High Diagnostic Yield of Whole Exome Sequencing in Participants With Retinal Dystrophies in a Clinical Ophthalmology Setting

High Diagnostic Yield of Whole Exome Sequencing in Participants With Retinal Dystrophies in a Clinical Ophthalmology Setting KRISTY LEE, JONATHAN S. BERG, LAURA MILKO, KRISTY CROOKS, MEI LU, CHRIS BIZON, PHILLIPS OWEN, KIRK C. WILHELMSEN, KAREN E. WECK, JAMES P. EVANS, AND SEEMA GARG
PURPOSE:

To assess the diagnostic yield and the practicality of implementing whole exome sequencing within a clinical ophthalmology setting.
DESIGN: Evaluation of a diagnostic protocol.
METHODS: SETTING: Patient participants were enrolled during clinical appointments in a university-based ophthalmic genetics clinic. PATIENT POPULATION: Twenty-six patients with a variety of presumed hereditary retinal dystrophies. INTERVENTION: Participants were offered whole exome sequencing in addition to clinically available sequencing gene panels between July 2012 and January 2013 to determine the molecular etiology of their retinal dystrophy. MAIN OUTCOME MEASURES: Diagnostic yield and acceptability of whole exome sequencing in patients with retinal disorders.
RESULTS: Twenty-six of 29 eligible patients (w90%) who were approached opted to undergo molecular testing. Each participant chose whole exome sequencing in addition to, or in lieu of, clinically available sequencing gene panels. Time to obtain informed consent was manageable in the clinical context. Whole exome sequencing successfully identified known pathogenic mutations or suspected deleterious variants in 57.7% of participants. Additionally, 1 participant had 2 autosomal dominant medically actionable incidental findings (unrelated to retinopathy) that were reported to enable the participant to take preventive action and reduce risk for future disease.
CONCLUSIONS: In this study, we identified the molecular etiology for more than half of all participants. Additionally, we found that participants were widely accepting of whole exome sequencing and the possibility of being informed about medically actionable incidental findings. (Am J Ophthalmol 2015;-:-–-. Ó 2015 by Elsevier Inc. All rights reserved.)

Supplemental Material available at AJO.com. Accepted for publication Apr 15, 2015. From the Departments of Genetics (K.L., J.S.B., L.M., K.C.W., J.P.E.), Pathology & Laboratory Medicine (K.C., M.L., K.E.W.), and Ophthalmology (S.G.), and The Renaissance Computing Institute (C.B., P.O., K.C.W.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. Inquiries to Kristy Lee, University of North Carolina at Chapel Hill, CB# 7462, Chapel Hill, NC 25799; e-mail: [email protected] 0002-9394/$36.00 http://dx.doi.org/10.1016/j.ajo.2015.04.026

Ó

2015 BY

T

HE PACE OF PROGRESS IN OPHTHALMIC GENETICS

has been exponential over the last decade. It is critical for ophthalmologists to understand emerging diagnostic technologies that may have clinical implications for their patients in the very near future. Whole exome sequencing (exome sequencing) and massively parallel sequencing gene panels are attractive new testing approaches for diagnosing genetic disorders that exhibit genetic heterogeneity and overlapping phenotypes. Few Mendelian disorders exhibit the degree of genetic heterogeneity demonstrated by retinitis pigmentosa (RP), one of the most common retinal dystrophies.1,2 Over 100 genes have been associated with this condition, yet only half of all patients with RP have an identifiable mutation.3,4 Moreover, other retinal dystrophies, including cone-rod dystrophy, cone dystrophy, and Stargardt disease, also exhibit genetic heterogeneity.3,5 Further complicating the clinical assessment of these disorders is the fact that retinal disorders also demonstrate significant phenotypic heterogeneity. For instance, mutations in the ABCA4 gene have been associated with several hereditary retinal dystrophies (Stargardt disease, cone-rod dystrophy, cone dystrophy, and RP).1 Prior to the advent of massively parallel sequencing, genetic testing for heterogeneous disorders was pursued 1 gene at a time or through limited and expensive gene panels via Sanger sequencing. The benefit of using a broader testing methodology in such circumstances is the potential to eliminate the guesswork inherent in choosing only a subset of genes to test. Another advantage is that many participants seen in ophthalmic genetics clinics report no family history of retinal dystrophy, complicating determinations of inheritance patterns that might otherwise guide diagnostic strategies. Since retinal dystrophies are thought to be almost exclusively hereditary in nature, one can assume that there are yet-unidentified genes associated with RP and other retinal dystrophies.2 Exome sequencing allows the clinician the flexibility of ordering a single test for all suspected heterogeneous disorders and allows the laboratory the flexibility to analyze newly reported genes without continuously updating testing platforms. A potential complication of exome sequencing testing vs targeted massively parallel sequencing gene panels is the prospect of patients receiving incidental findings unrelated

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to the retinal diagnosis. That is, when essentially all genes in an individual’s genome are sequenced, information will be potentially available regarding other genetic disorders unrelated to the indication for testing. The American College of Medical Genetics (ACMG) recommends that laboratories return selected medically actionable incidental findings as part of any genome-scale clinical test. Thus, a small but predictable subset of patients will have such additional findings.6 It is uncertain how patients might react to this possibility in the clinical setting; therefore, additional time is required to discuss the likelihood and examples of incidental findings as part of the informed consent process. The goals of this study were to investigate the use of exome sequencing to identify the molecular etiology of retinal dystrophies in a clinical ophthalmology setting and to determine the feasibility of using this novel and complex form of genetic testing, with regard to the potential discovery of incidental findings. Previous studies have shown the effectiveness of exome sequencing and targeted gene panels in determining the molecular etiology of retinal dystrophies.7–10 Here we demonstrate its high diagnostic yield, feasibility, and acceptability of exome sequencing for retinal dystrophy patients enrolled in a clinical setting.

SUBJECTS AND METHODS PATIENTS EVALUATED FOR RETINAL DISORDERS IN THE UNI-

versity of North Carolina Kittner Eye Center Ophthalmic Genetics Clinic between July 2012 and January 2013 were invited to participate. Participants were enrolled in the research protocol to undergo research genetic testing during their initial or follow-up clinical visits. Return patients were eligible if the molecular etiology of their retinal disorder was unknown. All potential participants were offered clinically available massively parallel sequencing targeted gene panel testing and research exome sequencing through this study. The University of North Carolina at Chapel Hill Institutional Review Board approval was obtained prior to patient enrollment, and this study adhered to the tenets of the Declaration of Helsinki. All participants were enrolled and consented by a certified genetic counselor and agreed to learn of any diagnostic-related findings as well as any medically actionable incidental findings. Known pathogenic mutations and variants of unknown clinical significance that could potentially explain their retinal disease were returned to participants. However, only clearly pathogenic medically actionable incidental findings were returned. Thus, variants of unknown significance within genes associated with medically actionable findings were not returned to participants given their uncertainty and low a priori risk of being pathogenic in presumably unaffected individuals. The list of conditions in the category of medically

