how amc (arthrogryposis) and cp (cerebral palsy) … · the “new genetics” and epigenetics...
TRANSCRIPT
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How AMC (Arthrogryposis) and CP (Cerebral Palsy) have to adjust to the “new genetics” and Epigenetics
Judith G. Hall, OC, MD Departments of Pediatrics and Medical Genetics
University of British Columbia Vancouver, BC, Canada
- No known conflicts of interest -
PLAN OF TALK
• Thank you for inviting me
• Clinical Geneticist
• Long standing interest in arthrogryposis (AMC)
• Common challenges
• Non-traditional inheritance
• Epigenetics
ARTHROGRYPOSIS (MULTIPLE CONGENITAL CONTRACTURES)
Congenital non-progressive
limitation of movement of
two or more joints in
different body areas
(lots and lots of things get included)
WHY SO INTERESTING?
• All about embryo/fetus developing movement
• All causes share decreased in utero movement
• Why develop contractures?
CONGENITAL CONTRACTURES IN THE NEWBORN
• Clubfoot……………………………................1/500 – 1/1000
• Congenital dislocated hips....................1/200 – 1/500
• Multiple congenital contractures…....1/3000 – 1/6000
• All congenital contractures………………...1/100– 1/250
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Arthrogryposis
is not a diagnosis
— it is a sign
Maternal Connective tissue
illness skeletal dysplasia
Fetal Vascular crowding compromise Neurologic Muscle deficits defects
LIMITATION OF FETAL JOINT MOBILITY
MULTIPLE CONGENITAL CONTRACTURES
(ARTHROGRYPOSIS)
FREQUENCY OF ARTHROGRYPOSIS - Many lethal types, miscarriages, stillborns
• VERY heterogeneous
• No proper ICD code(s)
• Need population base information
• Australia epidemic
• North America
– Washington State
– British Columbia registry
• Others
– Finland
– Sweden
OCCURRENCE OF ARTHROGRYPOSIS
• ~ 1/3000 – 5000….live births
• 1/3………………………Amyoplasia
• 1/3………………………CNS – newborn lethal
• 1-3………………………Heterogeneous group of disorders
Ways to approach a diagnosis – What are useful clinical discriminators?
APPROACH TO MULTIPLE CONGENITAL CONTRACTURES -
CLINICAL
• Mainly limbs
• Limbs and other body areas
• Limbs and CNS/lethal
AREAS OF INVOLVEMENT TOTAL STUDY GROUP
Primarily limbs
Limbs plus other body
areas
Limbs plus CNS
186
53%
129
37%
35
10%
33% 33% 33%
AMYOPLASIA “CLASSICAL ARTHROGRYPOSIS”
• Typical symmetric positions of limbs
• Usually “teratogenic” clubfoot
• Absent muscles with fibrotic replacement
• Mid facial hemangioma
• 10% abdominal structural anomaly (vascular accident) and other vascular compromise (lost fingers or toes)
• Apparent increase in one of monozygotic twins
• Surprisingly good response to early physical therapy
• No apparent recurrence risk or risk for other congenital anomalies
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AMYOPLASIA AMYOPLASIA AMYOPLASIA
AMYOPLASIA AMYOPLASIA AMYOPLASIA—UPPER ONLY
AMYOPLASIA—LOWER ONLY
AMYOPLASIA “CLASSICAL ARTHROGRYPOSIS”
• Typical symmetric positions of limbs
• Usually “teratogenic” clubfoot
• Absent muscles with fibrotic replacement
• Mid facial hemangioma
• 10% abdominal structural anomaly (vascular accident) and other vascular compromise (lost fingers or toes)
• Apparent increase in one of monozygotic twins
• Surprisingly good response to early physical therapy
• No apparent recurrence risk or risk for other congenital anomalies
AMYOPLASIA and NORMAL TWIN
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NORMAL TWIN and AMYOPLASIA
AMYOPLASIA “CLASSICAL ARTHROGRYPOSIS”
• Typical symmetric positions of limbs
• Usually “teratogenic” clubfoot
• Absent muscles with fibrotic replacement
• Mid facial hemangioma
• 10% abdominal structural anomaly (vascular accident) and other vascular compromise (lost fingers or toes)
• Apparent increase in one of monozygotic twins
• Surprisingly good response to early physical therapy
• No apparent recurrence risk or risk for other congenital anomalies
Ribera, 1642
APPROACH TO MULTIPLE CONGENITAL CONTRACTURES -
CLINICAL
• Mainly limbs
• Limbs and other body areas
• Limbs and CNS/lethal
CLASSIFICATION OF DISTAL AMCs Hall I Distal IIA Gordon (cleft palate, SS) IIB Ophthalmoplegia (fine muscle) IIC Cleft Lip IID Scoliosis + DA IIE Trismus + Unusual Hand + DA Freeman-Sheldon Syndrome Sheldon-Hall Sheldon-Hall Look Alike Deafness + DA Trismus Pseudocamptodactyly AD, Multiple Pterygium Contractural Arachnodactyly Absent Teeth + DA Chitayat, AR, DD X-linked Stavit, S. African, Naguib Moore-Weaver Distal MR, DD
Bamshad 1A 3 5
(10) 4
7B 2
2B 2C 6
7A 8 9
(11) (12) (13) (14) (15) (16)
Gene TPM2 MYH3 TNNT3, TNN12 MYH3 11q25 MYH8 FBN2
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CLASSIFICATION OF DISTAL AMCs Hall
I Distal IIA Gordon (cleft palate, SS) IIB Ophthalmoplegia (fine muscle) IIC Cleft Lip IID Scoliosis + DA IIE Trismus + Unusual Hand + DA Freeman-Sheldon Syndrome Sheldon-Hall Sheldon-Hall Look Alike Deafness + DA Trismus Pseudocamptodactyly AD, Multiple Pterygium Contractural Arachnodactyly Absent Teeth + DA Chitayat, AR, DD X-linked Stavit, S. African, Naguib Moore-Weaver Distal MR, DD
Bamshad 1A 3 5
(10) 4
7B 2
2B 2C 6
7A 8 9
(11) (12) (13) (14) (15) (16)
Gene TPM2 MYH3 TNNT3, TNN12 MYH3 11q25 MYH8 FBN2
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FETAL AKINESIA DEFORMATION SEQUENCE
PENA SHOKIER PHENOTYPE
• Intrauterine growth restriction
• Congenital contractures of the limbs
• Hypoplastic lungs
• Short umbilical cord
• Polyhydramnios – short gut
• Craniofacial anomalies
– Micrognathia +/- small mouth
– +/- cleft palate
– High bridge of nose
– Depressed tip of nose
• Lack of normal mechanical forces
may lead to secondary
deformations
• “Use” is essential for normal development
SECONDARY EFFECTS FROM LACK OF MOVEMENT IN UTERO
• IUGR – limbs are short
• Contractures with “collagenosis”, extra connective tissue, thick capsule
• Abnormal relationship of limb to weight bearing – joints at odd angles
• Muscle – disuse atrophy, decreased mass
• Dimples – attached to overlying skin
• Other changes of FADS – lungs, gut, craniofacial, etc.
