygous mutations
in ELOVL4 have been reported to manifest severe skin (termed as ichthyosis) and
severe brain dysfunction (intellectual disability and spastic quadriplegia) (Logan and
Anderson, 2014).
55
C) Stargardt Disease-4 (STGD-4):
Stargardt disease type-4 (OMIM 603786) phenotype is associated with
mutations in PROM1 (Prominin1;OMIM 604365). Clinically there is bilateral and
symmetrical macular atrophy, and yellow flecks in the fundus caused by accumulation
of lipofuscin pigments in the retinal pigment epithelial cells (Zhenglin Yang, 2008).
Zhang X et al studied 7 patients form 5 families with STGD-4 and he
concluded that patients with mutations in PROM1 have a late onset of the disease in
comparison to patients with ABCA4 mutations. The mutations in PROM1 can also
lead to other retinal disorders such as retinitis pigmentosa (RP41), autosomal
dominant cone-rod dystrophy (CORD12, CrD), macular dystrophy (MCDR2) and
autosomal recessive cone-rod dystrophy (CRD). Clinically all these above described
retinal disorders caused by mutations in PROM1 may be differentiated with full field
ERG and fundus appearance. The age of onset and night blindness may provide
additional useful information for diagnosis (Fan et al., 2015, Zhang et al., 2014).
1.3.2 Treatment of Stargardt Disease:
Currently no there is no treatment for Stargardt disease and still no
drug has been approved by Food and Drug Administration (FDA) in United States or
European Medicines Agency although various drug trials are undergoing in this
regard (Strauss et al., 2016).
It has been shown that there is progressive decline in levels of VL-
PUFA with increasing age and also in persons with age-related macular degeneration
which suggests that metabolism of VLC-PUFA may be affected due to lack of dietary
intake of the precursors for such lipids. It is hoped that genetic or dietary interventions
to elevate the levels of very long chain fatty acids in such patients may provide an
option in patients with age-related maculopathies and others for limiting their visual
disability. Furthermore, some relief to STGD-3 patients could be provided by
ribozyme or RNAi-mediated knock down to silence the dominant negative effects of
defective gene product on photoreceptors (Logan and Anderson, 2014).
56
1.4 Congenital Cataract
The term Cataract is defined as the opacification of crystalline lens of
the eye. The cataract is the most common treatable cause of visual impairment during
childhood. Childhood or congenital cataract may develop due to many causes such as
intrauterine infections, metabolic disorders or chromosomal abnormalities. It is also
inherited as an isolated ophthalmological abnormality either as non-syndromic
congenital cataract or it may be associated with some other systemic abnormalities
and manifest in the form of a syndrome. In non-consanguineous populations, the
congenital cataract is usually inherited in an autosomal dominant pattern although
autosomal recessive and X-linked inheritance is also seen (Francis et al., 2000).
1.4.1 Prevalence:
Congenital cataract is responsible for approximately one third cases of
blindness in infants. It can manifest as non-syndromic cataract or as a part of
syndromic cataract. In more than two third of the cases, the only manifestation is
involvement of lens alone. The prevalence of non-syndromic congenital cataracts
varies from 1-6 per 10,000 live births and nearly one-third of these cases are familial
with more than one member being affected (Chen et al., 2011). According to another
study, the prevalence depends upon the socio-economic condition and varies from 0.6
to six per 10,000 births in developed countries and from 5 to 15 per 10,000 births in
developing countries with one third of these cases having a genetic basis (Chen C,
2015). The condition is therefore more prevalent in developing countries (10 times)
than in developed countries (Lin, 2015). Some small population based studies
reported the prevalence of congenital cataracts as 5 per 10,000 births in China but a
recently published study has estimated its prevalence from 2.39—2.78% on the basis
of its 10 years hospital based serial study.
Chen J et al studied 12 Pakistani Families and more than 125 familial
cases of autosomal recessive cataract (arc) and found that gene named FYCO1 is
responsible for approximately 10% of total genetic cases of Congenital Cataracts in
the population including Pakistan. This suggests that in Pakistan, FYCO1 associated
mutations are the leading cause of inherited Congenital Cataracts (Chen et al., 2011).
57
Genetically congenital cataracts are very heterogeneous with variable
clinical manifestation. It has been estimated that 8.3-25% of non-syndromic
congenital cataracts are inherited in autosomal recessive, autosomal dominant or X-
linked pattern. So far more than 40 loci and 26 genes have been mapped for
congenital cataracts. Of these, 14 genetic loci are responsible for autosomal recessive
form of non-syndromic congenital cataracts (arc). Of all these loci, mutations in nine
genes have been found where as in six loci, no gene has been discovered so far (Chen
et al., 2011).
The development of eye lens begins during morphogenesis in
embryonic life with the formation of an embryonic nucleus. The lens fibers are then
continuously deposited during fetal life and after birth initially forming fetal nuclear
region followed by cortex around it (Francis et al., 2000).
1.4.2 Classification:
Cataracts may be classified in various ways.
(A) On the basis of Phenotype:
1. Isolated/Non-syndromic Congenital Cataracts:
When cataract is inherited as an isolated ophthalmological abnormality and the
opacification of the lens is the only clinical manifestation, it is termed as
isolated or non-syndromic cataract. It may be inherited in autosomal recessive,
dominant or X-linked pattern (Francis et al., 2000). So far more than 30 genes
have been identified in association with non-syndromic congenital cataracts.
Mutations in these genes could lead to structural and functional abnormalities
in various lens proteins including crystalline, gap junction, intermediate
filaments, membrane proteins and transcription factors (Ma, 2016) .
2. Syndromic Congenital Cataracts:
Here, the congenital cataract is one component in association with some other
systemic or chromosomal abnormalities or DNA repair deficiencies such as
Lowe syndrome, or Nance Horan Syndrome or ophthalmological
abnormalities such as micropthalmia, aniridia, or microcornea (Shiels and
Hejtmancik, 2016). Approximately 18% of congenital cataract patients also
58
manifest with microcornea (when corneal diameter is < 10 mm at the time of
birth in horizontal axis). Some patient also present with some other systemic
abnormalities including learning difficulties which are usually noticed in
childhood age (Ma, 2016). In some cases, it becomes quite difficult to
distinguish between non-syndromic (isolated) and syndromic congenital
cataract, for example, mutation in PITX3 gene associated with anterior
segment mesenchymal dysgenesis, may result in isolated cataract in some
individuals and with additional findings in others even within same family
(Shiels and Hejtmancik, 2016).
(B) On the basis of Age of Onset:
Although the age at which a cataract occurs, has no association with
etiology but placing a cataract in a particular age group is useful as each group shares
some common characteristics such as type of mutations in different genes which
might affect specific cellular processes or cataracts within the same group may have
same pathophysiology leading to their development. With this context, cataracts can
be classified according to their age of onset as follows:(Shiels and Hejtmancik, 2016)
1. Congenital Cataracts:
A cataract is called congenital or infantile if it occurs during first year after
birth. If it occurs after first year but during first decade of life, then it is termed
as Juvenile cataract. It has been estimated that approximately 8.3-25% of
congenital cataracts are hereditary in nature with the rest being caused by
either an intra-uterine infection (e.g.rubella) or they may occur due to some
pre-natal event.
2. Pre-Senile Cataracts:
Cataract occurring after first decade but before 45 years of age is called pre-
senile cataract.
3. Senile Cataracts:
They usually occur after 50 years or sometimes after 60 years of life.
59
(C) On the basis of Morphology:
The morphology of the opacification varies according to its location in
the eye lens and it may be either static or progressive. It has been a general rule that
posteriorly located or more dense opacities have a profound impact on visual loss. It
is very difficult to classify congenital cataract because of large varieties of
morphologies in the eye lens (Francis et al., 2000).
Fig-1.12: The Human Crystalline lens (Adapted from Francis PJ et al, 2000 CC)
Various categories of congenital cataracts can be summarized as follows on the basis
of their morphological pattern: (Francis et al., 2000)
1. Nuclear Cataract:
This is common type of cataract and is usually associated with some
abnormality in gene expression during lens development. Opacities may either
be confluent (merging with each other) or they may be discrete. It usually
affects both eyes and affected individuals may exhibit variable expressivity.
2. Pulverulent Cataract:
The pulverulent is derived from pulverized “dust-like” appearance of
opaciites. Such opacification may involve any part of the lens. This type of
cataract was first reported by Nettle and Ogilvie in 1906. The pulverulent
cataract may be limited to only central portion (Central Pulverulent) or it may
extend outward towards periphery (Zonular Pulverulent). There is
considerable variation occurs both in its distribution and degree which leads to
its distinguished phenotype from all type of cataracts.
60
3. Lamellar cataracts:
Marner’s cataracts, perinuclear, zonular or polymorphic cataracts are
synonyms for this type of cataracts. There is variable degree of opacification
and vision may be preserved or it may be affected to such an extent which
calls for its surgical removal. Common sites for this type of cataracts are
anterior or posterior Y-sutures. Sometimes opacities in cortex (or riders) may
be associated with lamellar cataracts.
4. Anterior-polar Cataracts:
These are symmetrical, well circumscribed and bilateral opacities and may be
inherited as dominant, recessive or X-linked phenotype. They are rarely
progressive and when large enough, they become pyramidal in shape with
their apex extending into the anterior chamber. The vision usually remain
well-preserved. Some studies have suggested their association with
microphthalmia an Astigmatism indicating the involvement of gene for
anterior segment development.
5. Posterior Polar Cataracts:
There is bilateral and symmetrical lens involvement and affected individuals
usually exhibit autosomal dominant mode of inheritance. Vision is greatly
affected and can progress to total cataract if posterior cortical opacities are
also present in the lens.
6. Cortical Cataracts:
This is a rare type and opacities are restricted only to outer cortical region,
usually superior pole adjacent to capsule of the lens. The nucleus is not
involved but its distribution is evident of its association with later stages of
lens development.
7. Blue dot (Cerulean) Cataracts:
It was first described by Vogt and is not truly congenital in nature. It is
developed later during childhood and continues to progress later in life. These
appear to be blue-white pin-head opacities hence named so. They are
distributed throughout the lens being more prominent in cortex where they
may coalesce to form larger wedge-shaped (cuneiform) opacities. In familial
cases, the expression may vary but phenotype remains the same within that
61
particular family. The vision remains preserved and surgery is rarely required
and is associated with excellent prognosis.
8. Coraliform or aceuliform Cataracts:
It was first described by Nettleship and resembles sea coral in a sense that
multiple finger-like projections extend outward from the lens nucleus towards
periphery. The impact on visual acuity varies and warrants an early surgical
intervention during infancy or childhood.
9. Total Cataract:
This phenotype has been exhibited both by families having autosomal
dominant and X-linked trait for congenital cataracts. It has been proposed that
total cataract may be the final outcome of any of above types of cataracts.
Some rare phenotypes of cataracts have been reported in sporadic cases but not in
families (Francis et al., 2000).
1.4.3 Genetics of Congenital Cataract:
Till date, 44 genetic loci (Table) have been mapped for primary
congenital cataracts and are associated with different morphological patterns
described previously. The causative genes have not been discovered at 11 of these 44
loci (See Table). The rest of the discovered genes at 33 loci have led to identification
of critical biological processes in the lens of eye. Among reported cataract families
with known mutations, 45% have shown association with gene for lens crystallins);
around 12% for mutations in genes for various growth and transcription factors; 16%
for connexins and about 5% each have shown their association for membrane
proteins, intermediate filament proteins or protein degradation apparatus and
approximately 8% are found to be associated with functionally divergent genes
including genes involved in the metabolism of lipids (Shiels and Hejtmancik, 2016).
62
Table 1.3: Loci and their corresponding genes for syndromic and non-syndromic
Congenital Cataracts. Adapted from (Shiels and Hejtmancik, 2016) with
some modification.
Cataract Morphological
pattern
Gene Locus Inheritan
ce
Associated phenotypes
CTRCT-1 Multiple GJA8 1q21.1 AD/AR Microcornea
CTRCT-2 Multiple CRYGC 2q33.3 AD Microcornea
CTRCT-3 Multiple CRYBB2 22q11.23 AD Microcornea
CTRCT-4 Multiple CRYGD 2q33.3 AD Microcornea
CTRCT-5 Multiple HSF4 16q21 AD/AR -
CTRCT-6 Multiple EPHA2 1p36.13 AD/AR Age-related cortical
CTRCT-7 ? 17q24 AD -
CTRCT-8 Multiple ? 1pter-p36.13 AD -
CTRCT-9 Multiple CRYAA 21q22.3 AD/AR Microcornea
CTRCT-10 Multiple CRYBA1 17q11.2 AD -
CTRCT-11 Multiple PITX3 10q24.32 AD Anterior segment
mesenchymal dysgenesis,
microphthalmia,
neurodevelop-
mental abnormalities
CTRCT-12 Multiple BFSP2 3q22.1 AD Myopia?
CTRCT-13 - GCNT2 6p24 AR Adult i (blood group)
phenotype
CTRCT-14 Multiple GJA3 13q12.1 AD -
CTRCT-15 Multiple MIP 12q13.3 AD -
CTRCT-16 Multiple CRYAB 11q22.3 AD/AR Myopathy, cardiomyopathy
CTRCT-17 Multiple CRYBB1 22q12.1 AD/AR -
CTRCT-18 FYCO1 3p21.31 AR -
CTRCT-19 LIM2 19q13.41 AR -
CTRCT-20 Multiple CRYGS 3q27.3 AD -
CTRCT-21 Multiple MAF 16q22.23 AD -
CTRCT-22 Multiple CRYBB3 22q11.23 AD/AR -
CTRCT-23 CRYBA4 22q12.1 AD -
CTRCT-24 Anterior polar ? 17p13 AD -
CTRCT-25 ? 15q21.22 AD -
CTRCT-26 Multiple ? 9q13-q23 AR -
CTRCT-27 Nuclear
progressive
? 2p12 AD -
CTRCT-28 ? 6p12-q12 Complex Age-related cortical
CTRCT-29 Coralliform ? 2pter-p24 AD -
CTRCT-30 Pulverulent VIM 10p13 AD -
CTRCT-31 Multiple CHMP4B 20q11.21 AD -
CTRCT-32 Multiple ? 14q22-q23 AD -
CTRCT-33 Cortical BFSP1 20p12.1 AR -
CTRCT-34 Multiple ? 1p34.3-p32.2 AR Microcornea
CTRCT-35 Congenital
nuclear
? 19q13 AR -
CTRCT-36 TDRD7 9q22.33 AR -
CTRCT-37 Cerulean ? 12q24.2-q24.3 AD -
CTRCT-38 AGK 7q34 AR Senger’s syndrome
CTRCT-39 Multiple CRYGB 2q34 AD -
CTRCT-40 NHS Xp22.13 X-linked Nance-Horan (Cataract
dental) syndrome
CTRCT-41 WFS1 4p16.1 AD Wolfram syndrome
CTRCT-42 CRYBA2 2q34 AD -
CTRCT-43 UNC45B 17q12 AD -
CTRCT-44 LSS 21q22.3 AR -
63
1.4.3.1 Role of Various Genes in Cataract Development:
In its simplest form, the cataract is defined as any opacity within the
lens of eye. When this occurs, the refractive index of the lens changes considerably in
order to adjust the image according to the light of different wavelengths passing
across it. Cataract occurs due to some variations in the lens which may include
changes both in its structure and proteins or sometimes both may co-exist. Due to
cataract, the natural geometric order of the lens and its membranes is altered, and
these effects can further enhance the above described changes in the lens structure and
proteins, leading to increased light scattering (Shiels and Hejtmancik, 2016).
The hereditary congenital cataracts are associated with considerable
phenotypic heterogeneity as same mutation among different families or among
different individuals within same family may exhibit variable clinical phenotypes.
Such type of phenotypic heterogeneity points toward the presence of some modifying
genetic or environmental factor affecting the expression of mutated protein primarily
responsible for cataract development. Likewise, congenital cataract also shows
genetic heterogeneity as mutations in genes apparently having no relationship among
them may result in cataracts having similar morphological pattern. Such phenotypic
and genetic heterogeneity suggests that whatever the triggering event is, the final
outcome is the clinically observed cataract (Shiels and Hejtmancik, 2016).
Collapse of micro-architecture of the lens is found to have been
associated with congenital cataracts. Vacuoles are formed causing profound
fluctuations in optical density together with increased scattering of light. It has been
suggested through various studies that congenital cataract usually results when
mutations affecting crystallins and other lens proteins are sufficient enough to cause
their direct and rapid aggregation within the lens. Furthermore, when such mutations
are benign, they only put the individuals at increased risk to environmental factors
(Diabetes, dietary, ultraviolet or oxidative stress) for age-related cataract
development. Based on this proposed genetic association, it is reported that hereditary
congenital cataracts tend to have highly penetrant Mendelian transmission (Shiels and
Hejtmancik, 2016).
64
Varying degree of consequences ranging from change in structure and
function may result from mutations in genes for lens proteins. For genes, for example,
PITX3 and MAF which code for transcription factors, the mutations may result in
complete absence of corresponding transcription factors for lens development leading
to abnormal lens proteins and structure. For genes for membrane and channel
proteins, mutations may lead to impaired ion or solute transport across membranes
(Shiels and Hejtmancik, 2016).
The major lens proteins in vertebrate eye lens are crystallins alpha (α),
beta (β) and gamma (α), comprising around 90% of total lens water soluble proteins.
There are two main classes of crystallin proteins i.e. α-crystallin and β-/γ- crystallin
superfamily. Both β- and γ-crystallins possess two domains composed of four motifs
and have similarity in their structure. The short range spatial order packing and
stability are thought to play an important role for maintaining transparency of the eye
lens. In humans, six genes for crystallins proteins are located together on
chromosome 2q33-35. Conversely, there is an additional crystalline gene CRYGS
located on chromosome 3q26.3-qter and is different from other crystallins in a sense
that it possesses an additional α-helix. γ-crystallins exhibit considerable internal
symmetry in their structure and it has been suggested that it contributes to provide
structural stability. γ-crystallins are located in nucleus of the lens and their proportion
in individuals less than two years of age has been found to be approximately 35% for
CRYGS, 45% for CRYGC and remaining 20% for CRYGD (Vanita et al., 2009).
Mutations in CRYGS cyrstallins have been reported both in congenital
cataracts and progressive juvenile cataracts. A novel mutation reported by Vanita et al
in 2009 (V42M) in CRYGS was found to be associated with congenital cataract with
bilateral symmetrical nuclear cataract and more dense in the central region than in the
periphery. This mutation appeared to cause abnormal folding in its corresponding
protein (i.e. γ-crystallins) due to exposure of internal hydrophobic residues to the
surface. Similar pathophysiological mechanisms explaining the increase in surface
hydrophobicity with subsequent decrease in their water solubility have been suggested
for other γ-crystallins associated with congenital cataracts as well (Pande et al., 2005)
& (Pande et al., 2010). Hence, any mutation in genes encoding γ-crystallins not only
cause self-aggregation but also make them extremely sensitive to chemical and
65
thermal denaturation ultimately leading to their precipitation and scattering of the
light (Shiels and Hejtmancik, 2016).
α-crystallins act as chaperones for γ-crystallins and others thus helping
them in proper folding. It has been suggested that the formation of mutant crystallins
may lead to their escape from binding with α-crystallins in inherited congenital
cataracts. Furthermore, increased accumulation of denatured proteins with time may
exceed the buffering capacity of the α-crystallins. Thus the presence of denatured
proteins in the eye lens not only leads to scattering of light but their presence within
the lens is also toxic for the lens homeostasis (Shiels and Hejtmancik, 2016).