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actionable incidental findings was based on a schema previously described by our group and further refined by a committee of medical and molecular geneticists, genetic counselors, a neurologist, a cardiologist, and an ethicist as part of the NCGENES Study currently being conducted at University of North Carolina at Chapel Hill.11,12 This list included all conditions recently recommended by the American College of Medical Genetics for return of incidental findings.6 Exome sequencing was performed using Agilent’s SureSelect XT Target Enrichment System (Agilent Technologies, Santa Clara, California) for Illumina (Illumina, Inc., San Diego, California) paired-end sequencing on the HiSeq 2000 instrument. The average depth of coverage for all participants across the entire region targeted for enrichment was 58.19. We used a custom pipeline developed for the NCGENES project to process raw sequence data from FASTQ files to generate variant calls.13 Variants were stored in a database and extensively annotated.14 To facilitate evaluation of variants possibly related to the participants’ retinal disorder, we filtered the exome data using a comprehensive list of 186 genes previously associated with syndromic and nonsyndromic retinopathies, which was curated using Online Mendelian Inheritance of Man (OMIM),15 GeneTests.org,16 relevant medical literature, and genes currently being evaluated in clinical laboratories. Participants’ exome data were reanalyzed using an updated gene list, including 214 genes, 1 year later. A complete list of these genes is available in Supplemental Table 1 (Supplemental Material available at AJO.com). Variants within this set of genes were then prioritized into computational classes by an informatics algorithm to select: (1) variants previously reported as mutations in the Human Gene Mutation Database17; (2) predicted truncating variants that demonstrated <1% minor allele frequency; (3) missense variants with <1% minor allele frequency; and several other categories with decreasingly likely pathogenicity. Variants were then analyzed for pathogenicity using a custom user interface.18 The total number of exome and filtered variants for each participant is available in Supplemental Table 2 (Supplemental Material available at AJO.com). Manual analysis of filtered variants entailed a combination of literature searches, publicly available variant databases queries, locus-specific database searches, Condel in silico modeling, and evolutionary conservation.19 The veracity of potential disease-causing variants identified by exome sequencing and passing manual curation were confirmed by Sanger sequencing on a duplicate sample in the CLIA-certified University of North Carolina McLendon Molecular Genetics Laboratory. All participants were asked to return for a follow-up research appointment to discuss results and were provided with a research report summarizing the yield of the exome sequencing analysis. Participants were not given the option of learning about non–medically actionable incidental results.

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TABLE 1. Summary of Participants’ Clinical Diagnoses and Presentation, Family History, and Previous Record of Genetic Testing Participant

Age at Enrollment (y)

Clinical Diagnosis

Age of Onset

Family History

Previous Testing Results

1 2 3

34 60 9

N/A N/A N/A

46 48 60 26 43 23 41 7

Early teens 20s Hearing loss - congenital Retinal dystrophy - w5 Early 20s Congenital 29 years 16 years 41 years 10 years 6 years w6 years

No No Yes - AD

4 5 6 7 8 9 10 11

No Yes - XL No Yes - AR No No No Yes - AD

N/A N/A N/A N/A N/A N/A ABCA4 c.161G>A (p.Cys54Tyr) Negative 4 adFEVR panel þ NDP gene

12 13 14 15 16

68 52 64 22 12

Retinitis pigmentosa Cone dystrophy Retinal dystrophy NOS þ hearing loss Cone-rod dystrophy Cone dystrophy Retinitis pigmentosa Stargardt disease Retinal dystrophy NOS Macular dystrophy Stargardt disease Familial exudative vitreoretinopathy Retinitis pigmentosa Cone-rod dystrophy Retinitis pigmentosa Retinitis pigmentosa Retinal dystrophy NOS

w62 years w42 years 34 years 15 years 11 years

No No Yes - AR No No

17 18

48 23

Retinitis pigmentosa Usher syndrome type 2

No Yes - AR

19 20 21

36 30 26

No Yes - AD Yes - AD

N/A RP1 c.2029C>T (p.R677X) N/A

22

29

Retinitis pigmentosa Retinitis pigmentosa Leber congenital amaurosis Retinitis pigmentosa

w38 years Hearing loss – 1 year retinitis pigmentosa – 13 years 35 years 30 years Congenital

N/A N/A N/A N/A Negative 13 recessive retinitis pigmentosa gene panel N/A N/A

15 years

Yes - AD

23 24 25 26

9 54 11 44

Retinitis pigmentosa Stargardt disease Retinitis pigmentosa Retinitis pigmentosa

6 years Mid-30s 9 years w42 years

Yes - AD Yes - AR Yes - XL No

Negative RHO, RDS, RP1 (c.1500-3200), PRPF31, PRPF8 (exon 42), PRPF3 (exon11), NR2E3 (c.150-210), TOPORS (c.1975-2820), IMPDH1 (exon 10), RP2, RPGR, SNRNP200 N/A N/A RPGR c.2323_2324delGA (p.Arg775fs) N/A

AD ¼ autosomal dominant; AR ¼ autosomal recessive; N/A ¼ not applicable; NOS ¼ not otherwise specified; XL ¼ X-linked.

RESULTS DURING THE ENROLLMENT PERIOD, 29 PATIENTS WERE

eligible (15 new and 14 return clinic patients). Twenty-six of the 29 patients (w90%) opted to undergo clinically available testing and/or research exome sequencing. Three new patients declined both clinical and research testing. All 26 patients who wished to proceed with genetic testing chose exome sequencing, and 3 of the new patients (Participants 11, 20, and 25) opted to have simultaneous clinically available genetic testing. Participant 11 had negative clinical testing and Participants 20 and 25 had pathogenic variants identified that were also detected via exome sequencing. The informed consent process for return patients opting for exome sequencing took 30 minutes or less. New patient

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appointments lasted 75 minutes, and the informed consent process took approximately 30 minutes. All questions about exome sequencing were addressed, and none of the participants shared any concerns regarding the possibility of learning medically actionable incidental findings. Participants represented a wide variety of ages and clinical indications, outlined in Table 1. Five of 26 participants (w19%) were under 18 years of age, and the adults ranged in age from 22 to 69 years. The majority of participants had a firm clinical diagnosis; however, 3 of 26 participants (w11%) had an uncertain clinical diagnosis at the time of enrollment. Fourteen of 26 participants (w54%) did not have a known family history of retinal dystrophy. Fifteen of 26 participants (w58%) had clearly deleterious mutations or highly suspicious variants of unknown

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4 TABLE 2. Summary of Whole Exome Sequencing Results for Each Participant and Descriptions of Previously Reported or Possibly Causative Variants Identified Within Genes Associated With Retinal Disease, Along With the Interpretation Provided to Participants Participant

Exome sequencing Diagnostic Results

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Minor Allele Frequencya

Conservation

Condel Scoreb

Interpretation

34

8.263e-06

Highly conserved

0.698 Deleterious

Known pathogenic

0.853 Deleterious 1.000 Deleterious 0.451 Deleterious 0.000 Neutral — —

Likely benign

1.000 Deleterious 0.619 Deleterious 0.007 Neutral

Known pathogenic

0.026 Neutral 0.922 Deleterious 0.026 Neutral 0.810 Deleterious

Uncertain variant

Previous Reports

1

NC_000003.11:g.129252554C>T

RHO c.1040C>T (p.Pro347Leu)

Pathogenic

2 3

Negative NC_000019.9:g.48343048G>A

CRX c.724G>A (p.Val242Met)c

Pathogenic38

0.001435

Not conserved

4

NC_000001.10:g.94528806A>G

ABCA4 c.1622T>C (p.Leu541Pro)

Pathogenic23

0.0001235

Highly conserved

NC_000001.10:g.94528265C>T

ABCA4 c.1805G>A (p.Arg602Gln)

Pathogenic23,24

1.68e-05

Highly conserved

NC_000001.10:g.94508969G>A

ABCA4 c.3113C>T (p.Ala1038Val)

0.001426

Mostly conserved

NC_000013.10:g.32911349G>T NC_000002.11:g.48026292_ 48026292delT Negative Negative NC_000001.10:g.94466624C>T

BRCA2 c.2857G>T (p.Glu593*) MSH6 c.1170delT (p.Phe391fs)

Polymorphic21 Pathogenic22 None None

0 0

— —

ABCA4 c.6320G>A (p.Arg2107His)

Pathogenic24

0.001872

Highly conserved

NC_000001.10:g.94496666G>A

ABCA4 c.4139C>T (p.Pro1380Leu)

Pathogenic24,32

0.0002012

Highly conserved

NC_000001.10:g.94520708A>G

ABCA4 c.2546T>C (p.Val849Ala)

Pathogenic24

0.001335

Not conserved

Negative NC_000007.13:g.33380565C>G

BBS9 c.1255C>G (p.Pro419Ala)

None

9.078e-05

Mostly conserved

NC_000007.13:g.33545217A>T

BBS9 c.2138A>T (p.Glu713Val)

None

0

Mostly conserved

NC_000001.10:g.94577135C>T

ABCA4 c.161G>A (p.Cys54Tyr)