GROWTH
• AMC affected limbs - short and small
• Final height ~ 5th centile for family
• Less muscle and less calcification of bone means less weight
• Avoid obesity – makes for more work
• Some limbs grow even less normally (like post-polio)
As organs begin to function muscles begin to contract
stretching developing tissues from inside
and outside
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EMBRYONIC/FETAL MOVEMENT Week 4 Heart beating begins 5-6 Head and trunk “stirs” 7 Shoulders “shrug” 8 Rhythmic “breathing” begins
even though larynx not open Jaw starts to move 9 Upper arms moving 10 Hips, lower arms moving 11 Lower limbs kicking 12 Hands open and ankles moving into
correct position
CLUBFOOT 16 WEEKS
Ultrasound 16 weeks
Clubfoot diagnosis
Amyoplasia
RS 517518 CH&MC
LTS3-85
PRENATAL DIAGNOSIS BY ULTRASOUND
(WHAT ARE THE CLUES, WHAT TO LOOK FOR)
• Usually not picked up without long careful real time US study – 45 min – 1 hr
• Nuchal edema
• Thin undercalcified bones
• Movement may start any time from 11 weeks to 34 weeks
• Small lungs
• Diaphragm defect or decreased movements
• Other structure or space constraints (amniotic bands, uterine fibroid, amount of amniotic fluid)
2 RETROSPECTIVE STUDIES OF AMYOPLASIA
2013 and 2015
Only 25% of 4 limb Amyoplasia diagnosed prenatally!
RECURRENCE RISK
Primarily limbs
Limbs plus other areas
Limbs plus CNS
Total
Estimated RR for overall group
Parents: 4.0%
Self: 6.5%
10.8%
10.4%
5.7% 6.8%
Estimated RR when knowns excluded
Parents: 4.7% 1.4% 7% 3%
If, and only if, a specific diagnosis cannot be made should a 5% recurrence risk estimation be given
Prognosis depends on the specific diagnosis and the natural history of that disorder
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WHAT DOES THIS HAVE TO DO WITH CEREBRAL PALSY?
AMC CP
Prevalence 1/3000 2/1000
Variability > 400 types 330 genes known
~ 50 genes known
Seizures/D/D 1/3 2/3
Prenatal Dx Check movement (only 25%) Genes better
Need gene
IUGR All – because of decreased muscle
With infection
PT/OT Essential mobilize Essential Prevention secondary
Drugs help No Yes
Both AMC (CNS group = 1/3) and CP (2/3) with DD and/or seizures…
• Look for chromosomal deletion
• Specific gene(s) mutations
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GENE ONTOLOGY
• AMC – 330 genes placed in 27 pathways
• Find pathway of genes in development
• Possible therapeutic approach
Hall JG, Kiefer J. Arthrogryposis as a Syndrome: Gene Ontology Analysis. Mol
Syndromol 7:101-109, 2016.
ESCOBAR SYNDROME (ONE OF MANY MULTIPLE PTERYGIUM SYNDROMES)
• Deficiency of one of the three embryonic components of the neuroreceptor
• 3 adult neuroreceptor components genes are normal
• Natural history – recurrent respiratory infections die in 20s
ESCOBAR SYNDROME (ONE OF MANY MULTIPLE PTERYGIUM SYNDROMES)
• It is possible to “wake up” the adult receptor and “cure” the syndrome
• Therapy already available
• Tensilon-like drugs used in Myasthenia Gravis
So if you know the mechanism, you may be able to treat!
DEVELOPMENTAL BIOLOGY
• Time specific, tissue specific, gender specific gene expression
• Cascades, pathways, and networks – FGF, SHH, WNT, TGFβ, RTK-RAS
• Embryo, early fetus, viable fetus
STAGES OF AN INDIVIDUAL 1. Preimplantation 2. Embryo 3. Early fetus 4. Later fetus (viable) 5. Newborn 6. Infant 7. Young child 8. Older child 9. Adolescent (gender differences) 10. Adult (premenopause/postmenopause) A. Young B. Middle C. Mid-life 11. Old 12. Old Old
RECENT WORK • 22,000 genes – for 200,000 proteins
• Less than 2% of the genome contains protein coding genes
• Non-coding DNA highly conserved
• Commonality of animal species
(90% mice, 98% primates, 99.9% humans)
• At least 20% of the human genome has copy number variation (CNV) between individuals (which reflect how individual we are!)
• At least 100 “mutations” not in parents – 5 of which are “of unknown significance”
• There is no such thing as a “normal” human being
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RECENT GENETIC WORK
• Genes are part of pathways/networks/systems (i.e., housekeeping, skin, limb, etc.)
• Maybe 1000 pathways – used over & over with alternative splicing/slightly different proteins
• Crosslink, buffering, redundancy of pathways
• Interactions with environment sculpt the pathways
NON-TRADITIONAL MECHANISMS (revealed by studying congenital anomalies)
• Mosaicism
• Microchimerism
• Imprinting
• Epigenetics
• Microbiome
• Fetal programming
MOSAICISM • An individual comprised of two or more genetically distinct
populations of cells derived from a single zygote (conception)
CHIMERA • An individual who is a mixture of cells from two or more
genetically distinct populations of cells which have been derived from more than one zygote
MICROCHIMERISM • The presence of a small number of cells derived from another
individual (transfusion, transplantation, pregnancy, twin, etc.)