It has been reported that CRYGS mutations are also linked with
development of cataracts in mice. As γ-crystallins are highly expressed in the eye
lens, the mutations in their respective genes are usually associated with nuclear or
zonular cataracts although there are some variations as well. On the other hand,
mutations in BFSP2 are found to be linked with various phenotypes such as juvenile-
onset progressive cataract, pulverulent cataracts and spoke-like cortical opacities. For
CRYGC, four mutations have found to be associated with different phenotypes
including nuclear and lamellar cataracts whereas five mutations in CRYGD have been
reported to be linked with entirely different phenotypes. Functional assays of two of
these mutations revealed enhanced rate of formation of crystals in the lens due to
alteration in surface properties of proteins while in one mutation caused increased
susceptibility of respective lens protein to thiol-mediated aggregation (Vanita et al.,
2009).
GJA8 is found to be associated with more than a dozen mutations
expressing multiple phenotypes with extensive diversity in their pathogenesis. So
much so that the identical mutations in different families led to expression of entirely
different phenotypes suggesting the possible role of epigenetic factors (Vanita et al.,
2009).
Some mutations are associated with destruction of lens cells and their
microarchitecture as was reported in case of 5 base insertion (c.119_123dup,
c.238insGCGGC, p.C42Afs*63) in the CRYGC gene (Ma et al., 2016) (Ren et al.,
2000) & (Scott et al., 1994) resulting in the expression of a unstable hybrid having
66
identical 41 amino acids like that of γC-crystallin followed by 62 novel amino acids.
When it was made to express in mice, there was destruction of lens microarchitecture
due to degeneration of lens fiber cells. These and other findings have led to the
conclusion that the mutant proteins have a direct toxic effect on the lens histological
architecture and thus its structure and function (Shiels and Hejtmancik, 2016).
Chen J studied the role of FYCO1 associated mutations in 13 families
and then in 125 familial cases. They studied the expression of FYCO1 transcripts and
described the intracellular localization of the proteins. FYCO1 is located on
chromosome 3, harbors 18 exons spanning 79kb and encodes for a 167 kDa protein
containing 1478 amino acids. It has remained conserved through evolutionary
changes and analyses using various bioinformatics tools have shown that FYCO1 is
long coiled-coil protein and is similar to members of two families of Rab effector
proteins i.e. RUN and FYVE domain-containing protein (RUFY1-4) and early
endosome antigen 1 (EEA1). It has a long central coiled-coil region flanked at the N
terminus by an α-helical RUN domain or a zinc finger domain and at the C terminus
by a FYVE domain. It is unique in its structure in a sense that C terminus possesses an
extension in the form of GOLD (Golgi Dynamics) domain and there is an
unstructured loop which connects the FYVE and Gold domain with each other. Most
of the mutations which have been identified in FYCO1 gene result in truncation of the
protein resulting in termination of peptide chain before the formation of GOLD
domain structure with subsequent loss of protein function. Additionally, most of
truncated mutations have been reported to hit internal exons with resulting nonsense-
mediated decay of the mRNAs (Chen et al., 2011).
Autophagy has been implicated in the pathogenesis of many disorders
such as tumorigenesis, cardiomyopathy, Chron’s disease, Non-insulin dependent
diabetes mellitus, neurodegenerative disorders and longevity. Many proteins and
multimolecular complexes contribute to formation of autophagosomes such as PI(3)
binding proteins, P13 phosphatase, Rabs, the Atg1/ULK1 protein-kinase complex, the
Vps34-Atg6/beclin1 class III P13-kinase complex and the Atg12 and Atg8/LC3
conjugation systems. The process of fusion of autophagosome with lysosome to form
autolysosome requires Rab7. FYCO1 has been suggested as an effector of Rab7 and a
PI(3)P binding protein and is associated with exterior of autophagosomes via tis
67
FYVE domain. It has been reported through various studies that FYCO1 plays a role
in fusion of autophagosome with lysosomes and it functions as a platform for
assembly of vesicle fusion and trafficking factors. As autophagy is important for
degradation of dead cellular debris including misfolded proteins, so any disruption of
this activity could lead to loss of lens transparency. Whatever the mechanism may,
loss of FYCO1 as a cause of congenital cataracts is an ample evidence for the
importance of autophagy to maintain the lens transparency (Chen et al., 2011).
1.4.4 Role of Unfolded Protein Response (UPR) in Congenital Cataract:
This effect which has been named as Unfolded Protein Response
(UPR) is exerted by mutant or unfolded/abnormal proteins. The basic objective of
UPR is to reduce the stress within endoplasmic reticulum (ER) when considerable
quantity of misfolded or unfolded proteins has been accumulated within its lumen.
This response consists of a series of intracellular signaling pathways with the aim to
reduce the stress within ER lumen created due to accumulation of unfolded/misfolded
proteins due to mutations in their respective genes. The response is initiated by a heat-
shock 70kDa protein 5 HSPA5 (also known as BiP or GRP78). Under normal
conditions, this protein binds to three major ER-resident sensors (IRE1, ATF6 and
EIF2Ak3) and keeps them in inactive states. The HSPA5 protein gets activated by
dissociating from these three sensors described above, in response to accumulation of
large number of unfolded proteins, thus initiating the unfolded protein response. UPR
tries to reduce the stress by causing a decrease in protein synthesis by inducing
eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3) which leads to
inactivation of alpha subunit of eukaryotic translation initiation factor 2 through
phosphorylation and thus inhibiting the initiation of translation. Simultaneously it also
causes upregulation of endoplasmic reticulum associated degradation product
(ERAD) and increases the chaperone levels. If stress is severe beyond the limits of
UPR, it causes apoptosis of the cells through intrinsic and mitochondrial mediated
pathways. Although lens fibre cells do not possess nuclei and endoplasmic reticulum
or Golgi, but lens epithelial cells and cortical fibre cells may take part in UPR as they
possess these organelles. Various studies through experiments on animal models have
provided sufficient evidence in support of UPR (Shiels and Hejtmancik, 2016).
68
1.4.5 Age-related Cataract:
Two specific types of cells comprise the ultrastructure of lens i.e
anterior layer of cuboidal epithelial cells containing all cytoplasmic organelles and
fibre cells making the most of lens structure but they lack nuclei, endoplasmic
reticulum and Golgi. The architecture, arrangement and sutures of large fibre cells
within the structure of lens play a central role in maintaining its transparency and
therefore uninterrupted light transmission across it. A very extensive network of gap
junctions is responsible for maintaining the normal function and survival of cells in
avascular lens. Each gap junction channel comprises of two hemi-channels termed as
connexons, and these connexons adhere the adjacent cells with each other. Each
connexon in turn is made up of six (hexamer) polypeptide of connexins which are
formed by three isoforms of connexins i.e. Cx43, Cx46 and Cx50. Each of these
connexins contains four transmembrane domains (from M1 to M4), two extracellular
loops (E1 and E2) and three intracellular regions (the NH2 terminus, a cytoplasmic
loop and a COOH terminus). The gap junctions are specialized membrane transport
proteins and are permeable to various ions and solutes (such as K+, Ca+2, glucose
etc) and second messangers (such as inositol triphosphate, cAMP, cGMP). These gap
junctions play a central role in lens homeostasis and maintaining the transparency of
lens fibre cells (Chen C, 2015).
1.4.6 Treatment Options for Cataract:
Although surgical removal of lens followed by intraocular lens
implantation is the first line of treatment but it is associated with poor consequences
especially when it is associated with other abnormalities and also due to several intra-
operative and post-operative complications. The main factor that determines the
surgical outcome in congenital cataract patients is the age at the time of surgery. Early
diagnosis and surgical intervention plays a very important role to prevent such
patients from severe complications thereafter. It has been reported that in developing
countries the mean age of diagnosis of children with congenital cataracts is 3.2 years
although such patients could be diagnosed within first 3-4 months after birth through
various national screening programs (Lin et al., 2015).
69
Although there has been much progress in surgical treatment of
cataract, it is still a leading cause of low vision and blindness all over the world. Such
increased global burden of cataract has compelled researchers to look for non-surgical
methods to prevent or delay the process of cataract formation and there are some good
news in this regard. Researchers have developed a method that regenerates lens
regeneration from endogenous stem cells with no need for intraocular lens
implantation. Likewise there is some hope that causative mutation for congenital
cataract could be corrected through CRISPER/Cas-9 technology (Shiels and
Hejtmancik, 2016).
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SECTION IV
OPHTHALMOLOGICAL
EXAMINATION
71
1.5 Ocular Examination
Patients with PCG usually present with IOP>21 mmHg, cupping of
optic disc, lacrimation, photophobia, Haab’s Striae, corneal edema, corneal
opacification, decreased visual acuity and eye enlargement (bupthalmos) (Cascella et
al., 2015b). Accurate diagnosis of glaucoma requires examination by expert
ophthalmologists with latest equipment and include tests such as measurement of
intraocular pressure, optic nerve examination including cup-disc ratio (fundoscopy)
and visual acuity. The screening with either measurement of IOP or visual acuity
alone is not sufficient for diagnosis because of low sensitivity (Gupta and Chen,
2016). Patients with Stargardt disease manifest central visual loss with central
scotomata, macular atrophy and yellow white retinal flecks which can be seen through
funduscopic examination. During initial stages of the disorder, fundoscopy shows a
normal fundus or mild retinal abnormalities with or without visual field defects. The
diagnosis of Stargardt diseases can further be aided by optical coherence tomography
(OCT) showing loss of normal retinal architecture beginning at central macula
commonly known as bull’s eye maculapathy-like appearance (Tanna et al., 2017).
Congenital cataract, the primary cause of treatable blindness, can lead to irreversible
blindness due to decreased sensory information available to visual center during the
developmental period of a child (Wu et al., 2016). Patients with congenital cataract
can be identified through ophthalmological examination that includes visual acuity,
slip-lamp examination, biometry and funduscopic examination (Chen C, 2015).
Furthermore, positive Family history is important because of high prevalence of
consanguinity in Pakistan and its strong association with autosomal recessive nature
of inherited visual disorders (Adhi et al., 2009).
The diagnosis of all above mentioned inherited visual disorders
therefore require a comprehensive ophthalmological examination based on various
tests. The details of these tests is given below:
1.5.1 Visual Acuity:
Distance visual acuity (VA) is the distance dependent on minimum
angle of separation between two objects which permits them to be perceived as
distinct entities. It is part of a routine ophthalmological examination if there is some
problem in clear vision. It is most commonly done with the help of Snellen chart (see
72
Fig-2.1) containing black letters or symbols of different sizes against a white
background (Bowling, 2016).
This test may be carried out either in a hospital or clinic or outside in
the field as a screening test for detection of ophthalmological disorders. Patient needs
to remove the glasses or contact lenses and is asked to read the chart which is 20 feet
from him. Afterwards he is asked to read the chart with one eye only at a time while
covering the other eye. For illiterate people who cannot read, numbers or pictures are
used.
Fig-1.13: The Snellen Chart
Visual acuity is expressed as a fraction.
The top number or the number in numerator refers to the distance of the
patient from the chart. This is standard distance and usually 20 feet.
The bottom number or the denominator refers to the distance at which a
person having normal eyesight should read the same line a patient correctly
reads.
73
20/20 is considered to be a normal visual acuity whereas 20/40 shows
that the line correctly read by a patient sitting 20 feet away is easily read by a person
having normal vision from a distance of 40 feet. Abnormal results indicate that a
patient needs glasses or contact lenses or it may be a sign that patient’s eye needs
further evaluation by an ophthalmologist. Results are expressed in the following way
(Bowling, 2016):
Normal Monocular VA: Normally it is equal to 6/6 (or 20/20) on Snellen chart. In
young adults, it is usually superior to 6/6.
Best-corrected VA: It indicates the level of VA achieved with maximum refractive
correction using different lenses during examination.
Counting Fingers (CF): It shows the ability of patient to tell how many fingers of the
examiner holding up, he can count at a specified distance which is approximately 1
metre.
Hand Movements (HM): It is the ability of the patient whether he can perceive the
movements of examiner’s hand held just in front of the patient.
Perception of Light (PL): Here, a patient is only able to tell whether he can perceive
the lightness or not while properly covering the other eye.
1.5.2 Corneal Diameter:
In children without any visual disorder, the normal corneal diameter in
horizontal axis ranges between 9.5 to 10.5 mm for neonates and 10.0 to 11.5 mm for
children up to one year of age. Corneal diameter more than 1.0 mm above the normal
range needs to be investigated. Glaucoma should be considered in any child having a
corneal diameter greater > 13.0 mm. Horizontal diameter > 14mm is usually regarded
as megalocornea (Khan et al., 2011)
1.5.3 Corneal Edema & Haab`s Striae:
Corneal edema is due to elevated IOP (usually in PCG or Juvenile-
onset glaucoma) and may be gradual or sudden in onset. Corneal edema usually
occurs along with curvilinear breaks in Descemet’s membrane (Haab’s striae), which
remain for whole of the patient’s life (Bowling, 2016).
74
1.5.4 Corneal Opacity:
Corneal opacity occurs when cornea becomes looses its transparent
nature and becomes opaque. Cornea appears while or cloudy and interferes with
passage of light through it to retina leading to decreased visual acuity. In glaucoma
patients, corneal opacity is initially more marked peripherally but gradually it extends
to center of cornea. In bupthalmos (enlarged eye balls), cornea may appear completely
opaque and vascularized.Bilateral corneal opacities are also observed in an another
inherited disorder known as Peter’s anamoly (Bowling, 2016).
1.5.5 Tonometry:
Among all risk factors for glaucoma, elevated IOP is most important
risk factor. (Yorio, 2008). Tonometry is an ophthalmological procedure to determine
IOP especially in patients who are risk for development of glaucoma. Most of the
instruments for tonometry are calibrated to measure IOP in millimeters of mercury
(mm Hg).
1-Goldmann Tonometry:
All patients visiting an ophthalmological clinic should undergo
measurement of IOP as many patients with glaucoma do not have any symptoms of
the disorder. applanation tonometry for measurement of IOP is a gold standard
method for determination of elevated IOP (Stevens, 2012). It is highly accurate and
dependable instrument for measurement of IOP requiring no contact with corneal
surface. It is based on the principle to applanate (flatten) the cornea by a puff of air.
Because it does not come in contact with cornea, so there is no possibility of any
damage to cornea and cross contamination (Salim et al., 2009).
75
Fig-1.14: Goldmann Applanation Tonometer Adapted from (Stevens, 2012)
Normal Values:
Considerable variation occurs in IOP throughout day and night. It is
usually highest in morning and lowest in afternoon. This diurnal variation of IOP for
normal eyes ranges between 3-6 mmHg and is more marked in patients with glaucoma.
Normally IOP ranges between 10-21 mm Hg with an average of about 15-16 mm Hg
(Bowling, 2016).
1.5.6 Gonioscopy:
Gonioscopy is the procedure to evaluate the anterior chamber (AC) or
iridocorneal angle. There are other procedures which can also be used to view the AC
angle such as optical coherence tomography and high-frequency ultrasound
biomicroscoy (UMB) but gonioscopy offers best evaluation in terms of clinical
ophthalmological practice. AC angle cannot be directly visualized through the intact
cornea because of total internal reflection of light by pre-corneal tear film.
76
Goniolenses are used for this purpose as they replace the tear film-air interface with a
tear film-goniolens interface thus eliminating the total internal reflection. The
goniolens helps an ophthalmologist to view the anterior chamber angle through a
mirror. (Bowling, 2016).
Gonioscopy is considered as gold standard method for assessment of
anterior chamber angle (ACA) in ophthalmological examination for patients at risk
for glaucoma (Fig-1.15 & Fig-1.16) (Campbell et al., 2015).
There are two methods of gonioscopy (Bowling, 2016):
I- Indirect Gonioscopy:
It uses a mirror that reflects the light rays from the AC angle and provides a mirror
image of the angle when used in combination with slit-lamp. There are two further
sub-types of indirect gonioscopy on the basis of number of lenese or prisms used
during the procedures:
a) Non-indentation gonioscopy: The classical Goldmann lens consists of three
mirrors which include Magna view, Ritch trabeculoplasty and the Khaw direct
view
b) Indentation gonioscopy: Indentation gonioscopy include prisms which
contain four goniolenses such as Zeiss, Posner and Sussman.
II- Direct Gonioscopy:
This technique is called direct gonioscopy as it allows viewing light rays emiting
from the AC angle directly without reflection from inside the lens. Here slit-lamp
is not required with patient lying in supine position under general anesthesia for
evaluation and for surgical treatment of congenital glaucoma.
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Fig-1.15: The Normal Irido-corneal angle
Source: (https://en.wikipedia.org/wiki/Gonioscopy#/media/File:Gonio.png)
Fig-1.16: A- Irido-corneal angle in Open Angle Glaucoma, B- Irido-corneal angle in
Angle-Closure Glaucoma.
Source: (https://en.wikipedia.org/wiki/Gonioscopy#/media/File:Gonioview.png)
1.5.7 Ophthalmoscopy (Fundoscopy):
Ophthalmoscopy (fundoscopy) is an ophthalmological examination
technique used by an ophthalmologist to see the interior of fundus of an eye and other
structures in order to assess their status by using hand held instrument called
ophthalmoscope (or funduscope). It is usually carried out during routine eye
examination and may be performed during emergency procedures.
78
It is very important to assess and determine the health status of retina
and the vitreous humor. Usually a mydriatic agent is used before doing fundus
examination to dilate the pupil for better inspection. Recent advancements have led to
newer instruments like Scanning Laser Ophthalmoscope which can allow fundus
examination through pupils as small as 2 millimeters, so dilating pupils is no longer
required with these devices.
Ophthalmoscopy is of two major types:
Direct ophthalmoscopy: It produces an upright and real image which is
around 15 times magnified than the real image. Direct ophthalmoscope is a
device about the size of a small torch with multiple lenses. This type of
ophthalmoscope is most commonly used during a routine physical and
ophthalmological examination (See Fig-1.17).
Indirect ophthalmoscopy: Indirect ophthalmoscope produces an inverted and
inverted image which is approximately 2 to 5 times magnified than the real
image. An indirect ophthalmoscope consists of a light attached to a headband
whereas the fundus is examined through lens handled manually by other hand.
An indirect ophthalmoscope provides a better view of the fundus even if there
is a milder opacification of cornea or lens. It is available both in monocular
and binocular modes and is more suitable for peripheral viewing of retina.
Fig-1.17: A-Fundus in a normal person, B- Fundus in a glaucomatous patient showing
cupping of optic disc.
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An alternative or complement to ophthalmoscopy is to perform a
fundus photography, where the image can be analyzed later by a professional.
1.5.8 Cup to Disc Ratio:
Cup-Disc ratio (CDR) is a measurement being used in ophthalmology
for diagnosis and assessment of progression of glaucoma. Optic disc is an anatomical
landmark in posterior retina where optic nerve and accompanied blood vessels enter
the eyeball. Usually optic nerve is flat or has a certain amount of normal cupping, but
elevated IOP in glaucoma leads to further cupping of the disc which is pathological in
nature. As glaucoma progresses, the cup gradually enlarges until it occupies most of
the disc area (Bowling, 2016).
The cup-to-disc ratio is obtained by dividing the diameter of the “cup”
of the optic disc with total diameter of the disc. CDR of 0.1 indicates the cup fills 1/10
of total area of the disc whereas a ratio of 0.7 shows that 7/10 area of the disc is filled
by the cup. Normal values of CDR is up to 0.3, therefore any value greater than this
indicates pathology or glaucoma (Tatham AJ, 2013) (Elolia, 1998).
