ngs in neurodegenerative disorders our first...

NGS in neurodegenerative

disorders

Our first experience

Laboratory for genetic and molecular diagnostic of neurological disorders

Neurology Clinic, Clinical Center of Serbia

University of Belgrade

Milena Jankovic, PhD

Laboratory for genetic and molecular diagnostic of

neurological disorders

Established in 2008.

“Single gene” testing:

Direct sequencing

Fragment analysis

RT PCR

Gel electrophoresis

Dystonia type 1

Huntington's disease

Friedriech's ataxia

Wilson's disease

Amyotrophic lateral sclerosis

Alzheimer's disease

Achondroplasia

Triple A syndrome

Becker's muscular dystrophy

CADASIL

Charcot Marie Tooth type 1A

Dentatorubral-pallidoluysian

atrophy (DRPLA)

Dystonia type 5a (DYT5a)

Dystonia type 6

Dystonia type 8

Dystonia type 10

Dystonia type 11

Dystonia type 18

Frontotemporal dementia

Fragile X syndrome

Hereditary folate malabsorption

Hereditary neuropathy with

pressure palsies (HNPP)

Limb girdle muscular dystrophy

Leber's optic atrophy

MELAS

Niemann Pick type C

Parkinson's disease

Pantothenate kinase-associated

neurodegeneration

Spinal and bulbar muscular

atrophy

Spinocerebellar Ataxia Type 1






Laboratory for genetic and molecular diagnostic of

neurological disorders

Research focus:

Dystonia

Dementia

Parkinson's disease

Ataxia

Complex disorders

many genes may be involved in pathogenesis

Complex phenotypes

overlapping symptoms,

different phenotypes in the family

NGS approach

Family with complex dystonia-parkinsonism

phenotype

Specific phenotype:

segmental and multifocal dystonia

parkinsonism

psychiatric symptoms

age of disease onset range from

23 do 61 years

no data of consanguinity

autosomal-dominant pattern of

inheritance

Specific phenotype:

segmental and multifocal dystonia

parkinsonism

psychiatric symptoms

age of disease onset range from

23 do 61 years

Genetic

testing:

TOR1A -

THAP1 -

GCH1 -

SGCE -

GNAL -


phenotype

MiSeq Instrument

TruSight One Sequencing Panel

“Clinical exome”

Targets 4,813 genes associated

with known clinical phenotypes

Variant Studio software 2.2 Illumina Inc., San Diego, CA, USA


phenotype

Laboratory for Molecular Biomedicine

Institute of molecular genetics and genetic engineering

University of Belgrade

In colaboration with:

Analysis criteria:

Variant present in all affected members and absent in asymptomatic family

member,

Variant frequency is less than 1% in European population, and

Coding variant is predicted to be pathogenic by Polyphen-2 or SIFT software.

14 variants in 13 genes passed our criteria


results

Gene Ch. cDNA

change dbSNP ID

Protein

change Sift (score)

PolyPhen2

(score)

Allele

Freq.

Europe

Conserved

Sequence Protein Related Disorder (Gene Cards)

C5 9 NM_001735.2:

c.1060C>A rs34552775

NP_001726.2:

p.Leu354Met

deleterious

(0.01)

probably

damaging

(0.987)

0,4 yes Complement Component 5

Diseases associated with C5 include complement

component 5 deficiency, and rapidly progressive

glomerulonephritis.

CYP2C9 10 NM_000771.3:

c.539C>T rs200149294

NP_000762.2:

p.Ser180Phe

deleterious

(0.01)

probably

damaging

(0.987)

0 yes Cytochrome P450, Family 2,

Subfamily C, Polypeptide 9

Diseases associated with CYP2C9 include warfarin

sensitivity, and carbutamide toxicity.

FREM2 13 NM_207361.4:

c.4031G>A rs143044921

NP_997244.3:

p.Arg1344His

tolerated

(0.16)

probably

damaging

(0.961)

0,26 yes

FRAS1 Related

Extracellular Matrix Protein

2

Diseases associated with FREM2 include unilateral renal

agenesis, and frem2-related Fraser syndrome.

GIGYF2 2 NM_001103147.1:

c.2129G>T -

NP_001096617.1:

p.Gly710Val

deleterious

(0.01)

probably

damaging

(0.982)

0 yes GRB10 Interacting GYF

Protein 2

This gene was linked to Parkinson disease type 11.

However, more recent studies in different populations have

been unable to replicate this association.

HLA-A 6

NM_002116.7:

c.751G>C rs145046067

NP_002107.3:

p.Asp251His

deleterious

(0)

probably

damaging

(0.999)

0 yes Major Histocompatibility

Complex, Class I, A

Diseases associated with HLA-A include postherpetic

neuralgia, and birdshot chorioretinopathy. NM_002116.7:

c.750delG

rs45576436

rs66729206

NP_002107.3:

p.Asp251Thrfs*46 n/a n/a 0 yes

IDH1 2 NM_005896.2:

c.52A>G -

NP_005887.2:

p.Met18Val

deleterious

(0)

probably

damaging

(0.921)

0 yes Isocitrate Dehydrogenase 1

(NADP+), Soluble

Diseases associated with IDH1 include metaphyseal

chondromatosis with d-2-hydroxyglutaric aciduria, and

periosteal chondrosarcoma.

