Whole genome sequencing of 4,787 individuals identifies gene-based rare variants in age-related macular degeneration.

Genome-wide association studies have contributed extensively to the discovery of disease-associated common variants. However, the genetic contribution to complex traits is still largely difficult to interpret. We report a genome-wide association study of 2394 cases and 2393 controls for age-related macular degeneration (AMD) via whole-genome sequencing, with 46.9 million genetic variants. Our study reveals significant single-variant association signals at four loci and independent gene-based signals in CFH, C2, C3, and NRTN. Using data from the Exome Aggregation Consortium (ExAC) for a gene-based test, we demonstrate an enrichment of predicted rare loss-of-function variants in CFH, CFI, and an as-yet unreported gene in AMD, ORMDL2. Our method of using a large variant list without individual-level genotypes as an external reference provides a flexible and convenient approach to leverage the publicly available variant datasets to augment the search for rare variant associations, which can explain additional disease risk in AMD.

The landscape of submicroscopic structural variants at the OPN1LW/OPN1MW gene cluster on Xq28 underlying blue cone monochromacy.

Blue cone monochromacy (BCM) is an X-linked retinal disorder characterized by low vision, photoaversion, and poor color discrimination. BCM is due to the lack of long-wavelength-sensitive and middle-wavelength-sensitive cone photoreceptor function and caused by mutations in the OPN1LW/OPN1MW gene cluster on Xq28. Here, we investigated the prevalence and the landscape of submicroscopic structural variants (SVs) at single-base resolution in BCM patients. We found that about one-third (n = 73) of the 213 molecularly confirmed BCM families carry an SV, most commonly deletions restricted to the OPN1LW/OPN1MW gene cluster. The structure and precise breakpoints of the SVs were resolved in all but one of the 73 families. Twenty-two families-all from the United States-showed the same SV, and we confirmed a common ancestry of this mutation. In total, 42 distinct SVs were identified, including 40 previously unreported SVs, thereby quadrupling the number of precisely mapped SVs underlying BCM. Notably, there was no "region of overlap" among these SVs. However, 90% of SVs encompass the upstream locus control region, an essential enhancer element. Its minimal functional extent based on deletion mapping in patients was refined to 358 bp. Breakpoint analyses suggest diverse mechanisms underlying SV formation as well as in one case the gene conversion-based exchange of a 142-bp deletion between opsin genes. Using parsimonious assumptions, we reconstructed the composition and copy number of the OPN1LW/OPN1MW gene cluster prior to the mutation event and found evidence that large gene arrays may be predisposed to the occurrence of SVs at this locus.

Clinical sequencing of the retinitis pigmentosa gene RPGR in over 1,000 cases of vision loss.

RPGR pathogenic variants are the major cause of X-linked retinitis pigmentosa. Here, we report the results from 1,033 clinical DNA tests that included sequencing of RPGR. A total of 184 RPGR variants were identified: 78 pathogenic or likely pathogenic, 14 uncertain, and 92 likely benign or benign. Among the pathogenic and likely pathogenic variants, 23 were novel, and most were frameshift or nonsense mutations (87%) and enriched (67%) in RPGR exon 15 (ORF15). Identical pathogenic variants found in different families were largely on different haplotype backgrounds, indicating relatively frequent, recurrent RPGR mutations. None of the 16 mother/affected son pairs showed de novo mutations; all 16 mothers were heterozygous for the pathogenic variant. These last two observations support the occurrence of most RPGR mutations in the male germline.

Deciphering the genetic architecture and ethnographic distribution of IRD in three ethnic populations by whole genome sequence analysis.