1.5.9 Optical Coherence Tomography:
Optical Coherence tomography (OCT) is one of latest imaging
technologies for visualizing internal ocular structures. It is based on measurement of
time taken by infrared light to be reflected from internal structures. It can be used to
view both anterior segment structures as well as posterior segment including retina
and its layers (Ramos et al., 2009). It is considered a standard ophthalmological
investigative tool for diagnosis and monitoring of progression of all retinal disorders
including Stargardt disease. It is very rapid, non-invasive and non-contact method of
imaging internal ocular structures. OCT can provide such minute morphological
details of ocular structures that once were considered to be possible only through
histopathological examination. In today’s ophthalmological practice, surgical
intervention relies significantly on OCT imaging in case of retinal diseases. Because
of its simple operation, it has become a routine ophthalmological procedure usually
done by trained technicians. (Arevalo et al., 2013)
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SECTION-V
WHOLE EXOME SEQUENCING
81
Great developments have occurred in sequencing human DNA to study
variations present in it since the first reliable technique developed by Fredrick Sanger
in December 1977. Today newer technologies allow complete sequencing of human
genome for as low as 1000 USD. Sanger sequencing basically is based on the
principle of chain terminators specific for each nucleotide to read the sequence of a
short fragment of DNA. This method later put the foundation of development of first
automated DNA sequencer in 1980s. Although, it has very high accuracy but it is
limited to sequencing of only one fragment of a DNA at a single time with a
maximum length of 1000 bp rendering this technique unsuitable for large DNA
sequencing projects. The completion of human genome project some 15 years ago
paved the way for high throughput DNA sequencing techniques. In spite of all newer
developments, Sanger sequencing is still the preferred method of sequencing to study
the monogenic disorders all over the world (Petersen et al., 2017). All the sequencing
technologies in Post-Sanger era constitute Next-Generation Sequencing technologies
and include various methods ranging from sequencing whole human genome to some
targeted or specific areas of the genome (Morozova and Marra, 2008). Whole exome
sequencing is based on sequencing protein coding regions of human genome and has
gained popularity to study Mendelian disorders due to its relatively low cost and less
time consumption in terms of data analysis after sequencing. This method enables us
to not only sequence the coding regions of genome but it includes splice site variants
as well. It is estimated that more than 85% of mutations are present in protein coding
regions in the human genome. Since its introduction in 2005, Whole Exome
Sequencing has led to identification of a large number of mutations in Mendelian
disorders (Petersen et al., 2017).
Different platforms are available for Whole-Exome Sequencing, based
on different strategies but all share some common steps. All these platforms require
processing of genomic DNA into a library appropriate for sequencing. This requires
breakdown of high-molecular DNA into shorter-sized DNA fragments suitable and
specific for each platform. Afterwards, blunt-ended DNA fragments are generated by
end-polishing followed by specific A/T adapters (specific for each platform) ligation
to 3’ and 5’ ends of the fragments. These fragments are now ready to be loaded for
sequencing but some platforms require pre-amplification of these fragments before
loading (Buermans and den Dunnen, 2014).
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Whole-exome sequencing is used for identification of variants
responsible for single-gene disorders, to explain the nature and mechanism of
heterogeneous genetic disorders and to improve the diagnostic techniques. The
magnitude of data obtained through Whole-exome sequencing is comparatively
smaller than that obtained through Whole-genome sequencing. Whole-exome
sequencing data of a single DNA sample usually include 12000 variants in coding
regions only of which more than 90% are already present in online available
databases. Sequencing of DNA samples is followed by careful analysis of the
obtained data. The raw data include various sequencing artifacts including poor
quality reads, sequencing errors and the contaminations induced during adaptor
ligation. The raw data is improved by removing or trimming such low-quality reads
from the variants obtained through sequencing which may lead to incorrect biological
conclusion. The next step is alignment of remaining variants to online available
reference genome databases such as UCSC and Genome Reference Consortium
(GRC). The third and most important step of Whole –exome data analysis is
identification of candidate variants which may include germ line variants, Copy
number variants (CNVs), structural variants (SVs) and somatic variants. It is
important that variant identification must be supported by enough number of
sequencing reads. During analysis of the data, the variants which are supported by a
small number of reads should be discarded. Finally the data is further filtered by
annotating it to computer-aided variant databases which are publicly available. The
most common public variant database tool used for this purpose is dbSNP. Depeding
upon the approach used, these public variant databases not only provide region-based
analysis but functional prediction of variants on the structure of proteins as well. An
important task during WES data analysis is to filter and narrow down the number of
candidate variants which could further be tested for interpreted by direct Sanger
sequencing for segregation. The most commonly used method for prioritizing the
candidate variants is to exclude those variants which are present in public SNP-
databases as they usually represent harmless variants. Filtering variants can be aided
by careful analysis of the pedigree as well. Nonetheless, all prioritization methods
have the risk of filtering out the pathogenic variant, therefore annotation and
prediction tools should be used carefully during data analysis. (Pabinger et al., 2014).
83
During the last decade, enormous progress has been made in
sequencing technology techniques. With decreasing cost of sequencing by each year,
it is hoped that Whole-exome sequencing may be shifted to Whole-genome
sequencing which would be more helpful to find the causative mutations (Pabinger et
al., 2014). It can said without any doubt that in near future, we will have an ultimate
sequencing platform that would work on single DNA/RNA molecule, available at
lesser cost and without any requirement for pre-amplification steps. Furthermore it
would have much higher accuracy as compared to existing platforms for next
generation sequencing (Buermans and den Dunnen, 2014).
84
CHAPTER 2
MATERIALS & METHODS
85
2.1 Methodology
2.1.1 Place of study
This study has been carried out at Molecular Biology and Genetics
Department, Liaquat University of Medical and Health Sciences, Jamshoro and
Department of Otorhinolaryngology, School of Medicine, University of Maryland,
Baltimore, United States of America (USA). Written informed consent was obtained
from all participating individuals including affected members of the families. Consent
was obtained from the guardians in case of minors. Proformas were filled by asking
questions related to family history, mode of inheritance and clinical features of
corresponding inherited visual disorder running in the families. All affected members
were subjected to detailed ophthalmological examination for confirmation of the
diagnosis. The detailed methodology of the study is described as follows:
2.1.1 Inclusion Criteria:
Families with two or more than two members affected with various
inherited visual disorders (Primary Congenital Glaucoma, Congenital Cataract or
Stargardt’s Disease) have been included in the study.
2.1.3 Exclusion Criteria:
1. The families with only one member affected.
2. Patients affected with visual disorders due to secondary causes.
Study of Genetic and clinical basis of inherited visual disorders was carried out in two
parts:
1. Field Work:
Identification of families having two or more members affected with
inherited visual disorders.
Enrollment of selected families after obtaining written informed
consent from patients or their legal guardians to participate in the
study.
86
Pedigree drawing of affected families.
Taking detailed History of enrolled families
Collection of blood samples from affected and normal individuals of
enrolled families.
2. Bench Work:
DNA Extraction.
Genotyping for linkage to reported loci/genes for Primary Congenital
Glaucoma, Stargardt disease and Congenital Cataract.
Haplotype analysis for linkage to reported loci/genes for above
mentioned disorders.
Sanger Sequencing for mutational analysis.
Whole Exome Sequencing in case of non-linkage to reported
loci/genes.
2.1.4 FIELD WORK:
2.1.4.1 Identification and Enrollment of Families:
Families having two or more than two members affected with inherited
visual disorders were identified with the help of consultant ophthalmologists doing
practice in various parts of Sindh province and Tertiary Care Eye Hospital affiliated
with Liaquat University of Medical and Health Sciences, Jamshoro. Some hospitals
such as LRBT (Late Rehmattollah Benevolent Trust), a charity-based Eye hospital
also extended their co-operation in order to identify the families. Special schools
specific for special/blind children were also approached in this regard. Preliminary
information and family histories were obtained on a proforma (Appendix-II). On the
basis of the information, families were selected for participation in the research.
To ascertain mode of inheritance, a personal visit was made to each
identified family. During visit, detailed family history was taken along with drawing
87
of pedigree. During pedigree drawing, multiple individuals, both near and distant
relatives were interviewed in detail to get to the correct information. Pedigrees of all
identified families were subsequently re-drawn with the help of a software called
Cyrillic (Cyrillic for Windows, 3.1) and Adobe Illustrator.
Detailed medical history was taken from all identified families with
special reference to affected members of each family to ensure the familial nature of
inherited visual disorders. Medical history was taken on a proforma (Appendix-II)
especially designed for affected individuals so as to minimize the possibility of other
causes that may be responsible for pathogenesis of respective inherited visual
disorder. All affected individuals underwent detailed ophthalmological examination
by consultants in a tertiary care eye hospital affiliated with Liaquat University of
Medical and Health Sciences for confirmation of diagnosis and to obtain further
clinical evidence that may help in understanding the pathogenesis at molecular level.
The findings of the ophthalmological examination were recorded on a proforma
(Appendix-III).
Following points were emphasized while taking clinical history and
general examination.
Age of onset of respective inherited visual disorder.
Any consanguinity between parents.
Any associated illness present at the onset of the disease.
Extent of visual loss since onset of disease and its progression afterwards.
Number of affected members on paternal/maternal side.
2.1.4.2 History Recording:
Affected members were enquired in detail about their past medical and
surgical history including hospitalization and surgery (such as trabeculectomy in case
of primary congenital glaucoma, or cataract removal in case of congenital cataract
etc). They were also asked about other disorders that could have been responsible for
causing inherited visual disorders.
88
2.1.4.3. General & Systemic Examination:
General physical appearance and health
Height and weight
Systemic examination
2.1.4.4. Clinical Assessment of Inherited Visual Disorders:
Patients were evaluated clinically at Liaquat University of Medical and
Health Sciences Eye Hospital which is basically a tertiary care hospital fully equipped
with ophthalmological facilities needed to clinically evaluate the patients for correct
diagnosis and proper management of ocular disorders. The patients were evaluated
after confirming their diagnosis and following tests were performed and the findings
were recorded. Some of the following tests are specific only to glaucoma (such as
corneal diameter, corneal edema & Haab’s Striae, Tonometry, C/D ratio etc while
fundoscopy was performed in all affected individuals where cornea was clear) where
as some special investigations were performed in case of patients with Stargardt
disease. Some clinical laboratory investigations were also done with special reference
to patients with congenital cataract.
i) Visual Acuity Test:
Visual field defects were ascertained using Snellen chart for all for
affected patients in all enrolled families with inherited visual disorders.
ii) Measurement of IOP:
IOP was determined in all individuals with congenital glaucoma only
and most of affected had elevated IOP as compared to normal individuals of
respective families. Both applanation and non-contact tonometry methods were used
for determination of IOP.
iii) Measurement of Corneal Diameter:
Corneal diameter was measured in all affected patients with primary
congenital glaucoma. In children, it was determined during examination under
anesthesia.
89
iv) Fundoscopy:
Fundoscopy was carried out in patients with primary congenital
glaucoma to assess th condition of optic nerve by determining CDR (Cup to Disc
ratio). In Stargardt disease patients, it was done to determine the condition of retina
and its layers along with deposition of any material manifested by pigmentation in
retina. Fundoscopy or ophthalmoscopy was done using both direct and direct
methods. In PCG patients with advanced features, it was not possible to view retina
through fundoscopy due to corneal scarring or corneal edema.
v) Optical Coherence Tomography:
Optical Coherence Tomography (OCT) is an advanced imaging
technique used by ophthalmologists to obtain high resolution images of retina and
anterior segment. OCT was performed in all patients with Stargardt disease to assess
the condition of retina and its layers. OCT also provides retinal measurements in
micrometers and is beneficial in all types of retinopathies and macular degenerative
disorders.
2.1.5 Laboratory Work:
A) DNA Extraction:
Venous blood (10 milliliters) was obtained from affected and normal
members including parents of all identified families. It was collected in 50 ml Falcon
tubes already containing 400 µl EDTA (Ethyline Diamine Tetra Acetic acid). The
collected blood samples were kept at -80 oC for long term storage but they were
transferred to -20oC freezer for at least 24 hours before DNA extraction. Genomic
DNA was extracted from blood samples following a non-organic procedure
(Grimberg et al., 1989). The steps of non-organic method are as follows:
Before starting DNA extraction, the blood samples were thawed.
The volume of whole blood in each falcon tube was noted in a DNA extraction
sheet.
90
Tris EDTA (30-35 ml) buffer (Tris HCl 10mM, EDTA 2mM) was added to
each falcon tube containing blood for washing. The samples were centrifuged
(3000 RPM) for 20 minutes at 25 oC. After centrifugation, the supernatant
fluid was discarded carefully keeping the pellet. The washing with Tris EDTA
was carried out four times until the pellet became white (or light pink) in
color.
After washing with Tris EDTA, TNE buffer (Tris Sodium EDTA; Tris HCL
10mM, EDTA 2mM, NaCl 400mM) was added to the pellet in each falcon
tube. TNE buffer was taken in a quantity of 0.6ml/ml of original blood
volume. 10% sodium dodecyl sulfate (SDS-20 l/ml of blood) & 50g
proteinase K (5 l /ml of original blood volume) were also added to each
sample. The samples were then put in an incubator at 37°C for overnight or 12
hours.
After ensuring complete digestion of the pellet (the solution appeared
homogenized and there were no suspended or broken down particles of pellet),
proteins were precipitated out by adding NaCl (6M) in a quantity of 100 μl/ml
of original blood volume followed by vigorous shaking, placing the tubes on
ice for 10-15 min and then centrifuging at 3000 RPM for 15 min so that the
salts and proteins settle down in the form of a pellet at the bottom of the tubes.
The supernatant was carefully transferred to appropriately labelled falcon
tubes whereas falcon tubes containing pellet of salts and proteins were
discarded. An equal volume of isopropanol was added to the supernatant in
each falcon tube to precipitate out the DNA. Extracted genomic DNA was
then washed with 70% ethanol by centrifuging (3000 RPM) for 10 minutes at
25oC.
After discarding the ethanol and carefully preserving the DNA pellet, the tubes
were air dried and DNA was dissolved in low TE preservation buffer (10 mM
Tris, 0.2 mM EDTA) and subsequently heat shocked at 70oC in a shaking
water bath for one hour in order to inactivate the deoxyribonucleases.
91
The extracted DNA was then aliquoted in properly labeled stocked screw
tubes and stored at -80oC for long term storage where as their dilutions were
kept at -20oC for short term use.
B) Quantification of DNA:
Two methods were employed in order to quantify the extracted DNA.
1. Measurement of Optical Density Using Spectrophotometer
Quantification of DNA was carried out by measuring their Optical Density
(OD) at 260 nm wavelength in spectrophotometer. 1µl of genomic DNA was
diluted with 99 µl of distilled injection water to prepare 1/100 dilutions of
each sample. The dilutions were used to measure the optical density at 260 nm
and 280 nm. The purity of extracted DNA can be assessed from the ratio of
readings obtained at 260 nm and 280 nm (i.e. A260 /A280). Because the
absorbance of DNA at 260 nm is twice than that of measured at 280 nm,
therefore pure DNA samples should have a ratio of 2.0. contamination in any
form if present, such as proteins, there would be some additional OD280 which
will decrease the absorbance ratio. Ratios less than 2.0 indicate contamination
with proteins and could affect the subsequent research procedures (Glasel,
1995).
2. Quantification by Agarose Gel Electrophoresis Against a Known
DNA Dilution
This method is based on using Ultraviolet (U/V) induced fluorescence of
ethidium bromide dye which intercalates in deoxyribonucleic acid. Agarose
gel results are viewed in Ultraviolet Gel document. The amount of DNA
fluorescence is directly proportional to the amount of nucleic acid present. The
fluorescence of newly extracted DNA can be compared with that of known
amount of standard DNA visually. The integrity of the nucleic acid can also be
assessed using this procedure.
There are two methods of preparing samples for quantification of DNA using
gel electrophoresis:
92
o Taking 1 μl of genomic DNA with a pipette and running it in 0.8-1.0% Agarose
Gel in electrophoresis apparatus at 100 Watts for 4-5 minutes.
o Taking 1 μl of 25ng/ μl of DNA dilution (method of preparing 25ng/ μl of DNA
dilution is described below) and running it 0.8-1.0% agarose gel at 100 Watts for
4-5 minutes with a known standard dilution and comparing the results in Gel
document analyzer.
C) Preparation of working dilutions of Extracted DNA:
25ng/μl working dilutions of all extracted DNA samples are prepared
either in 100 μl or 200 μl low TE buffer. The amount of DNA to be taken for
preparing working dilutions is calculated according to a formula described in below.
Working DNA concentration was kept at 25 ng/μl for single marker amplification.
C1V1 = C2V2
C1 = Initial concentration
V1 = Initial volume
C2 = Required concentration (of the dilution)
V2 = Volume of original solution (Stock) DNA required to prepare the dilution
For example, if O.D value for any extracted DNA sample is 300ng/µl
and we need to prepare 100 µl volume DNA dilution having 25ng/ µl concentration,
then apply the values in above mentioned formula:
C1 = 300ng/ µl
C2 = 25ng/ µl
V2 = 100 µl
V1 = ? (Volume of Stock DNA required to prepare the dilution)
C1V1 = C2V2
(300ng/ µl) V1= (25ng/ µl) x (100 µl)
V1 = 25 x 100µl / 300
V1 = 8.33 µl stock DNA is required to prepare 100 µl dilution.
Subtract 8.33 µl from 100 µl
= 91.67 µl of Low T.E buffer.
Dissolve 8.33 µl of Stock DNA in 91.67 µl of Low T.E buffer to prepare 100 µl
DNA dilution.
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D) STR Markers Used for Genotyping:
Each family was genotyped to Microsatellite STR markers for each
reported locus or gene for PCG, Stargardt disease and Congenital Cataract. Closely-
linked pre-designed STR markers were obtained from UCSC genome browser
(https://genome.ucsc.edu/). The forward primers were labelled with one of the
fluorescent dyes such as FAM, VIC or NED which are commercially available from
ABI. The selected markers for each locus/gene were checked for Heterozygosity in
the range of 0.7-0.8 in Marshfield marker data base. The labelling of dyed primers
was done in a way that all primers could be pooled at a single time for each locus or
gene for respective inherited visual disorder.
Details of various STR markers used for Genotyping and linkage
analysis for PCG, Stargardt disease and Congenital cataract are shown in the tables.
Table 2.1: Reported Loci for Primary Congenital Glaucoma (PCG)
Locus
(Gene)
STR Markers Distance
(cM)
ASR Dye
D2S1346 59.36 249-267 FAM
GLC3A D2S177 59.36 275-302 VIC
(CYP1B1) D2S2163 59.26 257-267 VIC
D2S2331 59.31 119-135 FAM
D1S228 29.93 116-129 FAM
GLC3B D1S402 31.02 249 VIC
D1S507 33.75 193-203 FAM
D1S2672 33.75 134-158 VIC
D14S43 84.16 158-190 FAM
GLC3C D14S61 86.29 197-227 VIC
D14D59 87.36 99-109 FAM
D14S74 87.36 291-313 FAM
D1S1165 188.32 177-210 FAM
GLC1A D1S2851 188.32 169-199 VIC
(MYOC) D1S2815 188.85 210-237 VIC
D1S218 191.52 266-286 FAM
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Table 2.2: Reported Loci/Gene for Stargardt disease
Gene STR Markers ASR (bps) cM Dye
ABCA4 D1S188 149-173 126.16 FAM
D1S3361 106-107 - VIC
D1S236 190-218 128.73 NED
PROM1
D4S3048 233-253 29.14 FAM
D4S1601 124-146 29.68 FAM
D4S2960 233-249 29.14 VIC
ELOVL4 D6S2407 148-157 89.83 FAM
D6S460 144-166 89.83 VIC
Table-2.3: Common Genes/Loci for Congenital Cataract Screening
Locus/Gene STR Marker ASR (bps) cM Dye GJA8 D1S252 98-119 150.27 FAM
D1S498 183-205 155.89 FAM
D1S2635 135-159 165.62 VIC
FYCO1 D3S3527 103-119 63.12 FAM
D3S3685 177-221 67.94 FAM
D3S3559 173-196 67.94 VIC
HSF4 D16S3043 118-150 84.75 FAM
D16S3086 182-198 85.94 VIC
D16S421
206-216 85.94 FAM
LIM2 D19S246 185-229 78.08 FAM
D19S589 161-181 87.66 VIC
D19S254 110-150 100.61 NED
CRYAA D21S1411 239 51.49 FAM
D21S1259 208-228 52.5 VIC
CRYBB3 D22S427 96-110 8.32 FAM
D22S686 180-220 13.6 VIC
CRYBB2 D22S419 257-273 21.47 FAM
D22S1167 266-278 24.74 VIC
CRYBB1 D22S1144 177-199 27.48 FAM
D22S689 202-226 28.57 VIC
E) Typing STR Markers by PCR:
The microsatellite STR markers were amplified by the polymerase
chain reaction (PCR) program on a Gene Amp PCR system ABI 2720 (Applied
Biosystems). The thermocycler program used for optimization and subsequent
amplification of single STR markers were Touch down 64-54oC, MultiCemb 54oC or
95
MultiCemb 56oC. Various reagents of PCR reaction mixture used for amplification of
STR markers are described in the Table 2.4.