Pathogenic20,24,32

1.65e-05

Mostly conserved

NC_000004.11:g.16008270C>T

PROM1 c.1345G>A (p.Val449Met)

None

0.001475

Mostly conserved

Negative NC_000001.10:g.216108128T>C

USH2A c.7130A>G (p.Asn2377Ser)

0.003772

Mostly conserved

NC_000001.10:g.215901511G>A

USH2A c.11927C>T (p.Thr3976Met)

VUS30 Polymorphism29 VUS28

0.0005192

Highly conserved

5 6 7

8 9

10

11 12

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0.001 Neutral 0.966 Deleterious

Known pathogenic Known pathogenic Uncertain variant Likely pathogenic Likely pathogenic

Known pathogenic Uncertain variant

Uncertain variant Uncertain variant Likely benignd

Uncertain variant Uncertain variant

Negative Continued on next page

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TABLE 2. Summary of Whole Exome Sequencing Results for Each Participant and Descriptions of Previously Reported or Possibly Causative Variants Identified Within Genes Associated With Retinal Disease, Along With the Interpretation Provided to Participants (Continued ) Participant

Exome sequencing Diagnostic Results

14 15

Negative NC_000011.9:g.66293652T>G

16

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Minor Allele Frequencya

Conservation

Condel Scoreb

Interpretation

0.627 Deleterious 0.910 Deleterious 0.930 Deleterious

Known pathogenic

Pathogenic36

0.001484

Highly conserved

NC_000014.8:g.68195946G>C

BBS1 c.1169T>G (p.Met390Arg) Homozygous RDH12 c.697G>C (p.Val233Leu)

Pathogenic33

2.491e-05

Highly conserved

NC_000014.8:g.68200483T>G

RDH12 c.869T>G (p.Val290Gly)

None

8.246e-05

Highly conserved

USH2A c.923_924insGCCa (p.His308fs)

Pathogenic8

0.0001074

—

—

Known pathogenic

USH2A c.12152_12153insTT (p.Glu4051fs)

None

0

—

—

Likely pathogenic

USH2A c.2276G>T (p.Cys759Phe)

Pathogenic28

0.000784

Highly conserved

Known pathogenic

USH2A c.12295-3T>A RP1 c.2029C>T (p.R677X)

None Pathogenic39

0

— —

1.00 Deleterious — —

MERTK c.1787-2A>C Apparently homozygouse

None

0

—

—

Likely pathogenic

PRPH2 c.457A>G (p.Lys153Glu)

None

0

Highly conserved

Uncertain variant

RPGR c.2323_2324delGA (p.Arg775fs)

Pathogenic39

0

—

0.873 Deleterious —

ABCA4 c.1927G>A (p.Val643Met)

Pathogenic22,25

0.001618

Highly conserved

0.383 Neutral

Uncertain variant

19

Negative NC_000001.10:g.216498866_ 216498867insTGGC NC_000001.10:g.215853632_ 215853633insAA NC_000001.10:g.216420460C>A

20 21 22

NC_000001.10:g.215848961A>T NC_000008.10:g.55538471C>T Negative NC_000007.13:g.128034615C>T

23 24

Negative NC_000006.11:g.42689616T>C

25

NC_000023.10:g.38145928_ 38145929delCT NC_000001.10:g.94528143C>T

26

Previous Reports

Uncertain variant Uncertain variant

Uncertain variant Known pathogenic

Known pathogenic

Exome sequencing results summarized by participant. Variants are described using Human Genome Variation Society (http://www.hgvs.org) nomenclature. Variant interpretation was primarily based on variant type (ie, predicted truncating vs missense), previous reports in the literature, and minor allele frequencies from other large studies. Truncating and canonical splice site variants required less evidence for pathogenicity than missense and intronic variants. Evolutionary conservation and in silico model data were considered as supporting evidence, but were not major factors in designating pathogenicity. a Minor allele frequencies were obtained using the Exome Aggregation Consortium (ExAC) Browser.40 b The Condel19 score is provided as a reference only and was not taken into consideration in variant interpretation. c Not confirmed by Sanger sequencing owing to variant being likely benign and not reported back to participant. d Parental studies by Sanger sequencing identified both the ABCA4 and PROM1 variants in the unaffected father, suggesting that the PROM1 variant was likely benign. e An ad hoc analysis examining coverage data suggests that this patient has a partial deletion of the MERTK gene in lieu of being homozygous for the splice variant. The participant’s parents are deceased. Therefore, we are unable to perform parental studies.

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significance consistent with their phenotype. A complete summary of previous genetic testing and exome sequencing results for each participant is shown in Table 2. Each of these variants was confirmed using Sanger sequencing. Participant 4 had 2 different medically actionable incidental findings: an apparently novel nonsense variant in BRCA2, c.2857G>T (p.Glu593*), which is associated with hereditary breast ovarian cancer syndrome; and an apparently novel frameshift variant in MSH6, c.1170delT (p.Phe391fs), which is associated with Lynch syndrome. Both truncating variants were considered likely pathogenic, given their suspected effect on the protein. When questioned further about family history of cancer at results disclosure, he reported that his paternal grandfather had a history of colon cancer at an older age (Figure).

DISCUSSION THIS STUDY DEMONSTRATES THE HIGH DIAGNOSTIC YIELD,

feasibility, and acceptability of exome sequencing for retinal dystrophy patients in the clinical ophthalmology setting. In our study, exome sequencing was widely accepted by a heterogeneous clinic population, with w90% of those approached choosing to participate. Patients who chose not to participate also declined clinically available genetic testing. The informed consent process for exome sequencing was manageable in a clinic setting. Several participants were not naı¨ve to basic genetic information, which allowed for a brief review and then focused discussion on the differences between traditional genetic testing and exome sequencing along with possible results they might receive. The discussion was lengthier than for traditional clinical testing, since it was necessary to explain the nature of the genetic testing as part of a research study. During the consent process, none of the participants expressed concern about the possibility of learning medically actionable incidental findings. However, several participants requested examples of possible results to conceptualize what a medically actionable incidental finding might entail. Several participants expressed that learning this type of information would allow them to be proactive with their health care in the future. Just over half of the participants were found to have a definitive or possible molecular etiology defined as the cause of their retinal dystrophy, consistent with reports using massively parallel sequencing gene panels in a research setting.7–10 Previously reported pathogenic variants were identified in several participants, which aided interpretation of exome sequencing results. Novel variants were interpreted as likely pathogenic, if they were expected to result in a truncated protein. Novel missense variants, which are not expected to truncate proteins, and variants within genes that were not a 6

FIGURE. Selected pedigrees of participants with a variety of retinal dystrophies evaluated by whole exome sequencing. (Top left) Participant 4 with BRCA2 and MSH2 mutations with only paternal grandfather with a history of cancer. (Top right) Participant 24 with PRPH2 mutation with 2 sisters with Stargardt disease and mother with diagnosis of agerelated macular degeneration. (Bottom right) Participant 25 with RPGR mutation with family history consistent with X-linked inheritance.

definitive fit for the participant’s phenotype were more difficult to assess, and were reported back to the participants as variants of unknown significance. Participants 4 and 7 each had three ABCA4 gene variants identified with potential clinical significance. Each participant had 2 known pathogenic mutations and 1 variant that had been previously reported as a pathogenic mutation and as a variant of unknown significance.20–24 Since exome sequencing analysis cannot determine the phase of the 3 variants, these results are consistent with autosomal recessive disease provided that at least 1 pathogenic variant is on each allele. Participant 26 had only 1 known previously reported pathogenic variant identified in the ABCA4 gene, c.1927G>A (p.Val643Met).20,25 Since ABCA4 mutations are associated with autosomal recessive RP, the identification of 1 mutation does not fully explain the participant’s diagnosis. A current known limitation of exome sequencing is the inability to identify larger deletions and duplications and possibly smaller indels.26,27 Therefore, it is possible that this participant has a mutation on the other ABCA4 allele that was not detected by this methodology. Alternatively, he could be a carrier for this form of RP and have an unrelated molecular cause of his RP, or the variant could be erroneously classified as a pathogenic variant. We classified this variant as having uncertain significance owing to its minor allele frequency. Mutations within the USH2A gene are typically associated with Usher syndrome or autosomal recessive