Patients with inherited retinal dystrophies (IRDs) were recruited from two understudied populations: Mexico and Pakistan as well as a third well-studied population of European Americans to define the genetic architecture of IRD by performing whole-genome sequencing (WGS). Whole-genome analysis was performed on 409 individuals from 108 unrelated pedigrees with IRDs. All patients underwent an ophthalmic evaluation to establish the retinal phenotype. Although the 108 pedigrees in this study had previously been examined for mutations in known IRD genes using a wide range of methodologies including targeted gene(s) or mutation(s) screening, linkage analysis and exome sequencing, the gene mutations responsible for IRD in these 108 pedigrees were not determined. WGS was performed on these pedigrees using Illumina X10 at a minimum of 30X depth. The sequence reads were mapped against hg19 followed by variant calling using GATK. The genome variants were annotated using SnpEff, PolyPhen2, and CADD score; the structural variants (SVs) were called using GenomeSTRiP and LUMPY. We identified potential causative sequence alterations in 61 pedigrees (57%), including 39 novel and 54 reported variants in IRD genes. For 57 of these pedigrees the observed genotype was consistent with the initial clinical diagnosis, the remaining 4 had the clinical diagnosis reclassified based on our findings. In seven pedigrees (12%) we observed atypical causal variants, i.e. unexpected genotype(s), including 4 pedigrees with causal variants in more than one IRD gene within all affected family members, one pedigree with intrafamilial genetic heterogeneity (different affected family members carrying causal variants in different IRD genes), one pedigree carrying a dominant causative variant present in pseudo-recessive form due to consanguinity and one pedigree with a de-novo variant in the affected family member. Combined atypical and large structural variants contributed to about 20% of cases. Among the novel mutations, 75% were detected in Mexican and 50% found in European American pedigrees and have not been reported in any other population while only 20% were detected in Pakistani pedigrees and were not previously reported. The remaining novel IRD causative variants were listed in gnomAD but were found to be very rare and population specific. Mutations in known IRD associated genes contributed to pathology in 63% Mexican, 60% Pakistani and 45% European American pedigrees analyzed. Overall, contribution of known IRD gene variants to disease pathology in these three populations was similar to that observed in other populations worldwide. This study revealed a spectrum of mutations contributing to IRD in three populations, identified a large proportion of novel potentially causative variants that are specific to the corresponding population or not reported in gnomAD and shed light on the genetic architecture of IRD in these diverse global populations.

Tissue-specific genotype-phenotype correlations among USH2A-related disorders in the RUSH2A study.

We assessed genotype-phenotype correlations among the visual, auditory, and olfactory phenotypes of 127 participants with Usher syndrome (USH2) (n =80) or nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) (n = 47) due to USH2A variants, using clinical data and molecular diagnostics from the Rate of Progression in USH2A Related Retinal Degeneration (RUSH2A) study. USH2A truncating alleles were associated with USH2 and had a dose-dependent effect on hearing loss severity with no effect on visual loss severity within the USH2 subgroup. A group of missense alleles in an interfibronectin domain appeared to be hypomorphic in ARRP. These alleles were associated with later age of onset, larger visual field area, better sensitivity thresholds, and better electroretinographic responses. No effect of genotype on the severity of olfactory deficits was observed. This study unveils a unique, tissue-specific USH2A allelic hierarchy with important prognostic implications for patient counseling and treatment trial endpoints. These findings may inform clinical care or research approaches in others with allelic disorders or pleiotropic phenotypes.

Comprehensive variant spectrum of the CNGA3 gene in patients affected by achromatopsia.

Achromatopsia (ACHM) is a congenital cone photoreceptor disorder characterized by impaired color discrimination, low visual acuity, photosensitivity, and nystagmus. To date, six genes have been associated with ACHM (CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6), the majority of these being implicated in the cone phototransduction cascade. CNGA3 encodes the CNGA3 subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors and is one of the major disease-associated genes for ACHM. Herein, we provide a comprehensive overview of the CNGA3 variant spectrum in a cohort of 1060 genetically confirmed ACHM patients, 385 (36.3%) of these carrying "likely disease-causing" variants in CNGA3. Compiling our own genetic data with those reported in the literature and in public databases, we further extend the CNGA3 variant spectrum to a total of 316 variants, 244 of which we interpreted as "likely disease-causing" according to ACMG/AMP criteria. We report 48 novel "likely disease-causing" variants, 24 of which are missense substitutions underlining the predominant role of this mutation class in the CNGA3 variant spectrum. In addition, we provide extensive in silico analyses and summarize reported functional data of previously analyzed missense, nonsense and splicing variants to further advance the pathogenicity assessment of the identified variants.