Table-2.4: The components of PCR reaction mixture used for amplification of
STR markers.
Reagents Final Concentration Stock Required
Genomic DNA 50 ng 25ng/μl 3 μl
Primer (Forward) 0.4-0.8pM 4.0 pM 0.2-0.3 μl
Primer (Reverse) 0.4-0.8pM 4.0 pM 0.2-0.3 μl
dNTPs 200μM 1.25 mM 1 μl
PCR Buffer* 1X 10X 1 μl
Taq Polymerase 0.5 units 2 units/μl 0.3 μl
dH2O
Total 10 μl
*PCR Buffer 100mM Tris HCl, pH 8.0, 500mM KCl, 15-25 mM MgCl2, and 1%
Triton
Fig-2.1: Diagrammatic Representation of PCR Program 54oC.
Fig-2.2: Diagrammatic Representation of PCR Program Touchdown 64-54oC
96
F) Quantification of PCR Products for Genotyping:
For quantification of PCR products for subsequent genotyping, 1.2%
agarose gel was prepared as follows.
o Took 0.3 gm of agarose powder after weighing in an electronic balance very
carefully.
o Carefully took 25 ml of TBE (Tris Borate EDTA) buffer and put weighed 0.3gm
of agarose powder in it and heat the mixture in an oven till agarose powder got
completely dissolved in it.
o Cooled down the mixture to 60oC and then added 2 ul of Ethidium bromide to
every 25 ml of mixture.
o Put the mixture in a tray with comb in it and let it dry.
o Ran down 2.5 μl of amplified PCR products in wells of the dried agarose gel and
viewed under ultraviolet light in Gel document analyzer for proper amplification.
G) Designing DNA Plate Map for Genotyping:
When all microsatellite markers had been amplified, a plate map was
designed consisting of at least 2-3 affected individuals with 1-2 normal members and
parents of the same family.
H) Sample Preparation for Genotyping using ABI PRISM 3130 Genetic
Analyzer:
An amount of 1-1.5 μl of amplified PCR product labeled with any
fluorescent Dye i.e. VIC, FAM, or NED along with 11.8 μl of Hi-Di Formamide
(Applied Biosystems) and 0.2 μl of an internal size standard LIZ (Applied
Biosystems) were combined in 96 well plate for genotyping. Amplified PCR Products
having different sizes were aliquoted together with a difference of at least 30
nucleotides among amplicons labeled with the same dye in order to avoid overlapping
of data during analysis. The samples were heat shocked to denature them at 95 oC for
5 minutes followed by quick chilling on ice or ice pack for at least 5 minutes. The
plate is then loaded in an automated 4-capillary electrophoresis genetic analyzer 3130
(Applied Biosystems).
97
Genescan analysis and Genotyper 3.7 NT software (Applied
Biosystems) were used for allele assignment. The genotyping data is produced in the
form peaks proportionate to their product size along with dye color. The resulting
genotyping data is then transferred to data spread sheets for haplotype and statistical
analyses.
I) Haplotype Analysis:
A haplotype analysis is used to assess the inheritance pattern of a
segregating disease among affected individual. A haplotype is a set of genotyped
alleles that are arranged in accordance to cM distance on an individual chromosomal
segment. For a linkage, it is necessary that at least 3 STR markers (located in the
linkage interval of any glaucoma locus) show homozygosity. Linkage to a locus/gene
was considered to be established when homozygous data in all affected individuals of
a family correlated with that of disease pattern and inheritance within the same
family.
J) DNA Sequencing:
Sequencing is a process to determine the exact order in which
nucleotide nitrogenous bases are present in a particular DNA strand. Various
sequencing primers were used for DNA sequencing designed through primer 3
webtool. We performed DNA sequencing reactions on an automated ABI 3130
Genetic Analayzer (Applied Biosystems) using Big Dye Terminator Chemistry
(Heiner et al., 1998) for families found linked to any reported locus or gene. Genetic
analyzer first performs separation based on the principle of electrophoresis followed
by spectral detection of DNA fragments fluorescently labelled with dyes. There are
four different dyes which are normally used to identify A, G, T and C terminated
reactions.
K) Designing Sequencing Primers:
The primers were designed to sequence the exons of reported
loci/genes for PCG, Stargardt disease and Congenital Cataract using the website
www.ensemble.com, Primer3 web tool and EditSeq software.
98
L) Preparation of Stock Solutions of Sequencing Primers:
Lyophilized primers were dissolved in TE to make 100 μm stock
solutions.
M) Preparation of Working Dilutions of Sequencing Primers:
I took 8 μl of stock solution in the working dilution tube and 92 μl of
filtered low TE buffer to prepare a final working dilution of 8μM. Working dilution
tube is properly labelled and kept at either 4 oC for short term use or at -20 oC for
long-term storage. In this way 100 μl working dilution for each sequencing primer
was prepared.
N) Optimization of PCR Conditions for Sequencing Primers:
After preparation of working dilutions of sequencing primers, they
were optimized using different sets of temperature based on the melting temperature
(Tm) of oligonucleotide primers using controlled DNA. Once optimized, we
proceeded for PCR amplification of those primers with samples of families found
linked through genotyping and haplotype analysis. The sequence of primers for
amplification of CYP1B1 exons is described in the Tables.
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Table-2.5: Sequencing Primers used for CYP1B1 gene amplification
Primer ID Primer Sequence
CYP-E2a-F 5`-AGCCTATTTAAGAAAAAGTGGAAT TA-3`
CYP-E2a-R 5`-GAATCCAGCTGGATCAAAGTT-3`
CYP-E2b-F 5`-CTACCACATTCCCAAGGACAC T-3`
CYP-E2b-R 5`-AGAAGCAGCACAAAAGAGGAA CT-3`
CYP-E1a-F 5`-CCTTCTCTTCTCCAAGGGAGAGT-3`
CYP-E1a-R 5`-CTCGCCATTCAGCACCACTAT-3`
CYP-E1b-F 5`-TACGGCGACGTTTTCCAG AT-3`
CYP-E1b-R 5`-CTCTTCGTTGCTGAGCA-3`
CYP-E1c-F 5`-ACGTCATGAGTGCCGTGTGT-3`
CYP-E1c-R 5`-GTCTCTACTCCGCCTTTTTCAGAC-3`
Once sequencing primers are optimized, then there are two methods to
proceed for Sanger sequencing. First method we used at Genetics and molecular
biology laboratory, Liaquat University of Medical and Health Sciences and include
following steps:
O) Amplification of DNA Samples using Sequencing Primers:
In order to perform the sequencing reaction, the region of interest in
DNA was first amplified by performing a PCR program as described in next section.
The PCR programs were performed with 50 ng of template DNA in a 25 μl volume
reaction mixture.
Table-2.6: Reagents of Reaction Mixture for amplification of PCR Fragments for Sequencing
Ingredients Final
Concentration Stock Required
Genomic DNA 50 ng 25ng/ μl 3 μl
Forward Primer 0.4-0.8pM 8.0 pM 0.5 μl
Reverse Primer 0.4-0.8pM 8.0 pM 0.5 μl
DNTPs 200uM 1.25 mM 2.5 μl
PCR Buffer* 1X 10X 2.5 μl
Taq Polymerase 1-2 units 2 units/ μl 0.5-1.5 μl
dH2O 25μl
*PCR Buffer 100mM Tris HCl, pH 8.0, 500mM KCl, 15-25 mM MgCl2, and
1% Triton
100
P) Confirmation of Amplification through Agarose Gel Electrophoresis:
After PCR amplification with sequencing primers, 2 μl of PCR product
was observed in 1.5% agarose gel in order to confirm the proper amplification and the
purity of PCR product before Sanger sequencing. Remaining amplified PCR product
in PCR tubes is further treated to remove the impurities (if any) before proceeding for
sequencing reactions as follows.
Q) Purification of Amplified PCR Products by Ethanol Precipitation:
The amplified PCR product after PCR sequencing reaction was
transferred to labelled Eppendorf tubes and 80 μl of 100% filtered ethanol and 20 μl
of distilled injection water are added to respective eppendorf tubes. The mixture in
eppendorf tubes is kept at room temperature for 20 minutes before centrifuging it at
13000 RPM and 4OC for 20 minutes. After centrifugation, the supernatant was
discarded and the eppendorf tubes were heat dried in heat block at 60 OC. Distilled
injection water (20 μl) was then added to heat dried eppendorf tubes with gentle
tapping and kept at room temperature for some time before proceeding further.
R) Sequencing PCR:
The PCR tubes were labelled according to coded names and IDs of the
patients.
In these PCR tubes:
o 3 μl of deionized distilled autoclaved water (or Injection Water) was added
o 1 μl of forward (or reverse primer) was added of 3.2 μM
o 1 μl of PCR product was added to these labelled PCR tubes.
o This made a total volume of 5 μl in each PCR tube.
Table-2.7: The Components of master mix for Sequencing PCR
Sequencing Buffer (SB) 1.2 μl
Big Dye Sequencing Reaction 0.7 μl
Water 3.1 μl
Total (Master Mix) 5 μl
101
The 5 μl of master mix was added to each PCR tube for sequencing
PCR. This made a total volume of 10 μl in all PCR tubes. The sequencing reaction is
then carried out in thermocycler ABI 2710 (Applied Biosystems).
S) Precipitation of Sequencing PCR Products:
After sequencing PCR is done, the amplified PCR products are
transferred carefully to eppendorf tubes properly labelled. To this volume of 10 μl in
each eppendorf tube, we added following reagents:
1 μl EDTA (125 mM)
1 μl Na acetate (pH 5.6)
25 μl Distilled Absolute Ethanol
After adding all above ingredients, the samples were left at room temperature
for 45 minutes.
Then all samples were centrifuged at 3250 RPM for 20 minutes.
After centrifugation, the supernatant was discarded and 100 μl of 70% ethanol
was dispensed to all samples and again centrifuged at 3250 RPM for 13
minutes.
The samples were then allowed to air dried with their lids open.
Finally 15 μl Formamide was added to all samples and the plate was submitted
for Sanger sequencing.
T) Preparing Plate for Sanger Sequencing at University of Maryland:
1. After all sequencing primers have been optimized, I put the desired samples at
same optimized conditions without any addition of dyes (Vic, Fam or NED).
E.g I used the following recipe for optimization with various programs such as
MultiCemb or Touchdown 64-54:
1x
EconoTaq 5.0 μl
PF 0.5 μl
PR 0.5 μl
DNA 1.0 μl
H2O 3.0 μl
102
Total 10 μl
2. After PCR, all samples (3.5μl) were ran down on Gel electrophoresis for
amplification. When desired amplification is achieved, I proceeded for
ExoSAP purification.
U) PCR Purification by ExoSAP:
In ExoSap (Exonuclease-I # M0293L and Shrimp Alkaline
Phosphatase, # M0289L, New England BioLabs) procedure, Exonuclease-I removes
unincorporated dNTPs that may be left behind whereas SAP prevents circularization
and self-digestion of DNA and keeps it in linear. For this procedure, I prepared
Mastermix according to the number of samples as follows:
1x
Antartic Phosphate Buffer 1μl
Antartic Phosphatase Enzyme0.05
Exonuclease 0.075 μl
dH2O 1.375 μl
Total 2.5 μl
3. I added 2.5ul of this Exo-SAP mastermix to each sample.
4. Afterwards, I incubated all samples in PCR machine with a program at
following temperatures (Exosap Incubation).
Fig-2.3: Diagrammatic Representation of ExoSAP PCR Incubation
5. After ExoSAP incubation, I added equal volume (9.0 μl) of dH2O to all
samples. (all samples already contained 6.5 μl of PCR product + 2.5 μl of
Exosap mastermix= 9 μl), vortexed it for few seconds and short spinned them.
6. 2.0 μl of each sample was taken in two separate rows in plate (one each for
forward and reverse primers, for subsequent Seq PCR).
103
7. 1 μl of forward or Reverse primers was added in plates already containing 2.0
μl sample.
8. Sequencing mastermix was prepared according to following Recipe.
1x
5x Seq Buffer 2 μl
Big Dye 0.2 μl
dH2O 4.8 μl
Primer 1 μl
PCR Product 2 μl
Total 10 μl
9. To each sample (already containing 1 μl of either F or R primer and 2 μl of
PCR Product), 7 μl of the master mix was added. The plate was then covered
with septa or aluminum silver tape and processed at following conditions for
sequencing PCR:
1cycle 95oC 2 Mins
36cycles 95oC 10 seconds
55oC 10 seconds
60oC 4 Minutes
1cycle 72oC 5 Minutes
25oC 5 Minutes
V) Precipitation of Sequencing PCR Products:
It is important to precipitate out the unlabeled and dye-labeled
components during the sequencing process as they can interfere with electrophoretic
separation and thus affect the data analysis. For example fluorescent signals emitting
from unincorporated dye-labeled terminators that co-migrate with sequencing reaction
can obscure the desired signals. So precipitation of such products can reduce or
eliminate this interference and sodium acetate precipitation helps to get good signals
near the end of the product. After PCR sequencing reaction was done, precipitation of
sequencing products was carrioud out. The master mix protocol for precipitation is
(Mater mix: 1.5μl 3M NaOAc + 31.25 μl 100% ethanol +7.25 μl dH2O).
104
1. When above PCR was done, 40 μl of sequencing precipitate was added to 10 μl
sequencing pcr product.
2. The plate was tightly sealed with silver plate tape and vortexed for 10 seconds
and then let it stand at room temperature (in darkness) for 20 minutes.
3. The plate was spinned at 3500 RPM (at 4oC) for 30 minutes.
4. Silver tape was removed followed by inverting the plate on paper towel and
spinning it again in this inverted position on paper towel in centrifuge at 400
RPM for 15-20 seconds.
5. Then 75 μl of 70% ethanol was dispensed to each sample in the plate.
6. The plate was centrifuged at 3500 RPM (4oC) for 20 minutes.
7. The plate was inverted on folded paper towel and spinned at 500 RPM for 20
seconds.
8. Let the plate dry at room temperature and then added 10 μl Hi-Di Formamide to
each sample.
9. All samples in the plate were denatured at 95oC for 2 minutes by placing them in
PCR machine.
10. The plate was then put on cold block or ice for 2 minutes and centrifuged at 1500
RPM and 4oC for 2 minutes. It was then submitted for Sanger Sequencing to the
concerned section after proper labelling.
Sequencing was carried out on a genetic analyzer ABI 3730 system (Applied
Biosystems, USA). The sequence data was analyzed using DNA STAR software to
view the chromatograms and check for any pathogenic variant, as well as for known
and novel mutations annotated in various online genome browsers.
W) Analysis of DNA Sequences:
In Pakistan, sequencing data was analyzed using ABI sequencing
analysis (version 3.4.1). Sequences were analyzed manually by using Chromas
software version 1.45. The sequences were also compared against normal sequences
by using either Nucleotide-Nucleotide BLAST (blast) or BLAST 2 sequences (2.2.10)
on the NCBI web (www.ncbi.com). The resulting alignments of the sequencing data
are presented both in graphical and text format. Any change in DNA sequence
105
compared to the reference sequence was confirmed by sequencing both sense and
anti-sense strands.
Sequencing data at University of Maryland was analyzed using ProSeq
software. The sequencing data was compared with normal reference sequences of
corresponding exon which was saved while designing sequencing primers. Any
change observed is then confirmed using various online tools such as ExAC browser,
Human Gene Mutation Database (HGMD), ClinVar on pudmed etc.
2.1.6 Whole Exome Sequencing of Selected Families at University of
Maryland:
This technique was employed for some selected families already
screened for common genes/loci reported for corresponding disorder. DNA samples
(5μg) were prepared for Whole Exome Sequencing. After extracting data, we first
filtered it, as the data contained huge number of variants (often more than 90,000
variants) according to a strategy (Fig-2.9) devised on the basis of already published
literature. Initially attention was focused on homozygous variants and prioritized them
on the basis of pathogenicity (as predicted by various protein bioinformatics tools)
and CADD (Combined Annotation Dependent Depletion) score. After prioritizing,
possible pathogenic variants were sequenced by Sanger technique for segregation first
in affected individuals and afterwards in normal members of the family. Using this
strategy, a novel gene (ARL3) was found for Stargardt disease and for Congenital
Cataract (INPP5K, recently reported in March, 2017). The details of variants checked
for segregation in each of these disorders are mentioned in the Table-2.8 and Table-
2.10 along with various annotations.
106
Fig-2.4-Variants Filtration Scheme for Whole Exome Sequencing
107
Table-2.8: Variants Sequenced for Segregation in LUSG-03 and LUSG-04
Variant Site WT
Allele
Alternative
Allele
Gene CADD
Score
ExAC
Allele
Frequency
dbSNFP
Functional
Prediction
Voting
10:104449669 C A ARL3 35 0 4/5 as
Damaging
19:55341709 CAAA - KIR3DL1 35 0 -
15:32449875 TG - CHRNA7 34 0.00006893 -
10:126691951 C - CTBP2 0 -
Table-2.9: Sequencing Primers of ARL3
Primer Sequence Product Size
Forward
Primer GTGCATGCTAATTCCAGCTACTC
399 bp Reverse
Primer GGCCTTCATAAATGAGATGAGACT
Table-2.10: Variants Sequenced for Segregation in LUCC-15
Variant
Site
WT
Allele
Alternative
Allele
Gene CADD
Score
ExAC
Frequency
dbSNFP
Functional
Prediction
Voting
17:1417169 A G INPP5K 25.6 0 4/5 as
Damaging
6:25661807 - AAGG SCGN 35 0 -
4:6303786 - A WFS1 27.1 0 -
19:55341709 CAAA - KIR2DS4 35 0 -
1:22183825 C T HSPG2 33 0.0002553 4/5 as
Damaging
14:74340760 A G PTGR2 26.7 0.0005601 4/5 as
Damaging
20:33876688 C T FAM83C 25.9 0.0008154 4/5 as
Damaging
1:33794528 T C PHC2 25.7 0.0001894 3/5 as
Damaging
2:10059904 C A TAF1B1 25.1 0.00007413 4/5 as
Damaging
Table-2.11: Sequencing Primers of INPP5K
Primer Sequence Product Size
Forward
Primer ACTTTCAAGGCCTGAGTTCTGAT
390 bp Reverse
Primer TGCACTATCTTATCCTCCTTCCTC
108
CHAPTER-3
RESULTS
109
The present study was approved from Ethical Review Committee,
Liaquat University of Medical and Health Sciences, Jamshoro. Total twenty seven
families with inherited visual disorders including primary congenital glaucoma,
Stargardt disease and Congenital Cataract were enrolled from Liaquat University Eye
Hospital (a Tertiary Care Eye Hospital, affiliated with Liaquat University of Medical
and Health Sciences, Jamshoro) and Late Rehmatollah Benevolent Fund (LRBT) Eye
Hospital. Written informed consent was obtained from all participating individuals
and predesigned questionnaire filled to record family and clinical history (Appendix-I,
II & III). Seventeen families with primary congenital glaucoma, five families each
with Stargardt disease and Congenital Cataract were ascertained. Pedigrees were
drawn to assess the mode of inheritance and detailed family history was taken.