McCUNE ALBRIGHT SYNDROME
• Mosaic for mutation causing increased activity of Gs protein
• Many different mutations, but consistent within an individual
• Would be lethal if present in all cells
• The affected individual is rescued by “normal” cells
MOSAICISM OCCURS IN ALL HUMAN BEINGS
• Large multicellular organism
• 1013-1014 cells in our bodies lost (plus programmed cell death)
• Mutation rate is 1/6,000 – 1/60,000 for known single gene human disorders
• We are all a walking “bag” of mutations
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WHEN AND WHERE DOES/DID THE MOSAICISM ARISE?
A. Early – all tissue
B. Placenta – only (trisomy rescue)
C. Somatic – frequent
D. Germline (anticipation, multiple affected offspring) in normal appearing parent
NON-TRADITIONAL MECHANISMS (revealed by studying congenital anomalies)
• Mosaicism
• Microchimerism
• Imprinting
• Epigenetics
• Microbiome
• Fetal programming
CHIMERA
MICROCHIMERISM
MOSAIC
FETAL MATERNAL MICROCHIMERISM
• The presence of a small number of cells derived from another individual (transfusion, transplantation, twin, etc.)
• An individual comprised of two or more genetically distinct populations of cells derived from a single zygote (conceptus)
• An individual who is a mixture of cells from two or more genetically distinct populations of cells which have been derived from more than one zygote
EFFORTS AT NON-INVASIVE PRENATAL DIAGNOSIS
• Can find fetal cells in mother’s blood
• However, they are from all her pregnancies
• Somehow these cells are tolerated long term
Bianchi, AJMG 2000; 91:22
►
FETAL-MATERNAL CELL TRAFFICKING
• Goes both ways
• Carry with us for lifetime
• Stem cells called upon by injury/stress
• More cells cross when placenta in trouble
- Bianchi, AJMG 2000; 91:22-28
- Lo, AJHG 1999; 64:218-224
• Newborns have mother’s cells in many tissues
- Bianchi, J Peds 2003; 142:31-35
• Relation to stem cell therapy
• Fetus to mother
• Mother to fetus
• Fetus1 fetus 2 (DZ twins)
• Fetus 1 Mother Fetus 2, 3, 4, etc.
• Fetus becomes mother fetus F2
FETAL-MATERNAL CELL TRAFFICKING
• Goes both ways
• Carry with us for lifetime
• Stem cells called upon by injury/stress
• More cells cross when placenta in trouble
- Bianchi, AJMG 2000; 91:22-28
- Lo, AJHG 1999; 64:218-224
• Newborns have mother’s cells in many tissues
- Bianchi, J Peds 2003; 142:31-35
• Relation to stem cell therapy
MOTHER’S CELLS IN FETUS
• 4 male babies who died for other reasons
• Maternal cells present in
–Liver all++
–Spleen all+++
–Thymus all++
–Skin 2/4
–Thyroid 1/2
SRIVATSA et al. J Pediatr 142: 31-35, 2003
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FETAL CELLS IN MOTHER
• Fetal cells present in thyroid tissue (probably both male and female)
• Many types of thyroid disease (adenoma, Hashimoto’s carcinoma, multi-nodular goiter, thyroiditis)
• Fetal cells can integrate and contribute to repair
• Role of fetal cell depends on its origin (cell type)
SRIVASTA et al. Lancet 358: 2034-2038, 2001 Srivasta, 2001
Srivasta, 2001
SUMMARY OF MICROCHIMERISM
• Fetal maternal microchimerism is common and probably universal in mammals
• Pregnancy associated microchimerism is increased by complications of pregnancy
• These microchimeric progenitor/stem cells play a role in repair of many tissues
• May trigger or facilitated autoimmune reactions probably depending on the compatibility of the antigens inherited from father
• Transgenerational “memory” may be important in other ways
• Fetal maternal microchimerism may be the reason women live longer than men (fetal cells even found in mom’s brain)
NON-TRADITIONAL MECHANISMS (revealed by studying congenital anomalies)
• Mosaicism
• Microchimerism
• Imprinting
• Epigenetics
• Microbiome
• Fetal programming
GENOMIC IMPRINTING
• Germ line specific modification which produces functional difference in expression of genetic material depending on whether the genetic material is maternally or paternally derived
Genome imprinting implies there are specific pieces of genetic information that must come from mother and others that must come from father to have normal development
Genome imprinting involves both an imprintable gene and imprinting of that gene (i.e., turning off, associated with methylation)
EVIDENCE OF GENOMIC IMPRINTING
• Pronuclear transplantation
• Triploid phenotypes
• Deletions and duplications depending on parent of origin (P of O)
• Uniparental disomies
• See changes in size, behaviour and survival ANGELMAN PRADER-WILLI
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• Both associated with 15p –
– PW-paternal
– AS-maternal
• Also both associated with 15UPD
– PW-paternal
– AS-maternal
• 2 separate critical regions and imprinting centres
WHY IS THERE IMPRINTING?
• Conflict regarding whose genome control
– Father optimize fitness of offspring
– Mother survivor
• Defense against transposable elements & virus
– Primitive protection system that affects gene expression
• Rheostat response to environment
– Fine tuning
Methylation of the imprinted gene is associated with non-expression
This was a key to looking at gene expression
Methylation requires a methyl group (role of folic acid)
NON-TRADITIONAL MECHANISMS (revealed by studying congenital anomalies)
• Mosaicism
• Microchimerism
• Imprinting
• Epigenetics
• Microbiome
• Fetal programming
NOW THE CHALLENGE IS TO UNDERSTAND HOW GENES ARE
TURNED ON AND OFF (i.e., control of gene expression)
• DNA methylation
• Histone acetylation
• Non-coding RNAs – small, micro, long, circular
• Telomeres
• What else?
EPIGENETICS
EPI = on or over – therefore modulates gene expression without modifying DNA sequence
1. Heritable states in gene function that are not explained by change in DNA sequence
2. Heritable and reversible modifications of chromatin which modulate chromosome/gene function and expression and thus the resulting phenotype
EPIGENETICS
EPI = on or over – therefore modulates gene expression without modifying DNA sequence
3. Meiotically and mitotically heritable changes in gene expression that are not coded in the DNA itself
4. Of interest because they are a window into control and regulation of gene expression and are relevant to tissue specificity and timing of expression
• What “tells” the genes to turn “on” and “off”?