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nonsyndromic RP. Participant 19 with RP had 1 known pathogenic mutation, c.2276G>T (p.Cys759Phe),28 and 1 possible splice site variant, c.12295-3T>A, previously reported as a variant of unknown significance.29 However, a similar noncanonical splicing variant, USH2A c.75953C>G, has been described as pathogenic.28 Participant 12 with late-onset RP had 2 missense variants of unknown significance, c.7130A>G (p.Asn2377Ser) and c.11927C>T (p.Thr3976Met), which have both previously been reported.28–30 Participant 25 had an extensive X-linked family history of RP (Figure). A known pathogenic mutation in the RPGR gene, c.2323_2324delGA (p.Arg775fs), was identified.31 After enrollment of this patient, eyeGENE (program through the National Eye Institute) also confirmed this exact mutation by Sanger sequencing. Approximately one third of patients with autosomal recessive Stargardt disease have only 1 identifiable mutation in the ABCA4 gene.23,32 Participant 10 previously had testing that identified a single known pathogenic mutation in the ABCA4 gene, c.161G>A (p.Cys54Tyr).32 Exome sequencing identified the known ABCA4 mutation and revealed a variant of unknown significance in the PROM1 gene, c.1345G>A (p.Val449Met), which has been associated with autosomal dominant Stargardt disease. To our knowledge, this variant has not previously been reported. It is highly evolutionarily conserved and was predicted as deleterious by Condel, supporting its pathogenicity.19 However, parental samples were collected after results disclosure and revealed that both the ABCA4 and PROM1 variants were inherited from the participant’s unaffected father, making the pathogenicity of the PROM1 variant unlikely. Participant 18 had 2 apparently novel frameshift mutations in USH2A, c.923_924insGCCA (p.His308fs) and c.12152_12153insTT (p.Glu4051fs), which explained his clinical diagnosis of Usher syndrome type 2. Three participants in this cohort had an unclear clinical diagnosis at the time of enrollment, and exome sequencing helped identify a probable cause in 1. Participant 16 was an 11-year-old male subject with retinal features consistent with a rare diagnosis of pigmented paravenous chorioretinal atrophy or an RP-like syndrome. Exome sequencing identified the 2 missense variants in the RDH12 gene, c.697G>C (p.Val233Leu) and c.869T>G (p.Val290Gly). The Val233Leu variant was previously reported in an individual with retinopathy who had a frameshift mutation on the other allele.33 To our knowledge, the Val290Gly has not previously been reported. Two participants had results suggesting a different inheritance pattern than inferred by their family history. Participant 1 is a 34-year-old man with a personal history of RP and no known family history who was found to have a previously reported pathogenic variant in the RHO gene, RHO c.1040C>T (p.Pro347Leu).34 Mutations within the RHO gene are associated with autosomal dominant RP, VOL. -, NO. -

which is not consistent with this participant’s family history. However, de novo dominant mutations have been reported in the RHO gene.33,34 Participant 24 is a 55-year-old man with a personal and family history of Stargardt disease who was found to have an apparently novel heterozygous variant in the PRPH2 gene, c.457A>G (p.Lys153Glu). The PRPH2 gene is associated with autosomal dominant Stargardt disease. Two of the participant’s siblings were reported to have the same phenotype (Figure). Neither the parents nor any other relatives carry a clinical diagnosis of Stargardt disease; thus, an autosomal recessive form of this condition was expected. Interestingly, his mother reportedly was diagnosed with age-related macular degeneration (AMD). The mother also likely has this variant and has been diagnosed with AMD owing to the later onset of symptoms in this family. The mother was not available for testing. One rationale for using exome sequencing vs massively parallel sequencing gene panels is to determine the mutation(s) within a gene that typically caused a syndromic form of a retinal disease. For example, there are reports of individuals with nonsyndromic RP having mutations in at least 3 genes originally described as associated with Bardet-Biedl syndrome (BBS): BBS1, TTC8, and ARL6, an autosomal recessive disease characterized by truncal obesity, postaxial polydactyly, cognitive impairment, genitourinary malformations, renal dysfunction, and cone-rod dystrophy or RP.35–37 Participant 9 is a 22-year-old woman diagnosed initially with Stargardt disease around 10 years of age. However, when she was evaluated in the ophthalmic genetics clinic, her electroretinogram demonstrated normal cone and rod photoreceptor function, and her examination revealed bilateral macular atrophy consistent with a macular dystrophy. She was found to have 2 missense variants in the BBS9 gene, which is associated with BBS. Thus, this participant appears to have nonsyndromic cone-rod dystrophy possibly caused by BBS9 mutations. We were unable to locate a previous report linking BBS9 pathogenic variants with nonsyndromic cone-rod dystrophy; thus, this would represent an expansion of the phenotype if these variants are indeed pathogenic. Variants of unknown significance were identified in 7 participants. These participants were informed that these variants could be disease causing or simply human variation. Some variants of unknown significance are more likely to be pathogenic than others, and this information was also shared with participants. For instance, the USH2A variant of unknown significance in Participant 19 is likely pathogenic, since this individual has a second known pathogenic variant within the same gene that fits the phenotype. Alternatively, participants with 1 variant of unknown significance identified along with 2 known pathogenic variants within a gene that fits the phenotype suggests that the variant of unknown significance is less likely to be pathogenic, as in the case of Participants 4 and 7. Participants

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9, 12, and 16 had 2 variants of unknown significance within genes that fit their phenotype, which are usually inherited in an autosomal recessive fashion. These variants rose to the level of suspicion to report to participants; however, they could undoubtedly be distractions rather than disease-causing variants. Participant 26 had only 1 variant of unknown significance within a gene that fits the phenotype, which suggests that he has a deletion on the other allele that was undetected, that he is simply a carrier for that form of RP and has other disease-causing variants, or that this variant is not disease causing, contrary to previous reports in the literature. Each of these possible scenarios were discussed with participants. All participants with variants of unknown significance were offered family studies, which, to date, 1 family has accepted. Parental testing in Participant 10 allowed the downgrade of a variant of unknown significance in the PROM1 gene to a likely benign variant. If the PROM1 variant was pathogenic, we would have expected that this variant would have been de novo, given that both parents were unaffected and pathogenic variants in this gene are associated with autosomal dominant Stargardt disease. Exome sequencing, with analysis focused on genes with known associations with retinal disorders, can be considered a ‘‘virtual’’ massively parallel sequencing gene panel, significantly decreasing the number of variants to analyze. However, unlike employing an actual gene panel, exome sequencing allows flexibility to reanalyze data as new gene candidates are identified without the need to develop a new physical test dependent on gene capture. This will facilitate the identification of novel gene candidates and reanalysis of exome data as additional retinal disease genes are described in participants with as yet negative results and in participants with variant(s) of unknown significance. Thus, for the time being, a whole-exome approach is readily justifiable as a diagnosis is sought in those with likely genetic retinal pathology. However, as the pace of gene discovery plateaus, there may well come a point at which a captured panel of genes is scrutinized, given the superior analytic performance of such panels owing to increased depth of coverage and the lack of concerns about secondary findings in that context. Participant 4 had medically actionable incidental findings in both the BRCA2 and MSH6 genes. The lack of personal or significant family history was surprising and illustrates the complexity of returning medically actionable results. During results disclosure, it was emphasized that these were novel variants that have not been directly associated with disease, although most known pathogenic mutations within these 2 genes are rare or private to families. It was also important to note that previous testing of these 2 genes has largely been in individuals with a personal and/or family history of cancer. Therefore, as larger numbers of unaffected individuals are tested in the general population through exome sequencing, current estimates of penetrance rates of well-known diseases may be found to be 8