Environmental factors or secondary causes responsible for vision impairment were
excluded. Venous blood (10 ml) was obtained from all affected and normal
individuals and genomic DNA was extracted. All families were subjected to
homozygousirty mapping for linkage to known loci of respective disease. The
families linked to known genes were sequenced to find out the mutation; whereas, the
unlinked families were further studied to find out the disease causing gene.
110
SECTION-I
GENETIC CHARACTERIZATION OF
FAMILIAL PCG
111
3.1- Homozygosity mapping of common PCG Loci:
Total seventeen families with two or more than two members affected
with PCG were enrolled for this study. All families were visited after confirmation of
the diagnosis. Written informed consent was obtained from all participating
individuals (consent was obtained from guardians in case of minors) and family
history was recorded on a predesigned questionnaire (Appendix-I & II). Pedigrees
were drawn to establish the mode of inheritance for each family and blood samples
were taken under aseptic conditions in EDTA (Ethylene Diamine Tetra acetic acid)
containing tubes. After DNA extraction, all families were genotyped for linkage with
STR markers for four reported loci of PCG i.e. GLC3A on 2p21 (OMIM 231300),
GLC3B on 1p36 (OMIM 600975), GLC3C on 14q24.3 (OMIM 613085) and GLC3D
on 14q24.2-14q24.3 (OMIM 613086). Thirteen families (76.5%) were found linked to
CYP1B1 gene on GLC3A locus on linkage analysis whereas four families remained
unlinked to any of the four loci of PCG. On mutational analysis, seven families
segregated with p.R390H with disease phenotype (7/17; 41%) whereas four reported
variants (p.A115P, p.E229K, p.P437L and p.R290fs*37) were found once each in
four PCG families. Two novel variants were also revealed in CYP1B1 gene, a
missense mutation i.e. p.G36D and an in-frame deletion i.e. (p.G67-A70del).
3.1.1- Reported CYP1B1 Mutations:
A) p.R390H
On linkage analysis followed by sequencing, p.R390H was
predominant mutation found to be present in seven families (41%). Description of all
these PCG affected families is given below:
i) PCG-02:
This family belonged to Khaaskheli caste and was enrolled from
district Shahdaad pur, Sindh (Fig-3.1). The family included seven affected individuals
in two generations with age ranging from 14 days to 35 years. Age of onset of PCG
varied from birth to 2 years of age. p.R390H was found to be segregated with the
disease phenotype in all affected individuals. All available normal samples were also
sequenced to confirm the segregation.
112
Fig-3.1: Haplotype analysis of PCG-02 linked to GLC3A. CYP1B1 gene lies between STR
markers D2S1346 and D2S2331. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage. “-” symbol indicates diseased allele whereas “+” symbol
indicates normal allele.
ii) PCG-03:
This family with four affected individuals belonging to Nohri caste
was identified in a tertiary care eye hospital (Fig-3.2). There were four affected
members in the family in a single generation with age ranging from 7 months to 16
years. All affected individuals had onset of PCG symptoms during first 2 ½ years of
age. Homozygosity mapping showed linkage to GLC3A locus. On sequencing of
CYP1B1 gene, p.R390H was found to be segregating in the family.
113
Fig:-3.2: Haplotype analysis of PCG-03 linked to GLC3A. CYP1B1 gene lies between STR
markers D2S1346 and D2S2331. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage. “–” symbol indicates diseased allele whereas “+”
symbol indicates normal allele.
iii) PCG-04:
This family was enrolled from Golarchi, district Badin in Southern
Sindh. It was a Sindhi speaking family with Khaaskheli caste. Five individuals were
affected in this family with age ranging from 8-38 years (Fig-3.3). The affected
individuals manifested PCG symptoms during their first two years of life. Haplotype
analysis showed linkage to CYP1B1 gene and when sequenced, p.R390H mutation
was found to segregate in all affected individuals of the family.
114
Fig:-3.3: Pedigree of PCG-04 family. Squares indicate males and circles indicate females
whereas affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage. “–” symbol indicates diseased allele whereas “+”
symbol indicates normal allele.
iv) PCG-15:
This family was enrolled from Matyari district in lower Sindh and
belonged to Syed caste. All three affected individuals with ages between 5-17 years at
the time of enrollment in this study manifested PCG symptoms during their first year
of life (Fig3.5). Haplotype analysis and subsequent Sanger sequencing showed
p.R390H mutation segregating in the family (Fig-3.4).
115
Fig:-3.4: Haplotype analysis of PCG-15 linked to GLC3A. CYP1B1 gene lies between STR markers
D2S1346 and D2S2331. Squares indicate males and circles indicate females whereas affected are
shown by filled squares and circles. The double line between individuals represents consanguineous
marriage. “–” symbol indicates diseased allele whereas “+” symbol indicates normal allele.
Fig-3.5: Photographs showing affected individuals of PCG-15
v) PCG-16:
This family with Chaachar caste belonged to District Rahim Yar Khan
but and was enrolled from LRBT tertiary care eye hospital at Gambat in upper Sindh.
Three individuals were affected in this family with ages between 5-9 years (Fig-3.6).
Age of onset was congenital in all three affected individuals with classical
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Bupthalmos (enlarged eye globes) and corneal opacification (Fig-3.7). Sequencing
analysis showed the presence of p.R390H mutation in CYP1B1.
Fig-3.6: Pedigree of PCG-16. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage. “–” symbol indicates diseased allele whereas “+”
symbol indicates normal allele.
Fig-3.7: Photographs of affected children of PCG-16
vi) PCG-17:
This family was identified at LRBT Gambat and belonged to Jamra
tribe at Khairpur district in upper Sindh. There were two affected individuals in the
family with congenital onset of PCG in both patients having age ranging from 6-10
years (Fig-3.8). p.R390H mutation was found to be segregating in the family on
mutational analysis.
117
Fig-3.8: Haplotype analysis of PCG-17 linked to GLC3A. CYP1B1 gene lies between STR
markers D2S1346 and D2S2331. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage. “–” symbol indicates diseased allele whereas “+”
symbol indicates normal allele.
vii) PCG-19:
This family was also identified at LRBT Gambat. The family belonged
to Malik caste and had four affected individuals in two successive generations with
age ranging between 3-36 years (Fig-3.9). All patients had onset of the disease during
first two years of the life (Fig-3.10). The haplotype analysis showed linkage to
CYP1B1 gene and sequencing results showed p.R390H mutation segregating with
disease phenotype within the family (Fig-3.11).
118
Fig-3.9: Haplotype analysis of PCG-19 linked to GLC3A. CYP1B1 gene lies between STR
markers D2S1346 and D2S2331. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage. “–” symbol indicates diseased allele whereas “+”
symbol indicates normal allele.
Fig-3.10: Photographs of affected children of PCG-19.
119
Fig-3.11: Chromatogram of p.R390H in CYP1B1 in PCG families
B) p.E229K (PCG-07):
The family belonged to Ghori caste and was enrolled from Jinnah
Colony, Latifabad, Hyderabad. There were three affected individuals in the family
having age between 16-20 years (Fig-3.12). There was no previous history of primary
congenital glaucoma in the family both on maternal or paternal side of affected
children. On sequencing, a reported mutation p.E229K segregated in all affected and
normal individuals.
120
Fig-3.12: Haplotype analysis of PCG-07 linked to GLC3A. CYP1B1 gene lies between STR
markers D2S1346 and D2S2331. Squares indicate males and circles indicate females whereas
affected are shown by filled square and circles. Double line between individuals show
consanguineous marriage. “–” sign indicates diseased allele where as “+” sign indicates
normal allele.
Fig-3.13: Ocular photographs of affected individuals of PCG-07
121
C) p.P437L (PCG-10):
This family was enrolled from Nasarpur, Distirct Tando Allah Yar,
Sindh and belonged to Khanzada caste. It had three affected individuals in two
generations with age ranging between 2-18 years (Fig-3.14). Age of onset was
congenital in all affected patients in the family. Sequencing analysis showed presence
of a p.P437L mutation in CYP1B1 gene confirmed through segregation in all affected
and normal samples of the family.
Fig:-3.14: Pedigree of PCG-10 family. Squares indicate males and circles indicate females
whereas affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage. “–” symbol indicates diseased allele whereas “+”
symbol indicates normal allele.
D) p.R290fs*37 (PCG-13):
This consanguineous Pakhtoon family was enrolled from Orangi town,
Karachi. All four affected individuals belonged to a single generation with no history
of primary congenital glaucoma in the family (Fig-3.15). All affected individuals had
ages between 11-32 years. STR genotyping was performed which showed linkage to
CYP1B1 gene and subsequent Sanger sequencing showed an insertion of a single
nucleotide i.e cytosine, between nucleotide 868 and 869 (c.868_869insC) which
resulted in reading frameshift and truncation of protein (p.R290fs*37). Previously no
frameshift mutation in CYP1B1 associated with PCG has been reported from Pakistan.
122
Fig-3.15: Haplotype analysis of PCG-13 linked to GLC3A. CYP1B1 gene lies between STR
markers D2S1346 and D2S2331. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage. “–” symbol indicates diseased allele whereas “+”
symbol indicates normal allele.
E) p.A115P (PCG-06):
PCG-06 family with PCG was enrolled from District Dadu in Sindh
province of Pakistan. There were three affected individuals in the family. The family
was identified having individuals affected with PCG at Eye Hospital affiliated with
Liaquat University of Medical and Health Sciences, Jamshoro. Haplotype analysis
showed homozygosity in all three affected individuals. Sequencing later revealed
substitution of Alanine at position 115 with Proline (p.A115P) which was shown to be
pathogenic using various Bioinformatics tools. Interestingly, haplotype analysis also
revealed homozygosity in four other phenotypically normal individuals with the same
change i.e p.A11P which was confirmed on subsequent sequence analysis (Fig-3.16).
123
Fig-3.16: Haplotype analysis of PCG-06 family linked to GLC3A. CYP1B1 gene lies between
STR markers D2S1346 and D2S2331. Squares indicate males and circles indicate females
whereas affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage. “–” symbol indicates diseased allele whereas “+”
symbol indicates normal allele.
3.1.1.1- Clinical Features of Patients with Reported CYP1B1 Mutations:
p.R390H in CYP1B1 was predominant mutation in our cohort of
seventeen PCG families as this mutation was present in seven PCG families (41%)
with 28 affected individuals. All affected patients had congenital bilateral glaucoma
with onset in first 3 years of life although there was variation in severity of clinical
features among affected individuals. The patients who underwent early
trabeculectomy after the onset of the disease had rescued vision in some cases while
who had not undergone any surgical procedure showed either classical bupthalmos or
pthysical eyes (Table-3.1). All affected patients without treabeculectomy had enlarged
cornea with maximum corneal diameter of 16mm. Corneal opacity was observed in
patients of PCG-02, PCG-04, PCG-15 (Fig-3.5) and PCG-16 (Fig-3.7).
Among families with p.R390H mutation in CYP1B1 gene, recorded
intraocular pressure was maximum among affected individuals of PCG-02 and PCG-
19 i.e. upto 40 mmHg. Three patients in family PCG-02 i.e. V:3, V:5 and V:6 showed
corneal opacity in their right eyes only as they went for early trabeculectomy in their
124
left eyes, whereas two patients in PCG-15 and PCG-17 showed clear cornea and
restored visual acuity after trabeculectomy (Table-3.1).
In PCG-07, all three affected individuals had ages between 16-20 years
at the time of enrollment, severe corneal opacities with virtually no perception of light
in patients IV:3 and IV:4 whereas patient IV:5 exhibited mild perception of light who
underwent bilateral trabeculectomy (Fig-3.13). The maximum IOP recorded was 41.5
mmHg in patients IV:3 and IV:4 (Table-3.1).
In PCG-10, the two affected individuals had ages between 2-18 years
with congenital onset of glaucoma. Patient IV:3 had enlarged cornea in both eyes with
classical bupthalmos and corneal opacities with virtually no perception of light.
Patient IV: 8 had corneal enlargement with edema and elevated IOP in both eyes. Cup
to disc ratio could not be determined due to non-visibility of retina on fundoscopy
(Table-3.1). Both patients had no history of any ophthalmological surgery.
In PCG-13, all four affected individuals had severe congenital
glaucoma with little variation among clinical features (Table-3.1). Patients exhibited
variable corneal opacities in their eyes. Patients V:3 and V:5 had bilateral corneal
opacities whereas patients V:2 had corneal opacity in his right eye only and patient
V:4 had corneal opacity in her left eye only. Patients V:3 and V:4 had visual acuity
upto 1/60 and 6/12 respectively. Patient V:5 who had bilateral corneal opacities also
exhibited megalocornea in both eyes measuring up to 16mm. Normal corneal
diameter was observed in patients V:3 and V:4 bilaterally. The maximum intraocular
pressure observed in affected individuals was up to 42 mmHg in patient V:5.
The clinical features of all three affected individuals and four
phenotypically normal individuals homozygous for the same change in PCG-06 are
shown in the Table-3.2. The patient IV:11 had no perception of light with complete
corneal scarring in both eyes (Fig-3.17). The patient IV:5 had corneal scarring in his
right eye only with classical bupthalmos appearance while he underwent
trabeculectomy in his left eye with preserved vision. The patient IV:10 had normal
appearing eyes with left trabeculectomy done for his left eye. The ages of all three
affected individuals ranged from 3-30 years at the time of enrollment and history
taking. The maximum intraocular pressure observed was 41.5 mmHg in patient IV:10.
125
All four phenotypically normal individuals homozygous for the same change also
underwent detailed and extensive ophthalmological examination but they did not
reveal any clinical features suggestive of congenital glaucoma.
Ages of all four clinically normal homozygous individuals in PCG-06
ranged from 8-20 years (Fig-3.17). Apparently normal phenotype in these individuals
could be due to either non-penetrance or variable expressivity which has been
mentioned earlier in studies from Iran and Saudi Arabia (Bejjani et al., 2000); (Suri et
al., 2009). There is a possibility that a modifier locus or gene may be present in these
individuals which is responsible for suppression of signs and symptoms of the
disease.
126
Table-3.1: Clinical Features of Affected Individuals of Families with Reported CYP1B1 Mutations.
Patient No. Age
(Years)
Visual
Acuity
OS/OD
Corneal
Diameter
OS/OD(mm)
Corneal
Edema
OS/OD
Haab’s
Striae
OS/OD
Corneal
Opacity
OS/OD
IOP
mmHg
OS/OD
CD
Ratio
OS/OD
Surgery
OS/OD
PCG-02 (p.R390H)
IV:4 25 NPL/NPL NR/NR NR/NR -/- +/+ NR/15 NR/NR OS Trab
V:3 05 6/36;NPL 14/11.5 -/NR -/- -/+ 17/29 NR/0.3 OS Trab
V:4 06 6/12;6/24 14/14 -/- -/- -/- 17/29 0.2/0.5 B/L Trab
V:5 01 NR/NR 15/14 +//NR +/NR -/+ 17/40 NR/NR OS Trab
V:6 1.5 FF/NR 14/14 +/NR -/- -/+ 35/35 0.4/NR OS Trab
V:7 2.5 NR/NR 13/11.5 NR/NR -/- +/+ 40/40 NR/NR B/L Trab
PCG-03 (p.R390H)
IV:2 2.5 NR/NR 13/11.5 NR/NR -/- +/+ 40/40 NR/NR B/L Trab
PCG-04 (p.R390H)
V:1 38 NR/NR NR/NR NR/NR -/- -/- NR/NR NR/NR -
V:2 28 NR/NR NR/NR NR/NR -/- -/- NR/NR NR/NR -
V:3 21 PL/PL 16/14 NR/NR -/+ +/+ 40/34 NR/NR -
V:5 23 NPL/NPL 15/14 NR/NR -/- +/+ 35/36 NR/NR -
V:7 08 6/36;6/24 13/11 +/+ -/- -/- 25/21 0.3/0.2 B/L
PCG-15 (p.R390H)
IV:1 16 NPL/NPL 16/15.5 NR/NR NR/NR +/+ 30/35 NR/NR -
IV:2 07 NPL/NPL NR/NR NR/NR NR/NR +/+ NR/NR NR/NR -
IV:3 19 6/36;6/36 15/15 +/+ +/+ +/+ 18/20 0.7/0.8 B/L Trab
PCG-16 (p.R390H)
IV:1 10 NPL/NPL 15/14 -/- -/- +/+ 35/40 NR/NR -
IV:5 07 NPL/NPL 16/15.5 -/- -/- +/+ 32/34 NR/NR -
IV:8 06 30/28 -
PCG-17 (p.R390H)
V:4 11 6/24;6/18 10.3/10.4 -/- -/- -/- 14/17 0.8/0.9 B/L Trabe
PCG-19 (p.R390H)
IV:5 18 NPL/NPL NR/NR NR/NR +/+ NR/NR 40/35 NR/NR -
127
IV:6 33 NR/NR NR/NR NR/NR NR/NR NR/NR NR/NR NR/NR B/L Trab
V:3 07 HM/HM 15/15 +/+ -/- -/- 28/32 NR/NR B/L Trabe
V:4 02 FF/FF 15/12 +/+ -/- -/- 40/25 NR/NR B/L Trab
PCG-07 (p.E229K)
IV:3 19 NPL/NPL 15/15 NR/NR NR/NR +/+ 41.5/NR NR/NR OS Trab
IV:4 20 PL/PL 15/15 NR/NR NR/NR +/+ 26.6/24 NR/NR B/L Trab
IV:5 16 NPL/NPL 15/15 NR/NR NR/NR +/+ 24.4/NR NR/NR OD Trab
PCG-10 (p.P437L)
IV:3 18 NPL/NPL 15/15.5 NR/NR NR/NR +/+ 29/25 NR/NR -
IV:8 02 NR/NR 13/13.5 +/+ NR/+ -/- 35/40 NR/NR -
PCG-13 (p.R290fs*37)
V:2 32 NPL/PL NR/NR -/+ -/- -/+ 41/ NPL/NPL OS Trab
V:3 17 NPL/NPL NR/NR +/+ -/+ +/+ 41/ 1/60;1/60 B/L Trab
V:4 11 NPL/NPL NR/NR +/- -/- +/- 41 NPL/6/12 -
V:5 19 PL/NPL 16/NR +/+ NR/NR +/+ 42/10 NPL/NPL - NPL: No perception of Light. PL: Perception of Light. HM: Hand Movements. FF: Fixation and Follow. CF: Counting Fingers. NR: Not
Recordable. IOP: Intraocular Pressure.
128
Table-3.2: Clinical Features of all individuals homozygous for p.A115P in PCG-06. *Phenotypically affected individuals.
NPL: No perception of Light. FF: Fixation and Follow. CF: Counting Fingers. NR: Not Recordable. IOP: Intraocular Pressure.
Fig-3.17: Photographs of affected individuals and normal homozygous of PCG-06
Patient
No.