• Who “tells” them to do it?!!!
Stevenson & Hall, 2006
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FEATURES OF TRANSCRIPTIONALLY ACTIVE & INACTIVE CHROMATIN
Transcriptionally Transcriptionally
ACTIVE chromatin INACTIVE chromatin
CHROMATIN Open, extended Highly condensed
conformation conformation, particularly apparent in heterochromatin
DNA METHYLATION Relatively Methylated including
unmethylated, at promoter regions
especially at
promoter regions
HISTONE Acetylated histones Deacetyled histones
ACETYLATION (Ubiquinization phosphorylation, etc)
Rodenhiser et al., 2006
Rodenhiser et al., 2006
WHOLE SETS OF PROTEINS AND ENZYMES THAT MODIFY CHROMATIN
• Histone acetylases
• Histone deacetylases
• Histone methyltransferase
• Adenosine 5’ di and triphosphate ribosylases
• DNMT (DNA methyltransferase) family
• MECP2 (time and tissue specific) Weichenhan & Plass, 2013
• Epigentic changes modulate gene expression without modifying DNA sequence
• Epigenome is the total epigenetic changes at a particular time and in a particular tissue that will be present on top of the genome
Kohli & Zhang, 2013
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NON-TRADITIONAL MECHANISMS (revealed by studying congenital anomalies)
• Mosaicism
• Microchimerism
• Imprinting
• Epigenetics
• Microbiome
• Fetal programming
MICROBIOME
• Live in harmony
• Actually need them to be healthy
• 27 different zones
• Present prenatally
• Get new ones at the time of birth
• More than 10x as many bugs as cells
CHANGES OVER AGING
NON-TRADITIONAL MECHANISMS (revealed by studying congenital anomalies)
• Mosaicism
• Microchimerism
• Imprinting
• Epigenetics
• Microbiome
• Fetal programming
• In order to survive, mammals developed capacity for flexibility (plasticisity)
• The evolution of mammals (humans) involved developing a spectrum of responses, survival advantage was to have flexibility in responses to a change in the environment
• Fetus particularly responsive and sensitive to maternal messages (potential mismatch)
• What are the types of signals from mom to fetus?
• Maternal diet seems to be a signal to the fetus, what else?
• Transgenerational effects
FETAL PROGRAMING (Development Origins of Health and Disease)
• Mammals evolved protective mechanisms
• Humans are mammals
• Transgenerational effects –– many tissues, at many times, and gender specific effects
• Related to regulating growth/size, behavior, and survival
BARKER HYPOTHESIS (Developmental Origins of Adult Health and
Disease – DOHaD)
• Epidemiologist in UK
• Looking for cause of early onset cardiovascular disease
• Males with heart attacks at lessthan 40 years old
• Had intrauterine growth restriction and low socioeconomic status
CRITICAL/SENSITIVE PERIODS
• Barker Hypothesis:
Critical/sensitive periods in development during which insult or stimulus causes long lasting effects on structure and/or function.
• Nutrition programs the structures and functions of the developing fetal organs resulting in susceptibilities which interact with environment to develop diseases later in life
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BARKER HYPOTHESIS • Undernutrition at different stages of pregnancy, and infancy
leads to increased risk of:
• Cardiovascular disease
• Hypertension
• Diabetes mellitus
• Abnormal cholesterol levels
• Abnormal coagulation factors
• Osteoporosis
• Cancer
(MUST BE TAKEN INTO ACCOUNT FOR GENOMIC ASSOCIATION STUDIES OF COMPLEX DISORDERS)
FETAL ORIGINS OF ADULT DISEASE – BIRTH SIZE IS IMPORTANT
• Animal studies
• WWII blockade (Dutch famine)
• Immigrants
• Scandinavian studies of famine
• Quebec ice storm
• Differences in male and female offspring
• Many human populations
THESE ARE 3 GENERATION EFFECTS
FETAL PROGRAMMING (Development Origins of Health and Disease)
• Evolutionary protective mechanism for mammals
• Humans are part of a mammalian species
• Transgenerational effects – probably several modes of communication – many organ specific effects
• Related to down regulating growth/size and behaviour
• Provides Darwinian fitness advantage adaptive responses
• Has potential for “mismatch” with later environment
“What gets inherited is not a deterministic genotype, but rather a genotype that encodes a potential range of
phenotypes.”
Gilbert 2000
GENES ARE EXPRESSED IN…
• Time in development specific
• Tissue specific
• Gender specific
• Parent of origin specific
• Pathways
• With transgenerational effects
And can be identified as silent or expressing
IS THIS COMPLEX OR WHAT?
DID WE REALLY THINK IT WOULD BE SIMPLE?
WERE YOU PAYING ATTENTION WHEN ALL OF THIS WAS HAPPENING?
WELL – YOUR TISSUES WERE!!
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AMC CP
Prevalence 1/3000 2/1000
Variability > 400 types 330 genes known
~ 50 genes known
Seizures/D/D 1/3 2/3
Prenatal Dx Check movement (only 25%) Genes better
Need gene
IUGR All – because of decreased muscle
With infection
PT/OT Essential mobilize Essential Prevention secondary
Drugs help No Yes
Sometimes it seems a miracle that anyone survives when you know all the things that can go wrong
Principle of clinical/medical/human genetics is to utilize the unusual/exceptional case to learn general principles of biology
Mother Nature is very clever, she tests things out, then reuses “good” pathways in additional ways, and she has built in the ability to adjust to different environments
Our challenge is to work with Mother Nature to achieve optimum health for all of us, because nobody is “normal”!
review article
T h e n e w e ngl a nd j o u r na l o f m e dic i n e
n engl j med 370;25 nejm.org june 19, 20142418
Elizabeth G. Phimister, Ph.D., Editor
Diagnostic Clinical Genome and Exome Sequencing
Leslie G. Biesecker, M.D., and Robert C. Green, M.D., M.P.H.