inflated. However, in the meantime, it was recommended that the participant’s relatives be tested for these presumed pathogenic mutations, in accordance with the National Comprehensive Cancer Network screening guidelines. This project was designed as a pilot study. Thus, the sample size is small; however, the yield of exome sequencing for this cohort is similar to other published studies carried out in a research setting. Glo¨ckle and associates reported a diagnostic yield of 50%–80% (depending on the phenotype) using massively parallel sequencing gene panels within an unselected set of participants with retinal dystrophies.10 Audo and associates reported a 57% detection rate in patients with a variety of retinal dystrophies using a massively parallel sequencing gene panel of 254 known and candidate genes.9 In this study, relatives were not enrolled at the onset, in order to more closely simulate a clinical ophthalmology setting. Not enrolling relatives limits the ability to determine the phase of variants identified in genes associated with autosomal recessive disorders, or to determine whether heterozygous variants in genes associated with dominant conditions are de novo vs incompletely penetrant. However, more often than not, informative family members do not attend clinic appointments with patients. In the clinical ophthalmology setting, family studies are initiated only when testing does not point to a clear causality. The same approach was used in this study. However, many informative relatives were unavailable. This is consistent with any clinic patient with genetic testing, and is a limitation that is not unique to retinal dystrophy patients or exome sequencing. The pace of progress in ophthalmic genetics has been exponential over the last decade; it is critical for ophthalmologists to understand emerging diagnostic technologies, such as exome sequencing, which may have widespread clinical applications in the very near future. With genetic therapies on the horizon, an accurate molecular diagnosis will be a prerequisite for patients seeking enrollment in clinical trials, confirmation of clinical diagnoses, and prognostic information for patients and their family members. In this study, exome sequencing was highly acceptable to the cohort and implementation was manageable in a clinical ophthalmology setting. Such broad testing proved useful in participants with a history of features overlapping among different retinal dystrophies and in participants with no clear clinical diagnosis. Using 1 test for heterogeneous disorders reduces the need to rely exclusively on family history information for characterizing an individual’s disease, and exome sequencing may increase diagnostic yield by allowing review of potentially pathogenic variants within genes that are associated with retinal disorders with somewhat different but similar phenotypes. Exome sequencing is emerging as a powerful tool in determining the molecular etiology for participants with a variety of heterogeneous retinal dystrophies, even in the clinical ophthalmology setting.

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ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST. Financial Disclosures: Jonathan Berg receives research support from the National Institutes of Health (1U19HD077632-01, 1U01-HG007437-01). Kristy Crooks is a part-time employee of Laboratory Corporation of America and ILS Genomics, Inc. Kirk C. Wilhelmsen receives research support from the National Institutes of Health (5U01HG006487-02, 1U01HG007437-01, 1U19HD077632-01, 5R01DA030976-04). Karen Weck (1U01HG00648701) receives research support from the National Institutes of Health. Dr Weck also serves as a consultant for Laboratory Corporation of America and BlueCross BlueShield of North Carolina, and serves as a lecturer for the Association for Molecular Pathology. James P. Evans receives research support from the National Institutes of Health (1U01HG006487-01). Funding/Support: This study was also supported by the Clinical Translational North Carolina Tracs grant (550KR21216), Unrestricted Grant from Research to Prevent Blindness (RPB), New York, New York to the University of North Carolina Department of Ophthalmology, Bryson Program for Human Genetics, Renaissance Computing Institute, and the National Institutes of Health (1R01DA030976-01, 1U01-HG006487-01, 5UL1-RR025747-03, 1U19-HD077632-01, 1U01-HG007437-01) and the University of North Carolina Lineberger Comprehensive Cancer Center. The authors would like to acknowledge the Wilmer Biostatistics Core Grant EY01765. All authors attest that they meet the current ICMJE requirements to qualify as authors.

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27. Schrijver I, Aziz N, Farkas DH, et al. Opportunities and challenges associated with clinical diagnostic genome sequencing: a report of the Association for Molecular Pathology. J Mol Diagn 2012;14(6):525–540. 28. Baux D, Larrieu L, Blanchet C, et al. Molecular and in silico analyses of the full-length isoform of usherin identify new pathogenic alleles in Usher type II patients. Hum Mutat 2007;28(8):781–789. 29. Le Quesne Stabej P, Saihan Z, Rangesh N, et al. Comprehensive sequence analysis of nine Usher syndrome genes in the UK National Collaborative Usher Study. J Med Genet 2012; 49(1):27–36. 30. Garcia-Garcia G, Aparisi MJ, Jaijo T, et al. Mutational screening of the USH2A gene in Spanish USH patients reveals 23 novel pathogenic mutations. Orphanet J Rare Dis 2011;6:65. 31. Breuer DK, Yashar BM, Filippova E, et al. A comprehensive mutation analysis of RP2 and RPGR in a North American cohort of families with X-linked retinitis pigmentosa. Am J Hum Genet 2002;70(6):1545–1554. 32. Lewis RA, Shroyer NF, Singh N, et al. Genotype/phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt disease. Am J Hum Genet 1999;64(2):422–434. 33. Mackay DS, Dev Borman A, Moradi P, et al. RDH12 retinopathy: novel mutations and phenotypic description. Mol Vis 2011;17:2706–2716.

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34. Booij JC, Florijn RJ, ten Brink JB, et al. Identification of mutations in the AIPL1, CRB1, GUCY2D, RPE65, and RPGRIP1 genes in patients with juvenile retinitis pigmentosa. J Med Genet 2005;42(11):e67. 35. Estrada-Cuzcano A, Koenekoop RK, Senechal A, et al. BBS1 mutations in a wide spectrum of phenotypes ranging from nonsyndromic retinitis pigmentosa to Bardet-Biedl syndrome. Arch Ophthalmol 2012;130(11): 1425–1432. 36. Riazuddin SA, Iqbal M, Wang Y, et al. A splice-site mutation in a retina-specific exon of BBS8 causes nonsyndromic retinitis pigmentosa. Am J Hum Genet 2010;86(5): 805–812. 37. Aldahmesh MA, Safieh LA, Alkuraya H, et al. Molecular characterization of retinitis pigmentosa in Saudi Arabia. Mol Vis 2009;15:2464–2469. 38. Rivolta C, Berson EL, Dryja TP. Dominant Leber congenital amaurosis, cone-rod degeneration, and retinitis pigmentosa caused by mutant versions of the transcription factor. CRX. Hum Mutat 2001;18(6):488–498. 39. Pierce EA, Quinn T, Meehan T, McGee TL, Berson EL, Dryja TP. Mutations in a gene encoding a new oxygenregulated photoreceptor protein cause dominant retinitis pigmentosa. Nat Genet 1999;22:248–254. 40. Exome Aggregation Consortium (ExAC). Available at http://exac.broadinstitute.org. Accessed March 14, 2015.

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Biosketch Kristy Lee, is a Certified Genetic Counselor and Research Associate Professor in the Department of Genetics at the University of North Carolina at Chapel Hill. Ms. Lee received her undergraduate degree from North Carolina State University and a Masters degree in Genetic Counseling from the University of North Carolina at Greensboro. Her research interests involve examining the effectiveness and implementation of whole genome/exome analyzes and novel gene discovery for retinal dystrophies.