Age
Years
Visual
Acuity
OS/OD
Corneal
Diameter
OS/OD(mm)
Corneal
Edema
OS/OD
Haab’s
Striae
OS/OD
Corneal
Opacity
OS/OD
IOP
mmHg
OS/OD
CD Ratio
OS/OD
Surgery
OS/OD
IV:5* 2.5 FF/NPL 15.5/15 +/+ -/- -/+ 16/25 0.7/0.5 OS Trab
IV:10* 30 CF/CF 15.5/15 -/+ -/- -/- 41.5/18.9 TC/0.2 OD Trab
IV:11* 16 NPL/NPL 15.5/15 +/NR +/NR +/+ 24/40 NR/NR OS Trab
IV:3 8 6/9;6/9 11/11 -/- -/- -/- 10.5/11.2 0.2/0.2 -
IV:4 10 6/6;6/6 10.5/10.5 -/- -/- -/- 10.2/12.2 0.2/0.2 -
IV:12 20 6/6;6/6 11/11 -/- -/- -/- 12/12.5 0.2/0.2 -
IV:13 19 6/6;6/6 11.5/11.5 -/- -/- -/- 19/17 0.2/0.2 -
129
3.1.2- Novel Mutations in CYP1B1:
A) p.G36D (PCG-08):
This family from District Thatta belonged to Thaheem caste. There
were four affected individuals in the family including three males and one female
(Fig-3.18). On direct sequencing, electropherograms of affected individuals showed a
transition from G>A at nucleotide number 107 (c.107G>A) resulting in substitution of
Glycine (G) to Aspartic acid (D) at codon number 36 (p.G36D) (Fig-3.19). This is a
novel mutation, which has not been reported earlier (Sheikh et al., 2014).
Fig-3.18: Pedigree of PCG-08 family. Squares indicate males and circles indicate females
whereas affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage. “–” symbol indicates diseased allele whereas “+”
symbol indicates normal allele.
130
Fig-3.19: Chromatograms of Normal, mutant and carrier of PCG-08. The wild-type
nucleotide G at position 107 is substituted by A in homozygous mutants whereas carrier
shows peaks for both G and A at the same position.
Fig-3.20: Photograph of patient IV:3 of PCG-08
Ages of all four affected individuals in PCG-08 ranged between 3-35
years at the time of enrollment of the family for the study. Patient IV:1 and IV:3 had
visual acuity ranging from fixation to follow whereas patient IV:5 and IV:6 had no
perception of light (Fig-3.20). Corneal opacity was present in all patients except IV:6
who had pthisical eyes. Maximum IOP observed (patient IV:1) was up to 35 mmHg
(Table-3.3). All six parents and normal siblings were found to be heterozygous for the
change.
131
Fig-3.21: Multiple Sequence alignment in various species. It is clear that G at 36 is conserved
in all species showing its importance in normal functioning of CYP1B1 protein.
Fig-3.22: HOPE protein prediction based schematic representation of wild-type and mutant
amino acid residues. The backbone, which is same for each amino acid, is shown in red color
whereas side chain, which is unique, is shown in black color.
Multiple sequence alignment in various species shows conservation of glycine
indicating its importance in normal functioning of CYP1B1 protein (Fig-3.21).
According to HOPE online tool, the mutant amino acid is bigger in size than wild-
type (WT) residue (Fig-3.22). The WT residue is neutral and is more hydrophobic
whereas mutant residue is negatively charged and is less hydrophobic. The WT
glycine residue is most flexible of all amino acids so its replacement with aspartic
acid could lead to defective protein folding as flexibility rendered by glycine might be
essential for the protein’s function. According to Mutation taster, the change is
disease causing and protein function might be affected.
132
B) (p.G67-A70del) PCG-09:
This family was enrolled from Badin district in lower Sindh belonging
to Kaloi tribe. There were two affected in the family with age between 2-11 years
(Fig-3.23). Interpretation of sequencing results showed a deletion of 12 base pairs
(GGGCCAGGCGGC) from c.198 to c.209 nucleotide (c.198-209del12) resulting in
deletion of four amino acids (Glycine-Glutamine-Alanine-Alanine) from codon
number 67 to 70 in CYP1B1 protein (Fig-3.24). The deletion (p.G67-A70del) has
been detected first time through this study and had deleterious consequences in the
affected individuals. Multiple sequence alignment in various species shows
conservation of glycine at codon 67 and alanine at codon 70 and is suggestive of their
significant role for normal functioning of the gene product (Fig-3.25). Both affected
members in the family with bilateral congenital glaucoma had onset of the disease in
their early childhood between 3-4 years with no surgical intervention (Table-3.3). The
maximum intraocular pressure (40mmHg) measured was in patient IV:1.
Fig-3.23: Pedigree of PCG-09 family. Squares indicate males and circles indicate females
whereas affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage. – symbol indicates diseased allele whereas + symbol
indicates normal allele.
133
Fig-3.24: Chromatograms of normal, affected and carrier in PCG-09
Fig-3.25: Multiple sequence alignment of CYP1B1 proteins from various species. It is
evident that G67 and Ala69 are conserved in all above species indicating their importance for
normal functioning of the protein.
134
Table-3.3: Clinical features of affected individuals in CYP1B1 linked families with Novel mutations.
Patient
No.
Age
Years
Visual
Acuity
OS/OD
Corneal
Diameter
OS/OD(mm)
Corneal
Edema
OS/OD
Haab’s
Striae
OS/OD
Corneal
Opacity
OS/OD
IOP
mmHg
OS/OD
CD Ratio
OS/OD
Surgery
OS/OD
PCG-08 (p.G36D) – Novel Mutation
IV:1 04 NPL/FF 16/11 +/+ +/NR +/+ 35/29 NR/NR B/L Trab
IV:3 03 FF/NPL +/NR NR/NR +/+ 29/5 NR/NR OS Trab
IV:5 23 NPL/NPL NR/NR NR/NR NR/NR +/+ NR/NR NR/NR -
IV:6 38 NPL/NPL 16/15 +/+ -/- -/- 29/35 TC/TC -
PCG-09 (p.G67-A70del) – Novel Mutation
IV:1 02 FF/NPL 14.5/14 +/+ -/- +/- 40/40 NR/TC -
IV:3 11 6/12;6/24 14/11.5 -/- -/- -/- 17/19 0.5/0.6 B/L Trab
NPL: No perception of Light. FF: Fixation and Follow. NR: Not Recordable. IOP: Intraocular Pressure. TC: Total cupping. B/L: Bilateral
135
3.2-Unlinked PCG Families:
3.2.1- PCG-11:
This family was identified at LRBT, Karachi and belonged to Afridi
caste. There were only two affected alive members in the family (Fig-3.26) with no
previous history of glaucoma or any other genetic disorder in the family. Age of onset
of glaucoma was 3 years for daughter whereas it was during first decade for affected
father. Both underwent trabeculectomy at earlier age to control their intraocular
pressure. Visual acuity was reduced in patient III:1 whereas it was normal in patient
IV:6. All four reported loci for PCG were screened using STR markers for each locus,
but linkage could not be established for any reported locus of PCG.
Fig-3.26: Pedigree of PCG-11. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage.
3.2.2- PCG-12:
This family was also identified from LRBT Karachi and belonged to
Shaikh community. Two members were affected in the family but there was not any
consanguinity among their parents (Fig-3.27). The age of onset of PCG in both
affected members was during first five years of life and they underwent repeated
trabeculectomy to control their IOP. The cornea was clear in both patients with
reduced visual acuity. Both affected and normal individuals were screened for linkage
to reported loci for PCG but no homozygosity was found.
136
Fig-3.27: Pedigree of PCG-12. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage.
3.2.3- PCG-14:
This family of Kharl tribe was enrolled from district Khairpur Mirs
with only two affected members in the family (Fig-3.28). Both affected patients had
onset of PCG symptoms during first two years of life. Both had corneal opacities with
enlarged corneas and reduced visual acuity to perception of light only. There was no
consanguinity in the family and it was found unlinked to reported loci for PCG.
Fig-3.28: Pedigree of PCG-14. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage.
3.2.4- PCG-20:
This family was diagnosed at Tertiary care Eye hospital, Hyderabad
and belonged to Unar caste. It has two affected members in the family with
consanguinity among parents (Fig-3.29). Onset of glaucoma was congenital in both
137
affected patients with reduced visual acuity but controlled IOP due to early
trabeculectomy. Linkage could not be established in the family to any reported locus
of PCG.
Fig-3.29: Pedigree of PCG-20. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage.
138
SECTION-II
MOLECULAR CHARACTERIZATION
OF STARGARDT DISEASE
139
3.3 Linkage Analysis of Stargardt Disease:
Five families with two or more than two members affected with
Stargardt disease were enrolled for this study from different areas of Sindh province.
After DNA extraction, linkage studies were carried out for all three known genes for
Stargardt disease (i.e. ABCA4 at 1p22.1, ELOVL4 at 6q14.1 and PROM1 at 4p15.32)
using closely linked STR markers for the each gene (Table-2.2). All five families
remained unlinked to any of the reported gene for Stargardt disease on haplotype
analysis. It was therefore decided to perform Whole Exome Sequencing of selected
families with Stargardt disease to find out the possible pathogenic variant for the
disease phenotype.
3.3.1- Whole Exome Sequencing Revealed a Novel Gene for
Stargardt Disease:
Whole Exome Sequencing of two families with Stargardt disease was
performed. The exome data thus obtained was filtered on the basis of Combined
Annotation Dependent Depletion (CADD) score, pathogenicity and allele frequency.
The pathogenicity was predicted by bioinformatics tools (Mutation Taster, Mutation
Assessor, SIFT, Polyphen2) and allele fre quency data was obtained through
ExAC browser. The candidate variants were subsequently sequenced for segregation
with disease phenotype. The details of these two families with Stargardt disease are
given below:
3.3.1.1- LUSG-03:
This family was identified with Stargardt disease at Liaquat University
Eye Hospital, Hyderabad. The family belonged to Kaka caste and there were three
affected individuals in the family (Fig-3.30). Whole Exome Sequencing (WES) was
carried out for one affected from LUSG-03. The probable pathogenic variants were
filtered according to a devised strategy based on CADD score and pathogenicity as
predicted by annotated protein bioinformatics tools (Fig-2.4). Four possible
pathogenic variants were checked through direct Sanger sequencing for segregation
(Table-2.8). A transversion was found at nucleotide number 296 from cytosine to
adenine (c.296C>A) resulting in substitution of Arginine to Isoleucine at codon
number 99 (p.Arg99Ile) in ARL3 gene on chromosome number 10 (Fig-3.31). This is
140
a novel gene for Stargardt disease and is not known to be associated with Stargardt
disease previously.
Fig-3.30: Pedigree of LUSG-03 family. Squares indicate males and circles indicate females
whereas affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage. “–” symbol indicates diseased allele whereas “+”
symbol indicate
s normal allele.
Fig-3.31: Chromatograms of affected and carrier in LUSG-03
141
All three affected individuals complained of decreased visual acuity.
Visual acuity was more severely affected in patient IV:3 as compared to other two
affected patients. There was no night blindness (or delayed adaptation to darkness)
and color blindness when they were examined at a tertiary care eye hospital. Fundus
was examined bilaterally in all three affected individuals, whereas OCT (Optial
Coherence Tomography) was done in two individuals. Fundus examination revealed
hypopigmentation possibly due to thinning of retina around macular region in all
affected patients (Fig-3.32). OCT further confirmed this finding as the central retinal
thickness in all patients is greatly reduced which is the basis of reduced visual acuity
in these patients (Fig-3.33). The normal retinal thickness ranges between 250-300 μm
where as in these patients it ranged between 97 to 116 μm.
Fig-3.32: Fundus photographs of affected patients of LUSG-03. Hypopigmented areas can be
seen in all fundus photographs corresponding to thinning of retina which has become less
reflective causing reduced vision in these patients. Also note the macular region in
photographs A and C which is less prominent as compared to fundus in normal patients.
Note: A-Patient IV:3; B- Patient IV:4; C-Patient IV:6
142
Fig-3.33: Optical Coherence Tomography showing retinal thickness of affected patients of
LUSG-03. It is evident that retinal thickness is greatly reduced at the center corresponding to
Fovea and the region around it which is basis for reduced vision in patients with Stargardt
disease. Note: The normal retinal thickness ranges between 250-300 μm.
3.3.1.2- LUSG-04:
This family was identified as having Stargardt disease at Eye hospital,
affiliated with Liaquat University of Medical and Health Sciences. The family
belonged to Kaka caste and resided in the same areas as that of LUSG-03. It is quite
possible that this family had some relation with LUSG-03 few generations back.
The same change c.296C>A resulting in p.Arg99Ile segregated in this family as well
(Fig-3.35).
143
Fig-3.34: Pedigree of LUSG-04 family. Squares indicate males and circles indicate females
whereas affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage. “–” symbol indicates diseased allele whereas “+”
symbol indicates normal allele.
Fig-3.35: Chromatograms of carrier and affected individuals of LUSG-04
All three patients in LUSG-04 family had same complaints as those of
LUSG-03. Visual acuity was reduced greatly in two patients while the patient IV:5
had near normal visual acuity. They did not complain of reduced vision during night
or delayed adaptation to darkness and were able to differentiate among colors during
ophthalmological examination by a consultant. Fundus examination revealed
hypopigmented areas around fovea and macula rendering retina less reflective and
leading to reduced visual acuity (Fig-3.36). Macula has become obscured in fundus
144
photographs of patient IV:4 (Fig-3.36 B). Patient IV:4 fundus also revealed classical
“Beaten Bronze” appearance on direct ophthalmoscopy which is not seen in fundus
photographs due to less magnification power of the fundus camera, whereas patient
IV:5 revealed “Bull’s eye maculopathy” during fundus examination (Fig-3.36 C).
OCT of all three affected individuals revealed thinning of central retina from ranging
from 140 -172 μm (Fig-3.37).
Fig-3.36: Fundus photographs of affected patients of LUSG-04. Hypopigmentation can be
noted in all fundus photographs pointing towards thinning of retina which has become less
reflective. In fundus photograph C (patient IV:5), Bulls eye maculopathy can be seen.
Note: A-Patient IV:2; B-Patient IV:4; C-Patient IV-5
145
Fig-3.37: Optical Coherence Tomography showing retinal thickness of affected patients of
LUSG-04. It is evident that retinal thickness is greatly reduced at the center corresponding to
Fovea and the region around it which is basis for reduced vision in patients with Stargardt
disease. Note: The normal retinal thickness ranges between 250-300 μm.
146
Fig-3.38: Multiple sequence alignment of ARL3 gene in various species. It is clear that the
arginine at p.99 is conserved in all species indicating its importance in normal functioning of
ARL3 protein.
Table-3.4: Protein Prediction of ARL3 (p.Arg99Ile) by Various Bioinformatics Tools
Bioinformatics Tool Protein Prediction
SIFT Damaging
Polyphen2 Probably damaging
Mutation Taster Disease Causing
Mutation Assessor Predicted Functional (High)
ARL3 has six coding exons and is located on chromosome number 10.
The change p.Arg99Ileu corresponds to exon number 5 (Fig3.39). All six exons code
for ARL3 protein which contains a single domain known as small GTP-binding
protein domain (Fig-3.40). Multiple sequence alignment in various species showed
the conservation of arginine at p.99 in all the species which indicates that the
substitution of arginine with Isoleucine results in abnormal ARL3 protein leading to
Stargardt disease (Fig-3.38). The pathogenicity has further been verified through
various protein bioinformatics tools which predicted this changes as damaging for the
protein (Table-3.5). ARL3 protein modelling was determined through Phyre2 web tool
which showed abnormal folding of the protein as indicated by red area showing
mutated substituted amino acid (Fig-3.41).
147
Fig-3.39: Diagrammatic depiction of ARL3 gene with its Exons and showing the change in
Exon number 5.
Fig-3.40: ARL3 protein domain known as small GTP-binding protein domain.
Fig-3.41: ARL3 protein modelling obtained through Phyre-2 online tool. The mutated Red
area indicates the mutated amino acid that results in abnormal folding of ARL3 protein
3.3.2- Unlinked Stargardt Disease Families:
Three families with Stargardt disease remained unlinked to any of
three reported genes for the disease. The details of these families are given below:
3.3.2.1- LUSG-02:
This family of Kandhro tribe was enrolled from Kotri, district
Hyderabad. The diagnosis was confirmed at Liaquat University Eye hospital by
extensive ophthalmological examination. There were four affected individuals in the
family having age from 8-16 years (Fig-3.42). Genomic DNA samples were subjected
to genotype using STR markers for linkage to all three known genes for Stargardt
disease but family did not show homozygosity to any of the reported genes.
Mutates
148
Fig-3.42: Pedigree of LUSG-02. Squares indicate males and circles indicate females whereas affected
are shown by filled squares and circles.
3.3.2.2- LUSG-07:
This consanguineous family belongs to Chandio caste, enrolled from
district Nawab Shah in Sindh province. All three affected individuals were siblings
with age ranging from 3-18 years (Fig-3.43). Stargardt disease-related symptoms
started appearing during first decade in all affected members of the family. DNA
samples of all three affected and normal siblings including parents were genotyped for
linkage to the reported genes for Stargardt disease but haplotype data did not show
any homozygosity for any gene.
Fig-3.43: Pedigree of LUSG-07. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage.
149
3.3.2.3- LUSG-08:
This family belonged to Fareedi caste and was enrolled from
Hyderabad city and was diagnosed with Stargardt disease at Liaquat University Eye
hospital. There were two affected individuals in the family having age between 20-30
years (Fig-3.44). The parents were having consanguineous relationship between them.
Haplotype data when analyzed did not show linkage to any known gene for Stargardt
disease.
Fig-3.44: Pedigree of LUSG-08. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage.
150
SECTION-III
GENETIC STUDY OF
CONGENITAL CATARACT
151
3.4 Linkage Analysis of Congenital Cataract:
Five families with congenital cataract were enrolled for the present
study. After obtaining written informed consent, venous blood was drawn for DNA
extraction from all available affected and normal individuals of each family. All
families were screened for linkage to common known loci or genes for congenital
cataract, but no family found linked to any of these loci/genes (Table-2.3). The
selected families were subjected to Whole Exome Sequencing to find out the possible
molecular and genetic cause for the disorder in the family. The details of each of five
congenital cataract families are described below.
3.4.1- LUCC-15:
This family of Pusiyo caste was identified with congenital cataract at
L.R.B.T. Eye hospital and lived in Village Mehro Pusiyo near Tandojam in
Hyderabad district. The family had three affected individuals born to consanguineous
parents (Fig-3.45). Initially all affected and normal individuals were screened for
common loci for congenital cataract using STR markers closely linked to the
corresponding loci. Details of those loci and STR markers are given in the Table-2.3.
Whole Exome Sequencing was done for one affected individual. The
variants were filtered according to a devised scheme and were further prioritized on
basis of CADD score and pathogenicity as predicted by various protein prediction
tools (Fig-2.4). Prioritized variants were sequenced and checked for segregation and
finally a transition was found from Thymine to Cytosine at nucleotide number 149
(c.149T>C) in INPP5K (Inositol polyphosphate 5-phosphatase K) gene on
chromosome 17 resulting in substitution of Isoleucine to Threonine at codon number
50 i.e. p.Ile50Thr (Fig-3.46). Functional study was designed in Zebra fish for
characterization of this novel gene’s association with syndromic congenital cataract
but meanwhile it was reported by two studies published in March, 2017 (Osborn et
al., 2017) & (Wiessner et al., 2017).
152
Fig-3.45: Pedigree of LUCC-15 family linked to INPP5K. Squares indicate males and circles
indicate females whereas affected are shown by filled squares and circles. The double line
between individuals represents consanguineous marriage. “–” symbol indicates diseased allele
whereas “+” symbol indicates normal allele.
Fig-3.46: Chromatograms of wild-type, carrier and affected individuals of LUCC-15
153
Fig-3.47: Multiple Sequence Alignment of INPP5K protein in various species showing the
conservation of substituted amino acid in all species.