From the National Human Genome Re-search Institute, National Institutes of Health, Bethesda, MD (L.G.B.); and the Division of Genetics, Department of Medicine, Brigham and Women’s Hospi-tal and Harvard Medical School, and Partners Healthcare Personalized Medi-cine — all in Boston (R.C.G.). Address reprint requests to Dr. Biesecker at 49 Convent Dr., Rm. 4A56, Bethesda, MD 20892-4472, or at [email protected].
N Engl J Med 2014;370:2418-25.DOI: 10.1056/NEJMra1312543Copyright © 2014 Massachusetts Medical Society.
Sequencing of the genome or exome for clinical applications, hereafter referred to as clinical genome and exome sequencing (CGES), has now entered medical practice.1 Several thousand CGES tests have already
been ordered for patients, with the goal of establishing diagnoses for rare, clini-cally unrecognizable, or puzzling disorders that are suspected to be genetic in ori-gin. We anticipate increases in the use of CGES, the key attribute of which — its breadth — distinguishes it from other forms of laboratory testing. The interroga-tion of variation in about 20,000 genes simultaneously can be a powerful and effec-tive diagnostic method.2
CGES has been hailed as an important tool in the implementation of predictive and individualized medicine, and there is intense research interest in the clinical benefits and risks of sequencing for screening healthy persons3; however, current practice recommendations4 do not support the use of sequencing for this purpose, and for that reason we do not further address it here. We have also limited this overview of CGES to the analysis of germline sequence variants for diagnostic purposes and do not discuss the use of CGES to uncover somatic variants in can-cer in order to individualize cancer therapy.
Clinicians should understand the diagnostic indications for CGES so that they can effectively deploy it in their practices. Because the success rate of CGES for the identification of a causative variant is approximately 25%,5 it is important to un-derstand the basis of this testing and how to select the patients most likely to benefit from it. Here, we summarize the technologies underlying CGES and offer our insights into how clinicians should order such testing, interpret the results, and communicate the results to their patients (an interactive graphic giving an overview of the process is available with the full text of this article at NEJM.org).
Technic a l Ov erv ie w a nd Limi tations of CGES
Detailed technical descriptions of sequencing can be found elsewhere,6-9 and we provide a graphical summary of one method of CGES in Figures 1 and 2. Regard-less of the specific technology that is used, the process begins with the extraction of DNA from white cells, after which the DNA is broken into short fragments, the sequences of which are determined with the use of one of various sequencing tech-nologies. The sequencing instrument generates millions of short sequence reads, which are strings of data representing the order of the DNA nucleotides, or bases, in each fragment. These sequence reads are then aligned to specific positions in the human genome reference sequence (see Glossary) with the use of computers.10 Similarities and differences between the patient’s sequence and the reference sequence are tabu-lated, and a computerized determination of the specific genotype (A, C, G, or T) at each position in the exome or genome is performed, resulting in an output file along
An interactive graphic giving an
overview of the steps in CGES is available at
NEJM.org
The New England Journal of Medicine Downloaded from nejm.org at WELCH MEDICAL LIBRARY - JOHNS HOPKINS UNIVERSITY on August 18, 2017. For personal use only. No other uses without permission.
Copyright © 2014 Massachusetts Medical Society. All rights reserved.
Clinical Genome and Exome Sequencing
n engl j med 370;25 nejm.org june 19, 2014 2419
with information representing the number of se-quence reads generated (depth of coverage) and the accuracy of the genotype at each position. The output file is computationally filtered in ac-cordance with the clinical objective of the test and the preferences of the laboratory. Typically, the file is filtered for variants that are rare or have not previously been reported (because it is reasoned that a common variant cannot cause a rare disease), variants predicted to cause a loss or altered function of a gene, and variants previ-ously reported to cause disease.10,11
CGES is most useful for the detection of sin-gle-nucleotide substitutions and insertions or deletions of 8 to 10 nucleotides or smaller; it is less accurate for other types of genomic varia-tion (Table 1). The yield of sequence reads is inherently uneven across the exome (or genome) — typical results provide adequate coverage of 85 to 95% of the targeted sequence. With exome sequencing, there is also variable coverage of flanking intronic regions, which may include disease-causing variants that affect the splicing of messenger RNA encoded by the gene (splice variants).
Indic ations for Or der ing CGES
CGES is currently indicated for the detection of rare variants in patients with a phenotype suspected to be due to a mendelian (single-gene) genetic disorder, after known single-gene candidates have been eliminated from consideration or when a multigene testing approach is prohibitively ex-pensive. Patients can be of any age but are com-monly children, since many genetic conditions are manifested in childhood; evaluations are per-formed because parents are searching for the cause or for information to guide management and treatment and desire accurate information regarding the risk of recurrence, as noted below.
The preparation for ordering CGES should include four key elements: gathering informa-tion on family history, systematically evaluating the patient’s phenotype, searching medical lit-erature and databases, and obtaining informed consent. A thorough family history should be obtained to assess whether there are similar or related phenotypes in other family members, as well as to evaluate and assess the inheritance pattern. The patient (and other apparently af-fected family members) should be evaluated for
other potentially relevant manifestations. For example, if the primary presentation is autism and CGES is being considered by a neurologist, the patient should also be carefully examined for respiratory, cardiac, renal, skin, and dysmorphic abnormalities. With a family history and a com-prehensive phenotype in hand, a literature re-view or syndrome database search should be performed (Table 2) to determine whether the patient’s presentation matches a rare but estab-
Glossary
Exome sequencing: DNA sequencing that targets the exons of all genes in the genome. The exome makes up about 1% of the genome, primarily exons of genes that code for proteins. This type of sequencing is sometimes referred to as “whole-exome sequenc-ing,” even though coverage of the exons is not 100%.
Exons: Segments of genes that are spliced together after gene transcription to form messenger RNA, which, in turn, is translated into protein.
Expressivity: Variation in the severity of a genetic dis-order among persons with some features of the condition.
Filtering analysis: The process of excluding DNA vari-ants from further consideration because of various attributes, with the use of bioinformatics and manual curation. For example, most filtering analyses exclude synonymous variants (DNA variants that are pre-dicted not to change the amino acid sequence of a protein).
Genome sequencing: DNA sequencing that targets the entire genome. It is sometimes termed “genome shotgun sequencing” or “whole-genome sequenc-ing,” even though coverage is not 100%.