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SUPPLEMENTAL TABLE 1. List of Genes Used for Targeted Whole Exome Sequencing Analysis to Filter Out Variants Identified Within Genes With No Known Association to Retinal Disease Gene

Location

Phenotype MIM

ABCA4

1p22.1

ABCC6

16p13.11

ABHD12 ACBD5 ADAM9 ADAMTS18 AHI1 AIPL1

20p11.21 10p12.1 8p11.22 16q23.1 6q23.3 17p13.2

248200 604116 601718 153800 248200 264800 177850 612674 Abu-Safieh1 612775 608454 608629 604393 604393 604393 203800 612291 615434 209900 613575 221900 209900 209900 209900 209900 209900 209900 209900 209900 153700 611809 193220 193220 193220 605670 Abu-Safieh1 613428 614500 614500 600852 610427

ALMS1 ARL13B ARL2BP ARL6

2p13.1 3q11.1 16q13 3q11.2

ATOH7 BBS1 BBS10 BBS12 BBS2 BBS4 BBS5 BBS7 BBS9 BEST1

10q21 11q13.2 12q21.2 4q27 16q12.2 15q24.1 2q31.1 4q27 7p14.3 11q12.3

C1QTNF5 C21orf2 C2orf71 C8orf37

11q23.3 21q22.3 2p23.2 8q22.1

CA4 CABP4

17q23.1 11q13.2

CACNA1F

Xp11.23

CACNA2D4 CAPN5 CC2D2A

12p13.33 11q13 4p15.32

CCDC28B CDH23

1p35.1 10q22.1

300476 300600 300071 610478 193235 612285 216360 612284 209900 601067 601067

Presentation

Stargardt disease 1 Cone-rod dystrophy 3 Retinitis pigmentosa 19 Macular degeneration, age related 2 Fundus flavimaculatus Pseudoxanthoma elasticum Pseudoxanthoma elasticum, forme fruste Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract Cone-rod dystrophy, spastic paraparesis, white matter disease Cone-rod dystrophy 9 Knobloch syndrome 2 Joubert syndrome-3 Cone-rod dystrophy Leber congenital amarosis 4 Retinitis pigimentosa, juvenile Alstrom syndrome Joubert syndrome 8 Retinitis pigmentosa with or without situs inversus Bardet-Biedl syndrome 3 Retinitis pigmentosa 55 Retinal nonattachment, nonsyndromic congenital Bardet-Biedl syndrome 1 Bardet-Biedl syndrome 10 Bardet-Biedl syndrome 12 Bardet-Biedl syndrome 2 Bardet-Biedl syndrome 4 Bardet-Biedl syndrome 5 Bardet-Biedl syndrome 7 Bardet-Biedl syndrome 9 Best macular dystrophy Retinitis pigmentosa 50 Vitelliform macular dystrophy, adult onset Vitreoretinochoroidopathy Microcornea, rod-cone dystrophy, cataract, and posterior staphyloma Retinal degeneration, late-onset, autosomal dominant Cone-rod dystrophy Retinitis pigmentosa 54 Cone-rod dystrophy 16 Retinitis pigmentosa 64 Retinitis pigmentosa 17 Night blindness, congenital stationary (incomplete), 2B, autosomal recessive Cone-rod dystrophy Aland Island eye disease Night blindness, congenital stationary (incomplete), 2A, X-linked Retinal cone dystrophy 4 Vitreoretinopathy, neovascular inflammatory Joubert syndrome 9 COACH syndrome Meckel syndrome, type 6 Modifier of Bardet-Biedl syndrome Usher syndrome, type 1D Usher syndrome, type 1D/F digenic Continued on next page

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SUPPLEMENTAL TABLE 1. List of Genes Used for Targeted Whole Exome Sequencing Analysis to Filter Out Variants Identified Within Genes With No Known Association to Retinal Disease (Continued ) Gene

Location

Phenotype MIM

CDH3

16q22.1

CDHR1

10q23.1

CEP164 CEP290

11q23.3 12q21.32

CERKL CHM CIB2 CLN3 CLRN1

2q31.3 Xq21.2 15q25.1 16p11.2 3q25.1

CNGA1 CNGA3 CNGB1 CNGB3

4p12 2q11.2 16q21 8q21.3

225280 601553 613660 613660 614845 209900 610188 611755 611134 610189 608380 303100 614869 204200 614180 276902 613756 216900 613767 262300 248200 217080 604841 184840 267750 108300 614134 607426 613835 172870 600105 120970 613829 612199 610127 210370 611383 613861 Abu-Safieh1 126600 600110 Abu-Safieh1 133540 216400 602772 606068 609033 607921 133780 133780 615360 610444 613856

CNNM4 COL11A1 COL11A2 COL18A1 COL2A1 COL9A1 COQ2 CRB1

2q11.2 1p21.1 6p21.32 21q22.3 12q13.11 6q13 4q21.23 1q31.3

CRX

19q13.33

CTC1 CTSD CYP4V2 DFNB31 DHDDS DTHD1 EFEMP1 ELOVL4 EMC1 ERCC6 ERCC8 EYS FAM161A FLVCR1 FSCN2 FZD4

17p13.1 11p15.5 4q35.2 9q32 1p36.11 4p14 2p16.1 6q14.1 1q21.3 10q11.23 5q12.1 6q12 2p15 1q32.3 17q25.3 11q14.2

GDF6 GNAT1 GNAT2

8q22.1 3p21.31 1p13.3

Presentation

Ectodermal dysplasia, ectrodactyly, and macular dystrophy Hypotrichosis with cone-rod dystrophy Cone-rod dystrophy 15 Retinitis pigmentosa 65 Nephronophthisis 15 (w/Leber congenital amaurosis) Bardet-Biedl syndrome 14 Joubert syndrome 5 Leber congenital amaurosis 10 Meckel syndrome 4 Senior-Loken syndrome 6 Retinitis pigmentosa 26 Choroideremia Usher syndrome, type IJ Ceroid lipofuscinosis, neuronal, 3 Retinitis pigmentosa 61 Usher syndrome, type 3A Retinitis pigmentosa 49 Achromatopsia-2 Retinitis pigmentosa 45 Achromatopsia-3 Macular degeneration, juvenile Jalili syndrome Stickler syndrome, type II Stickler syndrome, type III Knobloch syndrome, type 1 Stickler syndrome, type I Stickler syndrome, type IV Coenzyme Q10 deficiency, primary, 1 Leber congenital amaurosis 8 Pigmented paravenous chorioretinal atrophy Retinitis pigmentosa-12, autosomal recessive Cone-rod retinal dystrophy-2 Leber congenital amaurosis 7 Cerebroretinal microangiopathy with calcifications and cysts Ceroid lipofuscinosis, neuronal, 10 Bietti crystalline corneoretinal dystrophy Usher syndrome, type 2D Retinitis pigmentosa 59 Leber congenital amaurosis with myopathy (ref PMID: 2310516) Doyne honeycomb degeneration of retina Stargardt disease 3 Retinitis pigmentosa Cockayne syndrome, type B Cockayne syndrome, type A Retinitis pigmentosa 25 Retinitis pigmentosa 28 Ataxia, posterior column, with retinitis pigmentosa Retinitis pigmentosa 30 Exudative vitreoretinopathy Retinopathy of prematurity Leber congenital amaurosis 17 Night blindness, congenital stationary, autosomal dominant 3 Achromatopsia-4 Continued on next page

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SUPPLEMENTAL TABLE 1. List of Genes Used for Targeted Whole Exome Sequencing Analysis to Filter Out Variants Identified Within Genes With No Known Association to Retinal Disease (Continued ) Gene

Location

Phenotype MIM

Presentation

GNPTG GPR125 GPR179 GPR98

16p13.3 4p15.2 17q12 5q14.3

GRK1 GRM6 GUCA1A

13q34 5q35.3 6p21.1

GUCA1B GUCY2D

6p21.1 17p13.1

HARS HMX1 IDH3B IFT140 IGFBP7 IMPDH1

5q31.3 4p16.1 20p13 16p13.3 4q12 7q32.1

IMPG2

3q12.3

INPP5E

9q34.3

IQCB1 JAG1 KCNJ13

3q13.33 20p12.2 2q37.1

KCNV2 KIAA1549 KIF11 KLHL7 LCA5 LRAT

9p24.2 7q34 10q23.33 7p15.3 6q14.1 4q32.1

LRIT3 LRP5

4q25 11q13.2

LZTFL1 MAK MERTK MFRP MFSD8 MKKS MKS1 MTTP MYO6 MYO7A NDP

3p21.31 6p24.2 2q13 11q23.3 4q28.2 20p12.2 17q22 4q23 6q14.1 11q13.5 Xp11.3

NEK2 NMNAT1 NPHP1

1q32.3 1p36.22 2q13

252605 Abu-Safieh1 614565 605472 605472 613411 257270 602093 602093 613827 601777 204000 614504 612109 612572 266920 614224 613837 180105 613581 613581 213300 610156 609254 118450 614186 193230 610356 Abu-Safieh1 152950 612943 604537 613341 613341 613341 615058 601813 259770 209900 614181 613862 611040 610951 209900 209900 200100 607821 276900 305390 310600 615565 608553 266900