Family-based exome sequencing identifies rare coding variants in age-related macular degeneration.

Genome-wide association studies (GWAS) have identified 52 independent variants at 34 genetic loci that are associated with age-related macular degeneration (AMD), the most common cause of incurable vision loss in the elderly worldwide. However, causal genes at the majority of these loci remain unknown. In this study, we performed whole exome sequencing of 264 individuals from 63 multiplex families with AMD and analyzed the data for rare protein-altering variants in candidate target genes at AMD-associated loci. Rare coding variants were identified in the CFH, PUS7, RXFP2, PHF12 and TACC2 genes in three or more families. In addition, we detected rare coding variants in the C9, SPEF2 and BCAR1 genes, which were previously suggested as likely causative genes at respective AMD susceptibility loci. Identification of rare variants in the CFH and C9 genes in our study validated previous reports of rare variants in complement pathway genes in AMD. We then extended our exome-wide analysis and identified rare protein-altering variants in 13 genes outside the AMD-GWAS loci in three or more families. Two of these genes, SCN10A and KIR2DL4, are of interest because variants in these genes also showed association with AMD in case-control cohorts, albeit not at the level of genome-wide significance. Our study presents the first large-scale, exome-wide analysis of rare variants in AMD. Further independent replications and molecular investigation of candidate target genes, reported here, would assist in gaining novel insights into mechanisms underlying AMD pathogenesis.

Real-world outcomes of voretigene neparvovec treatment in pediatric patients with RPE65-associated Leber congenital amaurosis.

PURPOSE: To investigate real-world safety and efficacy of voretigene neparvovec gene therapy administration in pediatric patients with biallelic RPE65 disease-causing variants. METHODS: A retrospective study of 27 eyes of 14 patients with RPE65-associated Leber congenital amaurosis examined postoperative complications and longitudinal changes in photoreceptor function following treatment with subretinal injection of voretigene neparvovec. Full-field stimulus threshold testing (FST), Goldmann visual fields (GVF), best-corrected visual acuity (BCVA), and central subfield thickness (CST) on optical coherence tomography (OCT) scans were collected preoperatively and up to 12 months posttreatment. RESULTS: Baseline through 6-12 month follow-up FST and GVF data were obtained for 13 eyes of 7 patients. FST improved for each eye after treatment with a mean improvement of 2.1 log-units (P < 0.001) and GVF improved for each eye with a mean improvement of 221 sum degrees (P < 0.001). BCVA improved from logMAR 0.98 at baseline to logMAR 0.83 at last follow-up (P < 0.001). Across 19 eyes of 10 patients included in CST analysis, there was a small but statistically significant 9-µ decrease in mean CST from baseline to last follow-up (P < 0.001). The most common postoperative issues included elevation in intraocular pressure (59%), persistent intraocular inflammation (15%), and vitreous opacities (26%) that resolved over a period of months. CONCLUSIONS: This report provides some of the earliest longitudinal real-world evidence of the pediatric safety and efficacy of voretigene neparvovec using multiple functional and structural measures of the retina. Outcomes demonstrate significant improvements in visual function consistent with clinical trial results.

Deep-intronic variants in CNGB3 cause achromatopsia by pseudoexon activation.