Germline variant: A DNA sequence variant that was transmitted by means of a gamete (sperm or egg) or that was caused by a mutation in the zygote or at a very early stage of fetal development and is pre-sumed to be present in all of a person’s nucleated cells.
Human genome reference sequence: A reference se-quence that provides a haploid mosaic of different DNA sequences from multiple donors, which is re-vised periodically and is not necessarily normal.
Penetrance: The likelihood that a person with a caus-ative variant in a gene has any recognizable symp-tom, sign, or laboratory feature of the disease asso-ciated with that variant.
Sanger sequencing: A method of sequence determina-tion, invented by Frederick Sanger, that uses dideoxy terminator nucleotide chemistry, with the reaction products separated by gel electrophoresis.
Variant: A difference in a DNA sequence in comparison with the normal reference sequence. A variant may be benign (sometimes referred to as a polymor-phism) or pathogenic (sometimes referred to as a mutation).
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A C C T T C C T C G T G G A C T GA
C G C C G G G G C T A G C C
A
T
G
T
C
C
A
G
T
A C C T T C C T C GA T G A T G G A C T G C G C C G G G GT C G A G C C A GT
C
Results of exome sequencing
B
A
Gene A1Intergenic noncoding
sequence
Gene B1
Exon Intron
Exon IntronIntron
Imag
e an
alys
isImaging
Denaturation
Denaturation
Sequence reads
Referencesequence
Exon Exon
Reverse strandFragment 1
Fragment 2 Fragment 3Fragment 1
DNA linkers
Fragment 1
Fragment 2
Fragment 3
Fragment 1
Fragment 2
Fragment 3
Intron
DNA
Exon Intron Exon ExonExonIntron
D
E
F
Replicationand
Amplification
T
A
GT
CC
G
G
C
T
A
G
sequence
Fragmentation
Fragment 3
Replication and Amplification of Exon Fragments
DNA Sequencing
Analysis
Fragment 3
Artificial DNA Linkers Added to End of Each Fragment, the Fragments Denatured, and Coding Sequences Selected
Patient sequence
Sequence reads
Reference sequence
Fragment 1Forward strand
Noncoding sequencesdiscarded
Primer
Primers
Base pairs
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Figure 1 (facing page). Schematic Overview of Exome Sequencing.
Exome sequencing targets the approximately 1% of the genome that is made up of exons, which encode protein sequence. The DNA from the patient (Panel A) is isolated and broken into fragments (Panel B); the DNA fragments are coupled to artificial DNA linker segments (Panel C), and the fragments are selected with the use of artificial DNA or RNA baits that are complementary to targeted DNA (not shown). The sequencing process starts with the binding of the end of each DNA fragment to a solid matrix and in situ amplification (Panel D), and the DNA frag-ments are then sequenced on the slide in a series of reactions in which a complementary nucleotide with one of four colored fluorescent dyes is added to each cluster of identical molecules (Panel E). The identity of the colored fluorescent indicator of each cluster is imaged with a laser and a camera coupled to a microscope, the fluorescent indicator is removed, and the cycle is repeated to generate a nucleotide sequence read that is 75 to 150 nucleotides in length. The sequence reads are aligned to a reference DNA sequence (Panel F), and a genotype call for each position is made. In this example, most of the positions are homozygous reference sequence, but one position is called as heterozygous A/T. This figure illustrates one widely used sequencing technology, but it is not intended to endorse that technology over other methods.
A C C T T C C T C G T G G A C T G C G C C G G G G C T A G C C
A C C T T C C T C GA T G A T G G A C T G C G C C G G G GT C G A G C C A GT
Results of exome sequencing
Advantages of Genome Sequencing:
Is more sensitive and accurate than exome sequencing for detecting structural variation, such as insertions, deletions, and translocations (although both have limited sensitivity)
Includes non-exonic regulatory regions, although these are usually irrelevant to mendelian variation
Identifies common variants in intronic and intergenic regions (most common complex-disease risk variants and some pharmacogenetic variants)
Advantages of Exome Sequencing:
Provides higher coverage of exons
Costs less
Is currently offered by more laboratories
Exon IntronIntron
Sequence reads
Referencesequence
A
Patient sequence
Patient sequence
Sequence reads
Reference sequence
A CG A C T T C C T C G T G G A C T G C G C C G G G G C T A G C C T A G
A C C T T C C T C GA T G AA C C T A C A G T G G A C T G C G C C G G G GT C G A G C C A GT G T A A G T C C
A C C T A C A G G T A A G T C C
Results of genome sequencing
Exon IntronIntron
Sequence reads
Referencesequence
B
A CC T A G A T G T A A G T C C
Sequence reads
Reference sequence
Analysis of Exome Sequencing
Analysis of Genome Sequencing
AT
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Figure 2. Schematic Comparison of Exome and Genome Sequencing.
Panel A shows the targeted nature of exome sequencing, with sequence reads concentrated over the coding portions of genes. This is in contrast to genome sequencing, shown in Panel B, in which the sequence reads are nearly ran-domly distributed over the entire genome. Each approach has advantages over the other, some of which are listed in the two panels.
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lished syndrome that could narrow the focus of genetic testing before CGES is undertaken. If CGES is performed, it may lead to the discovery of a known pathogenic variant or a previously undescribed and apparently pathogenic variant in a gene known to cause human disease. Some laboratories may also report novel, apparently pathogenic variants in genes not yet known to cause human disease. In such cases, additional clinical and laboratory research may be necessary to establish the pathogenicity of the variants.
The strategy for selecting family members to undergo the sequencing and analysis will be influenced by the expected inheritance patterns (e.g., dominant or recessive) and whether other family members with and without similar fea-tures are available for phenotyping and genetic testing. In some scenarios, it can be important to test unaffected relatives (Table 3). When a de novo variant is suspected, the relatives most commonly tested are the biologic parents of the patient; if neither parent has the variant and bio-logic parentage is confirmed, then the variant is indeed de novo.12 When a recessively or domi-nantly inherited condition is suspected, parents, siblings, or even distant relatives may be tested, depending on the particular family history and with the understanding that if two family mem-bers are more distant, the number of potential candidate variants will be reduced. Thus, sequenc-ing in two affected cousins will yield fewer candi-
date variants than sequencing in two affected siblings. In other situations, CGES performed in only the affected child, followed by genotyping of just a few variants in affected and unaffected relatives, may show cosegregation of the variant and the disease, which supports the pathogenicity of the variant. Some CGES laboratories can assist by offering advice about the testing strategy for a given clinical scenario, but this is no substitute for a robust understanding of these issues.