Mucolipidosis III gamma Retinitis pigmentosa Night blindness, congenital stationary (complete), 1E, autosomal recessive Usher syndrome type 2C Usher syndrome, type 2C, GPR98/PDZD7 digenic Oguchi disease-2 Night blindness, congenital stationary (complete), 1B, autosomal recessive Cone dystrophy-3 Cone-rod dystrophy 14 Retinitis pigmentosa 48 Cone-rod dystrophy 6 Leber congenital amaurosis 1 Usher syndrome type 3B Oculoauricular syndrome Retinitis pigmentosa 46 Mainzer-Saldino syndrome Retinal arterial macroaneurysm with supravalvular pulmonic stenosis Leber congenital amaurosis 11 Retinitis pigmentosa 10 Maculopathy, IMPG2-related Retinitis pigmentosa 56 Joubert syndrome 1 Intellectual disability, truncal obesity, retinal dystrophy, and micropenis Senior-Loken syndrome 5 Alagille syndrome Leber congenital amaurosis 16 Snowflake vitreoretinal degeneration Retinal cone dystrophy 3B Retinitis pigmentosa Microcephaly with or without chorioretinopathy, lymphedema, or intellectual disability Retinitis pigmentosa 42 Leber congenital amaurosis 5 Leber congenital amaurosis 14 Retinal dystrophy, early-onset severe Retinitis pigmentosa, juvenile Night blindness, congenital stationary (complete), 1F, autosomal recessive Exudative vitreoretinopathy 4 Osteoporosis-pseudoglioma syndrome Bardet-Biedl syndrome Retinitis pigmentosa 62 Retinitis pigmentosa 38 Microphthalmia, isolated 5 Ceroid lipofuscinosis, neuronal, 7 Bardet-Biedl syndrome 6 Bardet-Biedl syndrome 13 Abetalipoproteinemia Deafness, autosomal recessive 37 Usher syndrome, type 1B Exudative vitreoretinopathy, X-linked Norrie disease Retinitis pigmentosa 67 Leber congenital amaurosis 9 Senior-Loken syndrome-1 Continued on next page

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SUPPLEMENTAL TABLE 1. List of Genes Used for Targeted Whole Exome Sequencing Analysis to Filter Out Variants Identified Within Genes With No Known Association to Retinal Disease (Continued ) Gene

Location

Phenotype MIM

NPHP3 NPHP4 NR2E3

3q22.1 1p36.31 15q23

NRL NYX OAT OFD1 OPA1 OPN1LW

14q11.2 Xp11.4 10q26.13 Xp22.2 3q29 Xq28

OPN1MW

Xq28

OPN1SW OTX2

7q32.1 14q22.3

604387 606996 268100 611131 613750 310500 258870 300804 165500 303700 303900 303700 303800 190900 610125 610125 607236 234200 602083 601067 613810 613801 163500 613093 613582 610024 610024 605472 276901 214100 601539 614866 614867 614872 614873 614879 300653 266500 600977 228980 256730 610599 612657 608051 612095 603786 601414 600138 613983 600059

PANK2

20p13

PCDH15

10q21.1

PDE6A PDE6B

5q32 4p16.3

PDE6C PDE6G PDE6H

10q23.33 17q25.3 12p12.3

PDZD7

10q24.31

PEX1

7q21.2

PEX2

8q21.11

PEX26

22q11.21

PEX7 PGK1 PHYH PITPNM3 PLA2G5 PPT1 PRCD PROM1

PRPF3 PRPF31 PRPF6 PRPF8

6q23.3 Xq21.1 10p13 17p13.2 1p36.13-p36.12 1p34.2 17q25.1 4p15.32

1q21.2-q21.3 19q13.42 20q13.33 17p13.3

Presentation

Nephronophthisis 3 Senior-Loken syndrome 4 Enhanced S-cone syndrome Retinitis pigmentosa 37 Retinitis pigmentosa 27 Night blindness, congenital stationary (complete), 1A, X-linked Gyrate atrophy of choroid and retina with or without ornithinemia Joubert syndrome 10 Optic atrophy 1 Blue cone monochromacy Colorblindness, protan Blue cone monochromacy Colorblindness, deutan Colorblindness, tritan Retinal dystrophy, early onset, and pituitary dysfunction Microphthalmia, syndromic 5 HARP syndrome Neurodegeneration with brain iron accumulation 1 Usher syndrome, type 1F Usher syndrome, type 1D/F digenic Retinitis pigmentosa 43 Retinitis pigmentosa 40 Night blindness, congenital stationary, autosomal dominant 2 Cone dystrophy 4 Retinitis pigmentosa 57 Achromatopsia 6 Retinal cone dystrophy 3 Usher syndrome, type IIC, GPR98/PDZD7 digenic Modifier of Usher syndrome type IIA Peroxisome biogenesis disorder 1A (Zellweger) Peroxisome biogenesis disorder 1B (NALD/IRD) Peroxisome biogenesis disorder 5A (Zellweger) Peroxisome biogenesis disorder 5B Peroxisome biogenesis disorder 7A (Zellweger) Peroxisome biogenesis disorder 7B Peroxisome biogenesis disorder 9B Phosphoglycerate kinase 1 deficiency Refsum disease Cone-rod dystrophy 5 Fleck retina, familial benign Ceroid lipofuscinosis, neuronal, 1 Retinitis pigmentosa 36 Cone-rod dystrophy 12 Macular dystrophy, retinal, 2 Retinitis pigmentosa 41 Stargardt disease 4 Retinitis pigmentosa 18 Retinitis pigmentosa 11 Retinitis pigmentosa 60 Retinitis pigmentosa 13 Continued on next page

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SUPPLEMENTAL TABLE 1. List of Genes Used for Targeted Whole Exome Sequencing Analysis to Filter Out Variants Identified Within Genes With No Known Association to Retinal Disease (Continued ) Gene

PRPH2

Location

Phenotype MIM

Presentation

6p21.1

613105 608161 169150 608161 608133 608133 136880 615374 610381 615233 615147 610612 612712 136880 613769 608415 608415 613731 610445 136880 603649 607475 136880 607476 136880 608133 180100 613587 312600 180104 204100 613794 300029 304020 300834 300455 608194 613826 216360 312700 613758 258100 613615 610282 610283 613830 610359 604232 604232 604360 108985 136900 268130

Choriodal dystrophy, central areolar 2 Foveomacular dystrophy, adult onset, with choroidal neovascularization Macular dystrophy, patterned Macular dystrophy, vitelliform Retinitis pigmentosa 7 Retinitis pigmentosa, digenic Retinitis punctata albescens Cone-rod dystrophy 18 Cone-rod dystrophy 11 Retinitis pigmentosa 66 Retinol dystrophy, iris coloboma, and comedogenic acne syndrome Leber congenital amaurosis 12 Leber congenital amaurosis 13 Fundus albipunctatus Retinitis pigmentosa 44 Bradyopsia Bradyopsia Retinitis pigmentosa 4, autosomal dominant or recessive Night blindness, congenital stationary, autosomal dominant 1 Retinitis punctata albescens Cone-rod dystrophy 7 Bothnia retinal dystrophy Fundus albipunctatus Newfoundland rod-cone dystrophy Retinitis punctata albescens Retinitis pigmentosa 7, digenic Retinitis pigmentosa 1 Occult macular dystrophy Retinitis pigmentosa 2 Retinitis pigmentosa 9 Leber congenital amaurosis 2 Retinitis pigmentosa 20 Retinitis pigmentosa 3 Cone-rod dystrophy, X-linked, 1 Macular degeneration, X-linked atrophic Retinitis pigmentosa, X-linked, and sinorespiratory infections, with or without deafness Cone-rod dystrophy 13 Leber congenital amaurosis 6 COACH syndrome Retinoschisis Retinitis pigmentosa 47 Oguchi disease-1 Senior-Loken syndrome 7 Retinitis pigmentosa 35 Cone-rod dystrophy 10 Night blindness, congenital stationary (complete), 1D, autosomal recessive Retinitis pigmentosa 33 Leber congenital amaurosis 3 Retinitis pigmentosa, juvenile, autosomal recessive Spastic paraplegia 11, autosomal recessive Sveinsson choreoretinal atrophy Sorsby fundus dystrophy Revesz syndrome