Multiple sequence alignment of Isoleucine at codon 50 in various
species showed its conservation in all the species indicating its significance for
INPP5K protein (Fig-3.47). Various protein bioinformatics tools were used to assess
the effect of the substituted amino acid on protein functioning which described the
change as damaging and disease causing (Table-3.6).
3.4.1.1- Clinical Features of affected patients:
All three affected in this family manifested initially with bilateral
cataract between 2-2 ½ half years and they underwent surgery for cataract removal
and intra-ocular lens implant immediately after the diagnosis. The associated features
in affected individuals and their comparison with a normal individual of the same
family are given in Table-3.6. Patient V:5 was found to be moderately intellectually
disabled as was judged by family history and asking common questions from the
patient. The patient V:1 had delayed development of growth milestones such as he
was not able to walk properly without any support and he was also not able to speak
properly as well. All three patients affected with congenital cataract had elevated
serum levels of creatine kinase and aldolase as compared to a normal sibling from the
same family.
154
Table-3.5: Clinical Features of Affected Individuals of LUCC-15
S# Features (V:5) (V:1) (V:8) V:7
Phenotype Affected Affected Affected Normal
1. Intellectual Disability Moderate Mild Normal Normal
2. Eye Abnormality e.g.
Cataracts (Age of onset)
+ (2 years) +(2½years) +(2
years)
-
3. Low Birth Weight + +++ + -
4. Speech Normal Impaired Normal Normal
5. Contractures(knee/Ankle) - - - -
6. Spinal Rigidity - - - -
7. Spinal Deformity
e.g.Kyphosis/Lordosis
- - - -
8. Intention Tremors - - - -
9. Hypotonia - + + -
10. Abnormal Gait/ Ataxia - + - -
11. Microcephaly - - - -
12. Short Stature - - - -
13. Seizures - - - -
14. Meningitis - - - -
15. Hypogonadism - - - -
16. Cardiac features - - - -
17. Respiratory Features - - - -
18. Serum Creatine Kinase
Normal range:1-4 ng/ml
37.33 28.58 74.44 2.72
19. Serum Aldolase
Normal range: 5-20 U/L
27.7 21.8 25.2 18.4
20. Serum Alkaline Phosphatase
Normal range:54-368 IU/L
132 295 209 380
Fig-3.48: Photographs of affected children of LUCC-15.
155
Table-3.6: Protein Prediction of INPP5K (Ile50Thr) by Various Bioinformatics Tools
Bioinformatics Tool Protein Prediction
SIFT Damaging
Polyphen2 Probably damaging
Mutation Taster Disease Causing
Mutation Assessor Predicted Functional (High)
Fig-3.49: Diagrammatic depiction of INPP5K gene showing total number of exons with red-
colored arrow indicating the site of mutation.
Fig-3.50: INPP5K protein with its domain known as Inositol polyphosphate phosphatase
catalytic domain.
INPP5K gene has 12 exons and p.Ile50Thr is located in exon 2 (Fig-
3.49). INPP5K protein contains 448 amino acids and has only one protein domain
known as inositol polyphosphate phosphatase catalytic domain (Fig-3.50). The
change (p.Ile50Thr) has been predicted pathogenic by various bioinformatics tools
(Table-3.6).
3.4.2- Unlinked Congenital Cataract Families
3.4.2.1- LUCC-01:
This Shaikh family with congenital cataract was enrolled from Sukkur
city in Upper Sindh. Two brothers were affected with phenotypically normal parents
in a consanguineous relationship (Fig-3.51). Genomic DNA was genotyped using
STR markers for common loci/genes for congenital cataract but haplotype data was
not homozygous for any of these loci/genes (Table-2.3).
156
Fig-3.51: Pedigree of LUCC-01. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage.
3.4.2.2- LUCC-02:
This consanguineous family of Sehta caste was enrolled from
Mehrabpur in Sindh province. There were three affected individuals in the family.
(Fig-3.52). Genomic DNA was genotyped for linkage analysis to common reported
loci/genes for congenital cataract but haplotype data did not show any homozygosity
for screened genes/loci (Table-2.3).
Fig-3.52: Pedigree of LUCC-02 Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage.
157
3.4.2.3- LUCC-04:
This family with Qureshi caste was diagnosed with congenital cataract
at LRBT eye hospital. The family was visited to obtain all relevant information and
blood samples. Twelve affected individuals were present in the family all with
phenotypically normal parents in a consanguineous relationship (Fig-3.53).
Genotyping was done for common loci/genes known for congenital cataract but
linkage could be established for any locus or gene (Table-2.3).
Fig-3.53: Pedigree of LUCC-04. Squares indicate males and circles indicate females whereas
affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage.
3.4.2.4- LUCC-13:
This family of Kaleri tribe was enrolled from Hyderabad city. All three
affected individuals were siblings with consanguinity between unaffected parents
(Fig-3.54). DNA samples were genotyped for linkage to common loci/genes known
for congenital cataract (Table-2.3). The family remained unlinked for any of these loci
or genes.
158
Fig-3.54: Pedigree of LUCC-13. Squares indicate males and circles indicate females
whereas affected are shown by filled squares and circles. The double line between individuals
represents consanguineous marriage.
159
CHAPTER 4
DISCUSSION
160
Preface:
Twenty seven families with inherited visual disorders (PCG, Stargardt
disease and Congenital Cataract) having two or more than two affected individuals
were enrolled for this study. Exclusion studies based on linkage analysis were carried
out using highly polymorophihc microsatellite STR markers on DNA samples of all
affected and unaffected individuals of enrolled families for known loci or genes. The
results showed linkage of thirteen PCG affected families to GLC3A locus harboring
CYP1B1 gene whereas four PCG affected families did not show linkage to any known
locus or gene. All five Stargardt disease affected families remained unlinked to
known genes for the disorder. Whole Exome Sequencing was performed on genomic
DNA of two of these families which resulted in identification of a novel gene (ARL3)
previously not known to be associated with Stargardt disease. Likewise WES was
done for one family with Congenital Cataract and subsequent data analysis followed
by Sanger sequencing of candidate pathogenic variants revealed a new gene (i.e.
INPP5K) for that family which was a novel finding at that time in November, 2016. It
was decided to strengthen novel data through animal model study but the INPP5K
association with congenital cataract was reported by two studies published
simultaneously in March, 2017. Remaining four congenital cataract families remained
unlinked to any common locus or gene for the disorder (Table-2.3)
161
SECTION-I
GENETIC CHARACTERIZATION OF
FAMILIAL PCG
162
4.1. Genetic Characterization of Familial PCG
Seventeen consanguineous PCG affected families were enrolled from
different cities of Sindh province. All these families were subjected to homozygosity
mapping for reported loci and genes. Thirteen families (13/17; 76%) found to be
linked to GLCA3 locus harboring CYP1B1 gene. Prior to this study, highest CYP1B1
associated prevalence (70%) of congenital glaucoma has been reported from Iran
(Chitsazian et al., 2007). A recently published study from Pakistan has identified
mutations in 23 CYP1B1 linked PCG affected families. (Rauf et al., 2016). In 13
CYP1B1 linked families in our study, sequencing was carried out for all coding exons,
exon-intron boundaries and 5’ and 3’ UTR regions in CYP1B1 which led to
identification of two novel mutations i.e. a missense mutation p.G36D and a non-
sense mutation p.G67-A70del. In remaining 11 families, five reported variants were
identified which include p.R390H, p.E229K, p.P437L, p.A115P and a homozygous
1bp insertion i.e. c.868_869insC (p.R290fs*37).
p.R390H was the predominant mutation found in eight out of 13 (61%)
CYP1B1 linked families. This mutation was first reported by Stoilov et al in 1998 in a
PCG family from Pakistan (Stoilov et al., 1998). Thereafter this mutation has been
reported as the second most common mutation in PCG affected patients in studies
from India (16%) and Iran (19.2%). In our study, eight (PCG-02, PCG-03, PCG-04,
PCG-10, PCG-15, PCG-16, PCG-17 and PCG-19) families harbored this mutation
and its predominance in Pakistani PCG patients is further confirmed by a recent study
in which this mutation has been reported in 13 out of 23 (56%) PCG families, whereas
it is present only in a small fraction of Caucasians (Rauf et al., 2016).
All individuals with p.R390H mutation in our study were homozygous
with variable expressivity in phenotypes, disease severity and success in surgical rates
(Table-3.1). Arginine at 390 is located in alpha K helix domain and is involved in
forming a salt bridge. It is a part of Glu-X-X-Arg (387-390) motif and is conserved in
all members of CYP1B1 superfamily. Its exceptional conservation in all members of
the family CYP1B1 indicates that it is essential for normal functioning of this protein
(Stoilov et al., 1998) & (Hollander et al., 2006). It has been suggested that substitution
of arginine at 390 to histidine results in abnormal defective protein as arginine at 390
163
in association with Glu387 and Asn428 plays an important role in proper folding of
the protein (Su, 2012).
In our study, although all families harboring this mutation showed
homozygosity in affected individuals, but previously this mutation has also been
reported in heterozygous state in 8 Iranian patients with PCG. Furthermore, it was the
second most common mutation (14.9%) in PCG affected Iranian patients with p.G61E
being the leading mutation in 28.8% patients (Chitsazian et al., 2007). Presence of
p.R390H in compound heterozygous state was also reported by Hollander et al in
2006 along with p.R117P in a female patient of Asian ethnicity (Hollander et al.,
2006). Moreover, p.R390H has also been reported to cause Juvenile-onset open angle
glaucoma in two Taiwanese patients in a homozygous state (Su, 2012).
The mutation p.E229K in exon 2 was found to be segregating with
disease phenotype in three affected individuals of family PCG-07. The pedigree
pattern showed recessive mode of inheritance as both parents having consanguineous
relationship found to be phenotypically unaffected (Fig-3.13) but were carrier of the
diseased allele on haplotype analysis and subsequent direct Sanger sequencing. The
two other normal siblings were also carrier of the diseased allele. Glutamic acid (E) at
position number 229 in CYP1B1 protein has remained conserved in members of
cytochrome P450 family. It occupies a central position for Helix F (218-234) of
CYP1B1 protein and it has been suggested that its substitution could either result in
premature termination of the helix at 229 or some sort of conformational change in
the resulting protein leading to defective protein function and manifestation of
congenital glaucoma in the affected individuals (Panicker et al., 2004).
This mutation was first reported in 2001 in a 34-year old non-familial
Lebanese man with primary congenital glaucoma and bupthalmos in a compound
heterozygous state along with p.A443G in exon 3. In addition to this, the affected
patient in their study carried five homozygous benign sequence variants which were
found to be present in healthy controls and were therefore ruled out as causative
variants. They suggested that the presence of these mutations in a compound
heterozygous state along with multiple benign sequence variants could have resulted
in defective CYP1B1 protein due to improper folding (Michels-Rautenstrauss et al.,
2001).
164
Afterwards it was reported by Panicker et al in 2002 in a
consanguineous family of Indian origin with two PCG affected individuals in a
compound heterozygous state. Both affected patients exhibited mild phenotype of
primary congenital glaucoma bilaterally although the affected mother had a late-onset
phenotype in one of the two eyes. They were of the opinion that the Glutamic acid at
229 plays an important role in maintaining the structural integrity of the protein
(Panicker et al., 2002). In 2003, the same mutation (p.E229K) was again reported in
two unrelated patients from France in a heterozygous state. Both patients had onset of
PCG in first six months after birth and presented with ocular enlargement (Colomb et
al., 2003). It was then reported by another study in five unrelated patients in a
heterozygous state. In four out of five patients, the mutation was associated with
severe phenotype in at least one of the eyes (Panicker et al., 2004).
p.E229K has also been reported in association with Juvenile-onset
Primary open-angle glaucoma in an Indian family with family history of glaucoma.
The mutation was present in a single patient in a heterozygous state with diagnosis of
POAG at the age of 17 years. This study showed that mutations in CYP1B1 may be
responsible for pathogenesis of POAG besides their strong association with PCG
(Acharya et al., 2006).
Homozygous p.E229K in association with PCG was first reported in
2007 in study that included 104 unrelated patients with congenital glaucoma. Of two
Iranian patients who harbored this mutation, one was homozygous while other was
compound heterozygous along with p.R368C (Chitsazian et al., 2007). p.E229K in
homozygous state has also been reported by several other studies from Lebanon and
Pakistan (Al-Haddad et al., 2016) & (Rauf et al., 2016).
p.P437L was found in a consanguineous PCG-10 family having three
affected individuals. It was first reported in two patients of a Turkish origin by Stoilov
et al in 1998. This mutation is located in exon 3 of CYP1B1 gene within the meander
region preceding the heme-binding region of CYP1B1 protein (Stoilov et al., 1998).
This mutation has also been reported in patients from Brazil (Stoilov et al., 2002),
India (Reddy et al., 2004) and Spain (Campos-Mollo et al., 2009). The age of onset of
PCG in Spanish patients was 3 years and affected patients had elevated IOP with
165
reduced visual acuity (Campos-Mollo et al., 2009). All patients in PCG-10 family in
our study had severe PCG phenotype with congenital onset, elevated IOP and
complete loss of visual acuity. It has been shown that proline at amino acid number
437 of CYP1B1 protein was conserved in 19 of 22 different cytochromes sequences
analyzed (Stoilov et al., 1998). The enzymatic activity of the mutation as determined
by site directed mutagenesis in transfected HEK-293T cells was equal or less than 4%
of that of wild-type protein (Medina-Trillo et al., 2016).
p.A115P mutation in exon 2 of CYP1B1 segregated with disease
phenotype in three patients affected with primary congenital glaucoma in PCG-06.
Prior to our study, this mutation has only been reported once in a single patient of
Indian origin in a homozygous state (Reddy et al., 2004). This mutation is located
near the C-terminal end of β-helix adjacent to Heme-binding region of the protein.
Substitution of Alanine at amino acid position 115 by Proline results in loss of
conformational freedom together with hydrophobic interactions in the absence of
amide hydrogen (Achary et al., 2006).
In this family, there were four phenotypically normal individuals,
although homozygous for the same change (p.A115P) but not having any clinical
feature of PCG. The ophthalmological features of these individuals are described in
Table-3.3. The absence of PCG associated clinical features in these four normal
homozygous individuals could be due to non-penetrance or variable expressivity of
the diseased allele.
Non-penetrance of CYP1B1 mutation was first reported in 1998 in
eleven individuals who were phenotypically unaffected but their haplotypes and
mutations were identical to their affected siblings (Bejjani et al., 1998). Afterwards, in
2000, forty such individuals were identified in a sub-group of 22 families having
affected genotypes but were normal at the time of their participation in the study. It
was interesting to observe that non-penetrance of CYP1B1 has been found for nearly
all type of mutations (Bejjani et al., 2000). Non-penetrance of mutation in CYP1B1
has also been reported in a family of Hispanic ethnicity where two of three siblings
manifested the disorder while third was completely normal (Hollander et al., 2006).
166
To best of our knowledge, non-penetrance of CYP1B1 has not been reported
previously in any PCG family from Pakistan. Furthermore, these four individuals
would be closely monitored on yearly basis for next 10-20 years for possible
development of late-onset glaucoma.
Prior to this study, no frameshift mutation was reported from Pakistan
in patients with primary congenital glaucoma. An insertion of Cytosine at c.868_869
was found which resulted in frameshift and truncation of protein (p.R290fs*37) in a
Pakhtoom family (PCG-13) with four affected individuals in a single generation. This
mutation was first reported in 1998 in two American families (Stoilov et al., 1998) but
phenotype associated with this mutation has not been described in detail so far. The
patients harboring this mutation appeared to have less severe phenotype as compared
to patients with other mutations in our study. The patient V:3 underwent bilateral
trabeculectomy and had normal intraocular pressure with visual acuity of 1/60 in both
eyes. The patient V:4 although did not undergo any surgical procedure but had normal
intraocular pressure with visual acuity of 1/60 in right eye and corneal opacity in left
eye. The patient V:5 had bilateral corneal opacity with elevated intraocular pressure
without any trabeculectomy in both eyes.
A novel missense mutation (p.G36D) was found to be segregating with
the disease in three affected individuals of family PCG-08 due to transition of G to A
at c.107 resulting in replacement of Glycine at codon 36 to aspartic acid. Polyphen
web tool predicted this change as damaging with a score of -1 whereas mutation taster
described this substitution as disease causing. According to HOPE protein prediction
online web tool, mutant residue is bigger in size than wild-type residue. The
substitution results in loss of hydrophobicity and acquisition of negative charge which
leads to defective protein functioning as flexibility contributed due to glycine, being
most flexible among all amino acids, is lost. Although the structure of first 49 residues
in CYP1B1 protein from -NH2 has not been determined so far (Tanwar et al., 2009)
but glycine has remained conserved in CYP1B1 protein in different species (Fig-3.19).
All three affected patients in PCG-08 suffered from bilateral corneal opacities and had
uncontrollable intraocular pressure even after trabeculectomy and use of topical
medications.
167
A novel in-frame deletion was found in PCG-09 family where deletion
of 12 base pairs (GGGCCAGGCGGC) from c.198-209 resulted in deletion of four
amino acids i.e. Glycine, Glutamine, Alanine and Alanine from G67-A70 (Fig-3.22).
Using Clustal Omega for alignment of CYP1B1 protein among various species, it was
observed that Glycine at 67 and Alanine at 69 has remained conserved among various
species. To predict the effect of this deletion, PROVEAN web tool was used which
showed a score of -15.8 describing it as deleterious. All mutations with score less than
-2.5 are considered pathogenic according to PROVEAN. A heterozygous mutation
resulting in deletion of eight nucleotides (GGCCAGGC) from c.199-206 has been
previously reported in German patients with PCG (Weisschuh et al., 2009). The
patient IV:3 in this family underwent bilateral trabeculectomy and had controlled
intraocular pressure although with reduced visual acuity.
168
SECTION-II
MOLECULAR CHARACTERIZATION
OF STARGARDT DISEASE
169
4.2 ARL3- A Novel Gene for Stargardt Disease:
In two Stargardt disease affected families (LUSG-03 and LUSG-04), a
novel gene (ARL3) was found to be segregating with the disease phenotype. ARL3
(ARF like protein 3) belongs to ARF subfamily of Ras (Retroviruses-associated DNA
sequences) superfamily of genes. The superfamily Ras is composed of more than 100
gene products and is further subdivided into several sub-families such as Ras, Rho,
Rab, Rap and Arf (Cavenagh et al., 1994).
ARF (ADP-Ribosylation Factor) subfamily is the most divergent
among all subfamilies of Ras superfamily because of its characteristic structural and
functional aspects. It was first identified as a protein cofactor in toxin-catalyzed ADP-
ribosylation of adenylate cyclase. It is a 21-kDa GTP-binding protein and its cofactor
activity is exclusively dependent upon binding of GTP. Several studies have shown
that ARF proteins are localized to Golgi complex and are involved in regulation of
several steps in transportation of vesicles between endoplasmic reticulum and golgi
complex. ARL3 was first identified by Clark J et al in 1993 by PCR amplification of
cDNA of ARF proteins. They used oligonucleotide primers corresponding to
conserved region of ARF and ARF like proteins (Clark et al., 1993).
Although ARL3 is classified as Arf-like GTPase, but it does not
possess any ADP ribosylation factor activity (Fansa and Wittinghofer, 2016). ARL3 is
a small GTPase and is present in all ciliated organisms. It is expressed in some human
tissues including brain and retina and tumor cell lines. Experiments have suggested
that some ARF proteins such as ARL2 and ARL3 interact with PDE6D and UNC119
(Hanke-Gogokhia et al., 2016). PDE6D and UNC119 act as “Trafficking
Chaperones”, bind lipidated proteins cargo in the inner segments, help them diffuse
through the photoreceptor cytoplasm, and finally this cargo is unloaded in outer
segments during phototransduction, a process in which an electrical action potential or
signal is generated when photons of light are captured by the visual pigments
(rodhopsins and cones opsins) present inside outer segments of the photoreceptors
(Pearring et al., 2013).