E va luating a CGES R esult
The outcomes of CGES analysis vary widely. In some CGES reports, a single causative variant is asserted to be the likely cause of the disease, whereas in others, multiple candidate variants are identified that must then be evaluated by the ordering clinician or by consultants. In many cases, no plausible variants are identified.
The two main considerations for evaluating CGES results are their analytic validity and their clinical validity. Analytic validity is a measure of the likelihood that the patient actually has the particular genotype shown in the CGES results — that is, the accuracy of the test. Clinical valid-ity, which is much more complicated and chal-lenging to assess, is the determination that a particular disease is truly caused by variants in a particular gene and that the specific variant that has been detected is indeed pathogenic.
Positive CGES findings are highly accurate, but the false negative rate varies according to the genomic region. For this reason, CGES is not yet a substitute for targeted sequencing of suspected genes or gene panels that have been optimized for a particular condition.13 Most laboratories vali-date positive CGES results with well-established methods such as polymerase-chain-reaction am-plification and Sanger sequencing. Confirmatory Sanger sequencing should be considered when any major medical intervention is being contem-plated on the basis of a CGES result, if such confirmation is not routinely provided by the CGES laboratory.
As noted above, determining the clinical va-lidity of CGES results is more challenging than determining their analytic validity. The general approach is to compare variants implicated by CGES with databases of known variation, which are in turn based on reports that describe causal variants as well as associations between variants
Table 1. DNA Variant Types Currently Not Well Detected or Undetectable by Clinical Genome and Exome Sequencing (CGES), with Examples of Phenotypes Associated with Such Variants.
Variant Type Associated Phenotype or Phenotypes
Repetitive DNA, including tri-nucleotide repeats
Fragile X syndrome, Huntington’s disease
Copy-number variants DiGeorge syndrome (22q11.2 deletion syndrome), Charcot–Marie–Tooth disease type 1A
Long insertion–deletion variants* Resistance to human immunodeficien-cy virus infection
Structural variants Chromosomal translocations associat-ed with spontaneous abortions
Aneuploidy Down’s syndrome, Turner’s syndrome
Epigenetic alterations Prader–Willi syndrome, Beckwith–Wiedemann syndrome
* There is a spectrum of genomic variants, from nucleotide insertions and deletions of at least 8 to 10 bp through copy-number variants, that are less effectively assayed by current CGES technology.
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Table 2. Examples of Online Databases to Assist Clinicians in Differential Diagnosis or Candidate-Gene Identification for Rare Syndromic Disorders before CGES Is Performed.
Free access (or an available free-access version)
Genetic Testing Registry (www.ncbi.nlm.nih.gov/gtr)
HuGE Navigator (http://hugenavigator.net/HuGENavigator)
Human Gene Mutation Database (www.biobase -international.com/product/hgmd)
Online Mendelian Inheritance in Man (www.omim.org)
Phenomizer (http://compbio.charite.de/phenomizer)
SimulConsult (www.simulconsult.com)
Subscription or fee required for access
Isabel (www.isabelhealthcare.com/home/default)
London Medical Databases (http://lmdatabases.com)
POSSUM (www.possum.net.au)
and phenotypes.14 In the literature, however, there are many false attributions of disease to variants, a problem that is in part due to the conflation of association with causation.15 Clini-cians reviewing the results of sequencing should be aware of the possibility of a false attribution of pathogenicity to a variant16 and should realize that the chances of false attribution are in-creased in CGES because thousands of genes are tested simultaneously.
The clinical usefulness of identifying the variant that is the cause of a previously undiag-nosed syndrome or heritable disorder varies. In some cases, it can lead to a specific treatment or management strategy that dramatically changes the clinical outcome.17,18 In the majority of cases in which the finding does not change clinical management, treatment, or prognosis, it may still be useful because it can end an expensive, poten-tially invasive, and stressful diagnostic odyssey. The identification of the causative variant may provide accurate estimates of recurrence risk and facilitate preconception intervention or pre-natal diagnosis for the affected patient or affected or at-risk relatives. In adult-onset disease, one of the most useful outcomes of successfully identi-fying the causative variant is the subsequent detection of presymptomatic, at-risk siblings for whom screening or preventive therapy might improve the clinical outcome. Examples include enhanced surveillance or prophylactic surgery for patients found to have a genetic susceptibil-ity to cancer.
Pretest counseling is particularly important, to maintain realistic expectations for finding the causative variant and to alert the patient or fam-ily that in most cases, a positive result is un-likely to change treatment or management deci-sions or to improve the prognosis.19 In addition, the patient should be advised that incidental findings unrelated to the reason for testing may be found and reported, as described below. It may also be important to discuss the cost of the test with the patient. As is the case with many medical services, assessment of the cost is com-plicated by many factors. The published billing charge for CGES in most laboratories is in the range of $4,000 to $15,000 per patient, with some laboratories offering lower per-person charges for family testing. To put this in per-spective, the per-person charge for sequencing of an exome may be only two to four times the
published billing charge for some single-gene sequencing tests, which is why exome sequenc-ing can be more efficient in a number of clinical scenarios. Some laboratories have reported that third-party payers are reimbursing for this test-ing, but practices vary widely, and patients should understand this in advance.
In ter pr e ting a nd Communic ating CGES R esult s
Clinicians should review the CGES results deliv-ered by the laboratory geneticist and place the findings into context with other relevant medical considerations. Sometimes an identified variant will spur additional history taking or an addi-tional examination of the patient, which may re-veal clinical features of a previously unrecog-nized syndrome or lead to the conclusion that the variant is not related to the disorder in the patient (Table 4).