RAB28 RAX2 RBP3 RBP4 RD3 RDH12 RDH5 RGR RGS9 RGS9BP RHO

4p15.33 19p13.3 10q11.22 10q23.33 1q32.3 14q24.1 12q13.2 10q23.1 17q24.1 19q13.11 3q22.1

RIMS1 RLBP1

6q13 15q26.1

ROM1 RP1 RP1L1 RP2 RP9 RPE65

11q12.3 8q12.1 8p23.1 Xp11.23 7p14.3 1p31.3-p31.2

RPGR

Xp11.4

RPGRIP1

14q11.2

RPGRIP1L RS1 SAG

16q12.2 Xp22.13 2q37.1

SDCCAG8 SEMA4A

1q43 1q22

SLC24A1 SNRNP200 SPATA7

15q22.31 2q11.2 14q31.3

SPG11 TEAD1 TIMP3 TINF2

15q21.1 11p15.3-p15.2 22q12.3 14q12

Continued on next page

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SUPPLEMENTAL TABLE 1. List of Genes Used for Targeted Whole Exome Sequencing Analysis to Filter Out Variants Identified Within Genes With No Known Association to Retinal Disease (Continued ) Gene

Location

Phenotype MIM

TMEM216 TMEM237 TMEM67

11q12.2 2q33.1 8q22.1

TOPORS TREX1

9p21.1 3p21.31

TRIM32 TRPM1 TSPAN12 TTC19 TTC8

9q33.1 15q13.3 7q31.31 17p12 14q31.3

TULP1

6p21.31

608091 614424 610688 613550 209900 609923 225750 192315 209900 613216 613310 615157 209900 613464 613843 600132 Kobayashi2 615158 615159 276904 606943 606943 612632 276901 143200 153840 216550 193235 209900 614378 222300 Collin3 614844 613617

UNC119 UQCRB UQCRQ USH1C USH1G USH1H USH2A

17q11.2 8q22.1 5q31.1 11p15.1 17q25.1 15q22-q23 1q41

VCAN VPS13B VRNI WDPCP WDR19 WFS1

5q14.2-q14.3 8q22.2 11q13 2p15 4p14 4p16.1

ZNF408 ZNF423 ZNF513

11p11.2 16q12.1 2p23.3

Presentation

Joubert syndrome 2 Joubert syndrome 14 Joubert syndrome 6 Nephronophthisis 11 Modifier of Bardet-Biedl syndrome 14 Retinitis pigmentosa 31 Aicardi-Goutieres syndrome 1, dominant and recessive Vasculopathy, retinal, with cerebral leukodystrophy Bardet-Biedl syndrome 11 Night blindness, congenital stationary (complete), 1C, autosomal recessive Exudative vitreoretinopathy 5 Mitochondrial complex III deficiency, nuclear type 2 Bardet-Biedl syndrome 8 Retinitis pigmentosa 51 Leber congenital amaurosis 15 Retinitis pigmentosa 14 Cone-rod dystrophy Mitochondrial complex III deficiency, nuclear type 3 Mitochondrial complex III deficiency, nuclear type 4 Usher syndrome, type 1C Usher syndrome, type 1G Usher syndrome, type 1H Usher syndrome, type 2A Retinitis pigmentosa 39 Wagner syndrome 1 Cohen syndrome Vitreoretinopathy, neovascular inflammatory Bardet-Biedl syndrome 15 Cranioectodermal dysplasia 4 Wolfram syndrome Wolfram-like syndrome, autosomal dominant Familial exudative vitreoretinopathy Joubert syndrome 19 Retinitis pigmentosa 58

This is a comprehensive list of the Retina Gene List used for variant analysis. Variants within these genes were ascertained and sorted according to their likelihood of pathogenicity.

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SUPPLEMENTAL TABLE 2. Total Number of Exome Sequencing Variants Identified in Each Participant Along With the Number of Exome Variants Subdivided by Type and Class to Highlight the Filtering Schema

Participant

Total Exome of Variants

Bin 1 Gene Variants

Bin 1 Variants Analyzed

Retina Gene variants

Retina Class A Variants

Retina Class B Variants

Retina Class C Variants

Retina Class D Variants

Retina Class E Variants

Retina Class F Variants

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Mean values Min values Max values

82 070 82 982 101 225 84 206 79 971 79 922 96 338 80 973 83 557 80 399 79 185 80 772 78 816 79 808 82 368 90 547 95 731 75 782 81 246 85 818 78 303 81 512 99 532 85 214 80 308 102 973 84 983 75 782 102 973

808 880 987 832 747 796 1024 857 851 806 737 814 827 798 825 918 984 798 766 848 768 848 957 867 842 1013 854 737 1024

1 2 0 3 4 1 3 4 6 4 2 2 1 4 1 4 3 2 1 3 1 2 4 2 1 2 2 0 6

1283 1298 1648 1296 1154 1273 1572 1393 1394 1291 1302 1275 1372 1279 1190 1410 1541 1229 1304 1399 1269 1251 1733 1377 1233 1768 1367 1154 1768

8 4 6 5 7 4 8 2 5 5 3 5 5 5 4 5 8 4 2 9 6 4 8 4 3 10 5 2 10

3 3 4 2 4 3 1 2 2 4 3 2 1 2 2 2 2 4 10 4 4 4 2 7 4 3 3 1 10

23 22 37 28 25 25 20 22 22 18 26 27 17 27 22 29 19 20 19 22 23 20 34 24 17 24 24 17 37

145 137 255 144 113 124 206 119 126 131 114 135 143 116 124 173 181 105 154 181 130 137 251 157 132 276 154 105 276

31 36 120 52 33 47 111 35 46 38 37 44 62 40 38 75 121 34 48 41 38 39 161 35 40 123 59 31 161

1073 1096 1226 1065 971 1070 1226 1213 1193 1094 1118 1062 1144 1089 1000 1126 1209 1062 1071 1142 1068 1047 1277 1150 1037 1332 1122 971 1332

The exome filtering process was subdivided into filtering variants within Bin 1 genes (genes associated with medically actionable conditions) and variants within the Retina Gene List. Only Bin 1 gene variants previously reported in the Human Genome Mutation Database (HGMD) and presumably novel frameshifting or nonsense variants or variants within conical splice sites were analyzed.4 Variants listed as Retina Class A were reported as pathogenic variants in the HGMD. Retina Class B variants are rare (<1% minor allele frequency) predicted truncating (nonsense, frameshifting indel) variants or variants within conical splice sites. Rare (<1% minor allele frequency) missense variants were categorized as Retina Class C variants. Missense, nonsense, and frameshifting variants with 1%–5% minor allele frequency and rare (<1% minor allele frequency) synonymous or intronic variants were categorized as Retina Class D variants. Retina Class E variants consisted of missense, nonsense, and frameshifting variants with >5% minor allele frequency. Retina Class F variants were intronic variants with >5% minor allele frequency. Greater emphasis was placed on analyzing variants within Classes A-C.

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REFERENCES FOR SUPPLEMENTAL DATA 1. Abu-Safieh L, Alrashed M, Anazi S, et al. Autozygome-guided exome sequencing in retinal dystrophy patients reveals pathogenetic mutations and novel candidate disease genes. Genome Res 2013;23(2):236–247. 2. Kobayashi A, Higashide T, Hamasaki D, et al. HRG4 (UNC119) mutation found in cone-rod dystrophy causes

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retinal degeneration in a transgenic model. Invest Ophthalmol Vis Sci 2000;41(11):3268–3277. 3. Collin RW, Nikopoulos K, Dona M, et al. ZNF408 is mutated in familial exudative vitreoretinopathy and is crucial for the development of zebrafish retinal vasculature. Proc Natl Acad Sci U S A 2013;110(24):9856–9861. 4. Krawczak M, Cooper DN. The Human Gene Mutation Database. Trends Genet 2007;13:121–122.

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