170
Of two types of photoreceptors, rods are dominant, mostly located at
the periphery whereas cones are mostly concentrated in central retina to the extent
that the fovea in humans contains only cones and is responsible for high resolution
central vision. In outer part of retina, photoreceptors are aligned parallel to one
another and adjacent to retinal pigment epithelium (RPE). Light rays striking the
retina must pass through the transparent inner layers before they reach the outer part
where light sensitive photoreceptors are located. From structural point of view, each
of these photoreceptors contain four distinct parts, outer segment, inner segment,
nuclear region and a synapse (Pearring et al., 2013).
The outer segment of these photoreceptors is a specialized non-motile
cilium being distinct from other cilia due to presence of membrane discs stacked on
one another. These outer segments are constantly being renewed throughout life of
humans. It is important for two reasons, first it acts as a preventive mechanism to
remove oxidative radicals that are accumulated during phototransduction and second
it serves to maintain the constant length of outer segments with generation of new
discs in them. RPE plays a very important role in this regard by continuous
phagocytosis of these outer segments. Several proteins are present in the outer
segments including rhodopsins which are first synthesized in the inner segments and
are then transported inside vesicles to the outer segments via a narrow connecting
cilium acting as a bridge between these two compartments (Pearring et al., 2013). Of
these proteins, some are involved in phototransduction while others are involved in
signaling of this process. Proteins participating in phototransduction include
transmembrane proteins such as rhodopsin, and peripheral membrane (PM) proteins
such as transducin and PDE6 (cGMP phosphodiesterase 6). The PM proteins involved
in signaling include rhodopsin kinase, G-protein coupled Transducin and cGMP
PDE6 (Hanke-Gogokhia et al., 2016). Many of these signaling proteins undergo
various types of lipid modifications (such as acetylation or prenylation) which allow
their rapid diffusion together with their interactions with other proteins for a rapid
onset of light response during phototransduction (Pearring et al., 2013). This suggest
that ARF GTPases including ARL3 play an important role in transportation of proteins
involved in phototransduction.
171
ARL3 colocalizes with RP2 (Retinitis Pigmentosa protein 2), PDE6D
and UNC119 to photoreceptor synaptic terminal, the cell body, the inner segment and
the connecting cilium. Two proteins have been identified which regulate GTPase
activity of ARL3 i.e. RP2 functions as Guanosine nucleotide Exchange Factor (GEF)
whereas ARL13b acts as GTPase Activating Protein (GAP) enabling GTP/GDP
exchange at ARL3-GDP. In humans, null mutations in PDE6D have known to be
associated with Joubert syndrome whereas in mice, its deletion leads to retinal
degeneration due to trafficking defects of GRK1 and PDE6. Besides, a missense
mutation has been identified in UNC119 that causes cone-rod dystrophy (Hanke-
Gogokhia et al., 2016) and mutations in RP2 results in a severe form of X-linked
retinal degeneration involving rods. Moreover, ARL3 knock out mice when examined
histologically with various stains showed impaired photoreceptors’ development. The
changes that were observed on histological examination in retina of ARL3 knock out
mice included rudimentary development of inner and outer segments, connecting
cilium and discs in the outer segments. Furthermore, ARL3 knock out mice also
exhibited increased apoptic death of photoreceptors in outer nuclear layer when
compared with ARL-WT type mice on TUNEL histochemistry (Schrick et al., 2006).
In this study, fundus photographs of all affected patients revealed
hypopigmented areas in retina making retina less reflective and leading to reduced
central vision in these patients (Fig-3.32 & Fig-3.40). When retinal thickness was
measured using OCT (optical coherence tomography), it showed decrease in its
thickness which was more marked in the center in the region around macula and fovea
(Fig-3.31 & Fig-3.41). Furthermore, hypopigmentation in macular and peri-macular
region as revealed by fundus photographs and marked decrease in central retinal
thickness could be due to either degeneration of outer segments or defective
development of the outer segments and retinal pigment epithelium. As all patients had
decreased visual acuity and there was no complaint of reduced vision during night, its
association with retinitis pigmentosa can be ruled out. All patients were also subjected
to differentiate among colors during ophthalmological examination to rule out the
possibility of achromatopsia or color blindness. To conclude, it can be said that ARL3
is the novel gene for Stargardt disease and therefore it is recommended that all new
cases of Stargardt disease must be screened for any mutation in this gene as well.
172
It would be appropriate to add here that because of unavailability of previous medical
records in all affected individuals in these families together with varying clinical
symptoms, possibility of cone dystrophy cannot be ruled out completely. It is
therefore recommended that in future, advent of specific clinical investigations that
could clearly differentiate between the two phenotypes may be used in order to clearly
relate the genetic and molecular basis with that of the corresponding phenotype.
173
SECTION-III
GENETIC STUDY OF
CONGENITAL CATARACT
174
4.3 INPP5K- A Novel Gene for Congenital Cataract:
A novel gene was identified in association with a syndromic form of
congenital cataract in a family (LUCC-15) with three affected individuals. The
pedigree shows a recessive mode of inheritance as all three affected individuals have
parents with consanguineous marriages and were phenotypically unaffected. The gene
was identified by analysis of data obtained through Whole Exome Sequencing. Initial
WES data contained more than 90,000 variants which were subjected to a filtration
strategy to exclude all synonymous, intronic and intergenic variants. The remaining
variants were further filtered down to those with allelic frequency <0.5% in ExAC
browser for South Asian population. Ten non-synonymous homozygous pathogenic
variants were selected on the basis of above described strategy and were further tested
for segregation within the family through Sanger sequencing. Finally, INPP5K was
found to be segregated in all three affected individuals with biallelic missense variants
whereas it was heterozygous in parents of all three affected individuals.
INPP5K (Inositol polyphosphate-5-phosphatase K; OMIM 607875)
has not been previously reported in association with syndromic or isolated congenital
cataract. Two studies published simultaneously in March 2017 reported its association
with a syndromic congenital cataract along with Congenital Muscular Dystrophy
(CMD) and Marinesco-Sjogren syndrome (OMIM 248800) (Osborn et al., 2017)
(Wiessner et al., 2017). One of these studies identified INPP5K homozygous mutation
in 12 affected individuals of eight families from Pakistan, Bangladesh, Brazil and
Germany (Wiessner et al., 2017) and the other had INPP5K mutation in 5 affected
from four families (Osborn et al., 2017).
In our study, there were three affected individuals. All patients on
Sanger sequencing showed substitution of Thymine to Cytosine at c.149 of INPP5K
gene resulting in replacement of Isoleucine by Threonine (Fig-3.46). Bioinformatics
tools (SIFT, Polyphen2, Mutation taster and Mutation Assessor) described this change
as pathogenic. Furthermore, Isoleucine has remained conserved in various species
from Human to Zebra fish indicating its importance in normal functioning of this gene
(Fig-3.47). 160 ethnically matched individuals were also checked for this variant and
it was not present in any of them. The variant was also absent in Exome Aggregation
175
Consortium (ExAC) browser, dbSNP146 and Genome Aggregation Database
(gnomAD).
INPP5K gene is located on chromosome 17 with 12 exons and 448
amino acids. It is also known as skeletal muscle and kidney-enriched inositol
phosphatase (SKIP) as it is highly expressed in brain, eye and skeletal muscles. It
belongs to a group of phosphatases enzymes and is responsible for dephosphorylation
of inositol ring at position 5. It mainly functions in endoplasmic reticulum along with
HSPA5 and regulate insulin receptor signaling in association with other MSS related
gene SIL1. Osborn DBM et al developed an INPP5K loss of function model in Zebra
fish which exhibited almost similar features consistent with human phenotype
(Marinesco-Sjogren syndrome) such as microphthalmia, shoretened and curved body,
reduced touch-evoked response, lens disorganization and myopathy. They further did
the histologic examination of zebra fish eye which revealed defective lens
development. INPP5K Knock-out embryos of zebra fish also revealed reduced
synaptic formation in skeletal muscle as compared to wild-type. They concluded that
all these clinical features may be attributed to enzymatic functions of INPP5K protein
and its binding partner in endoplasmic reticulum (Osborn et al., 2017).
In LUCC-15, all three affected were born without any complications.
Early development was quite normal in all affected patients except one patient (V:1)
who had delayed motor development and speech. Patient V:1 had mild-moderate
intellectual disability where as patient V:5 suffered from moderate intellectual
disability. The most salient feature in all three patients was bilateral cataracts
manifested at an early age between 2 ½ years to 3 years and underwent surgical
removal followed bilateral lens implant thereafter. Hypotonia was present in two
patients (V:1 and V:8) although there was no ataxia, difficulty in rising from squatting
position or any other motor problem. High serum CK levels in all three affected and
normal levels in a normal sibling are consistent with those in patients with CMD than
that of a simple congenital myopathy where serum CK levels are either normal or
near-normal (Wiessner et al., 2017).
In Short, all three patients with biallelic pathogenic mutation in
INPP5K presented with bilateral cataracts at an early age and elevated serum CK
levels where as intellectual disability was present in two of the three patients (patient
176
V:1 and V:5). There was no myopathy or ataxia in two patients whereas the third
patient exhibited delayed motor milestones, inability to walk without support and
impaired speech (patient V:1). These data suggest that mutation in INPP5K cause
congenital cataracts together my muscular dystrophy and intellectual disability. These
patients with such variable clinical features therefore may be grouped together with
larger MSS spectrum of disorders.
177
Conclusion:
Limitation of medical resources and research at tertiary care institutes
have made it difficult to diagnose and cure inherited disorders at initial stage
rendering people to suffer for life-long disability. As vision is the most important of
all senses, reduced visual acuity or blindness have negative effect on quality of life in
people with inherited visual disorders. It is therefore essential to diagnose such
inherited visual disorders at early stage by predictive molecular testing. To achieve
this aim, data must be available regarding all loci/genes responsible for inherited
visual disorders leading to blindness. In this context, the present study was undertaken
to understand the molecular and genetic basis of inherited visual disorders.
This study provides some insight into genetic and molecular aspects of
inherited visual disorders especially Stargardt disease and congenital cataract. This
study reports two novel CYP1B1 mutations responsible for PCG in Pakistani
population. In addition, a novel gene has been identified in association with Stargardt
disease and syndromic congenital cataract. All these findings are strongly suggestive
of genetic heterogeneity of Pakistani population for inherited visual disorders. The
findings may be helpful for scientific community across the globe in general and
Pakistan in particular. Data can therefore be used for genetic counselling and future
genetic screening for early diagnosis among affected families to improve the
prognosis by taking early precautionary measures.
178
APPENDICES
179
Appendix-I
Consent Form for Participation in Clinical Research
Medical Record No: Adult Patient
Guardians(in case of Minor)
Institute: Molecular Biology & Genetics Department & Eye Hospital, Liaquat
University of
Medical & Health Sciences Jamshoro.
Case # of Study: _______________________
Investigators: Dr.Ali Muhammad Waryah, Dr.Ashok Kumar.
Title of Study: Inherited Visual Disorders
INTRODUCTION
We invite you to participate in a research study being conducted by Molecular
Biology & Genetics Department & Eye Hospital, Liaquat University of Medical &
Health Sciences, Jamshoro as either you or one of your family members has been
suffering from inherited visual disorder. This study is based on a research about
inherited visual disorders. It is important as this research provides new insights about
visual disorders. You do not have to pay anything for participation in this study.
WHAT WILL BE EXPECTED OF YOU?
We request you for donation of a small amount of blood (5-10 ml) from your arm. We
will perform genetic analysis after extracting DNA(Hereditary Material present inside
cells). The whole process will hardly take 10 minutes. Your identification will be kept
confidential from staff working on your DNA. We also request you to provide us
access to ophthalmological record that may include various ophthalmological
investigations.
VOLUNTARILY PARTICIPATION
Your participation in this study is voluntarily. This is your right to quit from this
study whenever your wish to do so. In addition, blood donated by you to us will
remain a part of our study.
ADVERSE OUTCOMES?
Your participation in the study is not associated with any adverse outcome associated
with your health except this that you may hesitate to provide us information regarding
your family. Blood drawing may cause a temporary bruise and brief pain due to
insertion of needle but it will vanish away within few days.
180
BENEFITS
You will not get any direct and immediate benefits for participation in this study.
However your participation helps us to better understand the cause and mechanism of
disease that you are suffering from and it will lead to better management of that
disease in future. This study may benefit you and other patients alike in future. We are
highly grateful to you for your blood donation and providing us the required
information about your family.
CONTACT US
If you have any queries regarding this study, do not hesitate to contact Dr.Ali
Muhammad Waryah, Dr.Ashok Kumar or other staff on this address—Molecular
Biology & Genetics Department, Liaquat University of Medical & Health Sciences,
Jamshoro.
CONSENT FOR PARTICIPATION: I have had this study explained to me in a way that I understand, and I have had the chance to
ask questions. I agree to take part in this study.
__________________________
Signature (or Guardians) Date Signed
___________________________
Signature of Investigator
___________________________
Signature of Co-Investigator
181
Appendix-II
Proforma for Identificaiton of Patients with Inherited Visual Disorders
Patient I.D:________________________
Sampling performed previously: Yes No
Name:_______________________________________________________________
Age:______________________________ Sex: M F
Sur (Family) Name:__________________ Ethnic group:___________________
F/Name:______________________________________________________________
Address:______________________________________________________________
_____________________________________________________________________
Contact No:___________________________________________________________
Consanguineous relationship b/w father and mother: Yes No
Visual loss present since birth: Yes No
If No, then at what age it was first noticed:__________________________________
Any significant medical event occurred when visual loss/Dec.vision was first
noticed:______________________________________________________________
_____________________________________________________________________
Any Associated abnormality(Goitre/Cushing’s syndrome/Deafness abnormality etc):
_____________________________________________________________________
_____________________________________________________________________
No. of affected family members:__________________________________________
Any family member affected on father’s or mother’s side:
_____________________________________________________________________
Any complication that might have occurred during pregnancy or delivery of affected
person: ______________________________________________________________
Past medical or surgical history: __________________________________________
Any other abnormality that might have affected large no. of family members:
_____________________________________________________________________
Department of Molecular Biology & Human Genetics
Liaquat University of Medical & Health Sciences, Jamshoro
www.lumhs.edu.pk
182
Appendix-III
Ophthalmological Examination & Assessment Proforma
M.R.No:
Name:
Age:
Sex:
Address:
Tel No:
A-Clinical History
Presenting Complaints:
D/V (RE/LE/Bilateral) Since
Painless/Painful
Sudden/Gradual
Field Loss (RE/LE/Bilateral) Since
Quadrant
Miscalleneous (Specify)
Past History:
Diabetes Mellitus
Hypertension
Ocular Disease
Ocular Surgery
Ocular Trauma
Spectacles (Hypermetrope/Myope)
Miscalleneous (Specify)
Drug History:
Topical/Systemic Steroids
Topical/Systemic Antiglaucoma Drugs
Miscalleneous (Specify)
Family History:
H/o glaucoma in relatives- Yes/No
B-Clinical Examination:
Vision:
VA (Uncorrected)
VA (Corrected)
Refractive Status
N/V (Corrected)
Anterior Segment Examination:
RE LE
Lids:
Conjuctiva
Cornea
183
A/C
Iris/Pupil
Lens
Intraocular Pressure:
Airpuff IOP:
AT:
Fundus Examination:
Disc Size RE LE
CDR:
NR Rim
RNFL
Any Other
Gonioscopy:
Grading:
Any Abnormal Vessels at angle:
C-Investigations:
HRT:
Visual Field (Specify Program) FDT or HFA
OCT (RNFL + Optic Disc)
CCT (Selected Cases)
D-Provisional Diagnosis:
Primary Congenital Glaucoma
POAG
Normotensive Glaucoma
Ocular Hypertension
PACG
Secondary open angle glaucoma
Secondary angle closure glaucoma
E-Treatment:
F-Follow up
184
Appendix-IV
DNA EXTRACTION SHEET
Performed By:______________________ Date:_______________
S.# Sample I.D Blood
Volume
(ml)
TNE
Buffer
0.6ml/ml
Blood
10% SDS
20µl/ml
Blood
Proteinase K
5µl/ml Blood
NaCl
(6M)
100µl/ml
Blood
Dilution with
Low TE
preservation
Buffer
DNA
Quantification
Remarks
185
Appendix-V
Optical Density, DNA Quantification & DNA Working Dilution Preparation Sheet Performed By: ____________________ Date: _____________
S.# Sample I.D. OD260 OD260/OD280 DNA Quantificaiton
X = OD260/5000
Y= 25 x 300*/X Z= 300-Y
X= DNA Quantification Y=Quantity of Extracted DNA to be taken to prepare the working dilution Z=Quantity of Injection water to taken to prepare the working dilution *If the working dilution is to be prepared in volume other than 300µl, replace 300 with that volume.
186
Appendix-VI
List of Soft wares / Websites Accessed Clustal Omega (www.ebi.ac.uk/Tools/msa/clustalo/)
EditSeq
Ensemble (www.ensembl.org)
ExAC Browser (exac.broadinstitute.org/)
Genecards (www.genecards.org/)
HGMD (www.hgmd.cf.ac.uk/)
HOPE (www.cmbi.ru.nl/hope/method/)
Interprot (https://www.ebi.ac.uk/interpro/)
Mutation Assessor (mutationassessor.org)
Mutation Taster (www.mutationtaster.org/)
NCBI BLAST (https://www.ncbi.nlm.nih.gov/BLAST/)
Phyre2 (www.sbg.bio.ic.ac.uk/~phyre/)
Primer3 (primer3.ut.ee/)
PROVEAN (provean.jcvi.org/)
Pubmed ClinVar (https://www.ncbi.nlm.nih.gov/pubmed/)
SeqMan
SIFT (sift.jcvi.org/)
SMART Protein Domain (smart.embl-heidelberg.de/)
UCSC (https://genome.ucsc.edu)
187
Appendix-VII
List of Publications: Sheikh SA, Waryah AM, Narsani AK, Shaikh H, Gilal IA, Shah K, Qasim M, Memon AI,
Kewalramani P, Shaikh N. Mutational spectrum of the CYP1B1 gene in Pakistani patients
with primary congenital glaucoma: novel variants and genotype-phenotype correlations. Mol
Vis. 2014 Jul 7;20:991-1001. eCollection 2014. PMID:25018621
Waryah AM, Narsani AK, Sheikh SA, Shaikh H, Shahani MY. The novel heterozygous
Thr377Arg MYOC mutation causes severe Juvenile Open Angle Glaucoma in a large
Pakistani family. Gene. 2013 Oct 10;528(2):356-9. doi: 10.1016/j.gene.2013.07.016. Epub
2013 Jul 23.PMID: 23886590
Yousaf S, Shahzad M, Kausar T, Sheikh SA, Tariq N, Shabbir AS; University of Washington
Center for Mendelian Genomics., Ali M, Waryah AM, Shaikh RS, Riazuddin S, Ahmed ZM.
Identification and clinical characterization of Hermansky-Pudlak syndrome alleles in the
Pakistani population. Pigment Cell Melanoma Res. 2016 Mar;29(2):231-5. doi:
10.1111/pcmr.12438. Epub 2015 Dec 18. PMID:26575419
Waryah A.M., Shahzad M., Shaikh H., Sheikh S.A., Channa N.A., Hufnagel R.B.,
Makhdoom A., Riazuddin S., Ahmed Z.M. A novel CHST3 allele associated with
spondyloepiphyseal dysplasia and hearing loss in Pakistani kindred. Clinical Genetics.2016.
PMID 26572954.
188
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