In some cases, the CGES report from the test-ing laboratory identifies a causative variant (or two variants for a recessive disorder) in a single gene that is considered sufficiently pathogenic and specific that a diagnostic association with a heritable disorder is strongly supported. Such a conclusion by the laboratory geneticist is typi-cally based on the integration of the submitted clinical information with information on dis-eases associated with the identified variant.5 In this case, as with all laboratory tests, the order-
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ing clinician should confirm that this conclu-sion is clinically appropriate before making the diagnosis by reviewing available data from trust-ed medical sources (e.g., PubMed, GeneReviews, or textbooks). The patient — and, with permis-sion, the patient’s family — should be provided with information on the inheritance, penetrance, expressivity, prognosis, and in some cases spe-cific treatment or management for the condi-tion, either by the ordering clinician or through
referral to a specialist, depending on the nature of the diagnosed disorder.
In other cases, the laboratory CGES report identifies one or more candidate variants that may or may not be the cause of the phenotype that triggered ordering of the test. Additional literature and database searching, additional phenotyping, and even functional studies may be needed to winnow these candidates down to the causative variant, if indeed it is among these candidates. This approach to post-test clinical evaluation21 requires a high level of expertise in genetics and informatics, as well as knowledge of the clinical features of the phenotype in the patient and of the phenotypes associated with the identified variants and genes. The details of this type of evaluation are beyond the scope of this review, but such evaluations may require consultation with geneticists or other disease-specific experts and the enrollment of patients and their families in studies.
If a CGES result is negative, subsequent im-provements in knowledge may lead to the recog-nition that a previously uninterpretable variant in a negative CGES result is in fact pathogenic. The methods and approaches for ongoing re-analysis of CGES results have not been estab-lished, but it should eventually be possible to regularly reanalyze such results with the goal of identifying previously unknown variants. How this will be done and how it will be reimbursed are not yet known. These issues aside, counsel-ing a family about a negative CGES result can be challenging, because the patient may be disap-pointed with an inconclusive outcome of such an extensive and expensive test. It is important to communicate to the patient or to the parents of a tested child that a negative result of CGES test-ing does not reliably rule out the presence of a causative variant.
Inciden ta l Findings
CGES can generate results unrelated to the indi-cation that prompted sequencing, and these re-sults may be clinically useful.19,22 As with all testing, it is important to constrain such evalua-tions to avoid unnecessary future testing and ex-pense. To this end, the American College of Medical Genetics and Genomics has recom-mended that the laboratories providing CGES routinely seek and report to the ordering clini-cian specific variants in a minimum set of 56
Table 4. Examples of How to Evaluate the Pathogenicity of a Variant.
Variant supported as pathogenic
A CGES report for a boy with apparently isolated intellectual disability identi-fied a novel, previously unidentified variant in the gene ATRX. This gene is associated with the α-thalassemia mental retardation syndrome. In re-sponse to this report, the clinician orders hematologic testing, which identifies a subtle thalassemia phenotype. This additional testing strong-ly supports the variant identified in the CGES result as the cause of the intellectual disability in the patient.
Variant not supported as pathogenic
A CGES report identified a variant in the gene NF2 in a 2-month-old infant with congenital, bilateral sensorineural hearing loss. The variant is pres-ent in the Human Gene Mutation Database as a disease-causing variant associated with neurofibromatosis 2. However, that database entry is based on a single report20 that did not specify whether the patient with the variant was a case patient, a patient with a suspected case, or a control. A review of neurofibromatosis 2 in GeneReviews (www.ncbi.nlm.nih.gov/books/NBK1201/) shows that this disorder typically causes unilateral hearing loss with an onset in young adulthood, not bilateral deafness with an on-set in infancy. This post-hoc assessment — the absence of support in the literature for causality of the variant combined with data from GeneReviews on the clinical features known to be caused by mutations in NF2 — sug-gests that the evidence for the pathogenicity of this variant is weak, and the variant is unlikely to explain the child’s phenotype.
Table 3. Genetic Considerations in Deciding Which Relatives Should Undergo CGES.
Autosomal dominant inheritance suspected
Testing is most valuable in the trio consisting of a child and both biologic parents if the disorder may be caused by a de novo variant of a gene as-sociated with a disorder that is caused by heterozygous (mono allelic) mutations.
If a phenotype with autosomal dominant inheritance within a family is being evaluated, sequences from two distant relatives with the phenotype will be more valuable than sequences from two close relatives with the phe-notype. Sequences from distant relatives are recommended because there will be fewer variants shared solely by chance in close relatives.
Autosomal recessive inheritance suspected
Testing can also be valuable in the trio of a child and both biologic parents if gene variants inherited in an autosomal recessive pattern are being con-sidered.
If there is consanguinity, then testing the trio may be less helpful, because the child’s sequence will already have large areas of homozygosity.
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genes representing 24 disorders that are highly medically actionable.22 It is estimated that 1 to 3% of patients undergoing CGES have such a finding,23 which may be outside the expertise of the clinician who ordered the test. A clinician with expertise in the identified disorder should review the personal and family history for other manifestations of the disorder. Such a result may necessitate referral for proper counseling and medical guidance. In cases in which the personal or family history may suggest a specific genetic disease but no etiologic or likely etiologic variant is found, it is essential to communicate to the patient that CGES is not a comprehensive test for all disease-associated variants and that an ab-sence of incidental findings does not mean that specific, indicated testing is unnecessary. Some laboratories routinely perform incidental-finding analysis, and others offer an opt-out for such an analysis; in either case, the ordering clinician should be aware of this issue and should obtain consent and counsel patients appropriately.
Summ a r y
CGES is a useful diagnostic test for a number of clinical situations, and it is already being used by clinical geneticists and other specialists. The in-dications and approaches we outline here are sure to evolve over time, as more data are gener-ated for various clinical disorders, data interpre-tation is improved, and CGES is studied in new clinical situations (e.g., in neonatal medicine). Clinicians can feel confident ordering this test if they become comfortable with the evaluations, both before and after testing, and the processes that are required to maximize its usefulness, and if they are familiar with its limitations.
The opinions expressed in this article reflect the views of the authors and may not represent the opinions or views of any in-stitutions with which they are affiliated.
Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.
We thank the members of the Clinical Sequencing Explor-atory Research Consortium, for valuable ideas and discussion on this topic, and Shamil Sunyaev, Ph.D., for helpful suggestions.
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