2 NM_001164507.1:

c.6076-6T>G - - n/a n/a 0 yes Nebulin

Mutations in this gene are associated with recessive

nemaline myopathy.

PIKFYVE 2 NM_015040.3:

c.2164A>G -

NP_055855.2:

p.Thr722Ala

tolerated

(0.42)

possibly

damaging

(0.473)

0 yes Phosphoinositide Kinase,

FYVE Finger Containing

Mutations in this gene cause corneal fleck dystrophy

(CFD); an autosomal dominant disorder characterized by

numerous small white flecks present in all layers of the

corneal stroma.

RGR 10 NM_002921.3:

c.841T>C -

NP_002912.2:

p.Trp281Arg

deleterious

(0)

probably

damaging

(1)

0 no Retinal G Protein Coupled

Receptor

This gene may be associated with autosomal recessive and

autosomal dominant retinitis pigmentosa.

RRH 4 NM_006583.2:

c.350C>T -

NP_006574.1:

p.Thr117Met

deleterious

(0)

probably

damaging

(0.996)

0 yes

Retinal Pigment Epithelium-

Derived Rhodopsin

Homolog

Diseases associated with RRH include retinitis pigmentosa,

and alcohol dependence.

RYR1 19 NM_000540.2:

c.9635A>G rs199738299

NP_000531.2:

p.Glu3212Gly

deleterious

(0.04)

benign

(0.205) 0 yes

Ryanodine Receptor 1

(Skeletal)

Mutations in this gene are associated with malignant

hyperthermia susceptibility, central core disease, and

minicore myopathy with external ophthalmoplegia.

SIRT1 10 NM_012238.4:

c.719T>C rs199618656

NP_036370.2:

p.Ile240Thr

deleterious

(0.01)

benign

(0.261) 0 yes Sirtuin

Diseases associated with SIRT1 include xeroderma

pigmentosum, group d, and aortic valve disease.

TPTE 21 NM_199261.2:

c.664C>T rs76723236

NP_954870.2:

p.Arg222Trp

deleterious

(0.01)

possibly

damaging

(0.841)

0 no

Transmembrane

Phosphatase With Tensin

Homology

TPTE protein may play a role in the signal transduction

pathways of the endocrine or spermatogenic function of

the testis. Alternative splicing results in multiple transcript

variants.


results

Variants present in all affected members and absent in asymptomatic family member

Dystonia Genes

(Locus)

Complex Dystonia-

Parkinsonism sy.

Genes (Locus)

Parkinson Disease Genes (Locus)

TOR1A (DYT1) ATP1A3 (DYT12) SNCA (PARK1) EIF4G1(PARK18)

THAP (DYT6) PRKRA (DYT16) LRRK2 (PARK8) GBA

GCH1 (DYT5a) TAF1 (DYT3) VPS35 (PARK17) GIGYF2 (PARK11)

TH (DYT5b) SLC6A3 Parkin (PARK2) HTRA2 (PARK13)

SRP PLA2G6 (PARK14) PINK1 (PARK6) MAPT

SLC2A1 (DYT18) DJ-1 (PARK7) PDXK

CIZ1 (DYT23) ATP13A2 (PARK9) POLG1

PRRT2 (DYT10) FBXO7(PARK15) SNCAIP

PNKD (DYT8) DNAJC6 (PARK19) SNCB

ANO3 (DYT24) ADH1C UCHL1 (PARK5)

GNAL(DYT25) DCTN1 CSF1R

List of genes related to dystonia, PD, or parkinsonism-dystonia syndromes


results


results

GIGYF2 is coding Grb10 interacting GYF protein 2

with potential involvement in insulin and insulin-like

growth factor (IGF) signaling in the brain.

This gene is located in a chromosomal region that

was genetically linked to Parkinson disease (PD) type

11 by genome wide linkage analysis in familial PD

patients.

GIGYF2 was proposed as the gene that is

defective at PARK11 locus.

GIGYF2 gene

Not present in dbSNP143 and

ExAC databases

In silico analysis:

MutationTaster and

MutPred

pathogenicity

I-Mutant 3.0

decreased stability

CADD

significant

functional effect

c.2129G>T p.Gly710Val

Mutations in one gene causing different

diseases

Guerreiro et al., 2014

homozygous/heterozygous variants in the same gene causing a severe

early-onset disease and increasing the risk for a different late-onset

disease

Guerreiro et al., 2014

Highlighting the role of NGS in uncovering pleiotrotropic events in neurodegenerative disorders

Mutations in one gene causing different

diseases

MiSeq instrument

in

Laboratory for genetic and molecular

diagnostic of neurological disorders

Since 2017...

Acknowledgement

Movement Disorders

Division

Laboratory for Molecular

Biomedicine

ngs in neurodegenerative disorders our first...

Documents