Our comprehensive cohort of 1100 unrelated achromatopsia (ACHM) patients comprises a considerable number of cases (~5%) harboring only a single pathogenic variant in the major ACHM gene CNGB3. We sequenced the entire CNGB3 locus in 33 of these patients to find a second variant which eventually explained the patients' phenotype. Forty-seven intronic CNGB3 variants were identified in 28 subjects after a filtering step based on frequency and the exclusion of variants found in cis with pathogenic alleles. In a second step, in silico prediction tools were used to filter out those variants with little odds of being deleterious. This left three variants that were analyzed using heterologous splicing assays. Variant c.1663-1205G>A, found in 14 subjects, and variant c.1663-2137C>T, found in two subjects, were indeed shown to exert a splicing defect by causing pseudoexon insertion into the transcript. Subsequent screening of further unsolved CNGB3 subjects identified four additional cases harboring the c.1663-1205G>A variant which makes it the eighth most frequent CNGB3 variant in our cohort. Compound heterozygosity could be validated in ten cases. Our study demonstrates that whole gene sequencing can be a powerful approach to identify the second pathogenic allele in patients apparently harboring only one disease-causing variant.

Genetic testing for inherited retinal degenerations: Triumphs and tribulations.

Inherited retinal degenerations (IRDs) are a genotypically and phenotypically diverse group of conditions. Great strides have been made toward identifying the genetic basis for these conditions over the last 30 years-more than 270 different genes involved in syndromic and nonsyndromic forms of retinal dystrophies have now been identified. The identification of these genes and the improvement of clinical laboratory techniques have led to the identification of the genetic basis of disease in 56-76% of patients with IRDs through next generation sequencing and copy number variant analysis. Genetic testing is an essential part of clinical care for patients affected with IRDs and is required to confirm the diagnosis, understand the inheritance of the condition, and determine eligibility for gene-specific treatments or clinical trials. Despite the success achieved in determining the genetic cause of these conditions, several challenges remain, which must be considered when providing genetic testing and genetic counseling to patients. For this reason, an integrated team of ophthalmic and genetic clinicians who are familiar with these challenges is necessary to provide optimal comprehensive care to these patients.

Implementation of a registry and open access genetic testing program for inherited retinal diseases within a non-profit foundation.

The Foundation Fighting Blindness is a 50-year old 501c(3) non-profit organization dedicated to supporting the development of treatments and cures for people affected by the inherited retinal diseases (IRD), a group of clinical diagnoses that include orphan diseases such as retinitis pigmentosa, Usher syndrome, and Stargardt disease, among others. Over $760 M has been raised and invested in preclinical and clinical research and resources. Key resources include a multi-national clinical consortium, an international patient registry with over 15,700 members that is expanding rapidly, and an open access genetic testing program that provides no cost comprehensive genetic testing to people clinically diagnosed with an IRD living in the United States. These programs are described with particular focus on the challenges and outcomes of establishing the registry and genetic testing program.

Surgical outcomes of Glaucoma associated with Axenfeld-Rieger syndrome.

BACKGROUND: The surgical management of glaucoma associated with Axenfeld-Rieger Syndrome (ARS) is poorly described in the literature. The goal of this study is to compare the effectiveness of various glaucoma surgeries on intraocular pressure (IOP) management in ARS. METHODS: Retrospective cohort study at a university hospital-based practice of patients diagnosed with ARS between 1973 and 2018. Exclusion criterion was follow-up less than 1 year. The number of eyes with glaucoma (IOP ≥ 21 mmHg with corneal edema, Haabs striae, optic nerve cupping or buphthalmos) requiring surgery was determined. The success and survival rates of goniotomy, trabeculotomy±trabeculectomy (no antifibrotics), cycloablation, trabeculectomy with anti-fibrotics, and glaucoma drainage device placement were assessed. Success was defined as IOP of 5-20 mmHg and no additional IOP-lowering surgery or visually devastating complications. Kaplan-Meier survival curves and the Wilcoxon test were used for statistical analysis. RESULTS: In 32 patients identified with ARS (median age at presentation 6.9 years, 0-58.7 years; median follow-up 5.4 years, 1.1-43.7 years), 23 (71.9%) patients were diagnosed with glaucoma at median age 6.3 years (0-57.9 years). In glaucomatous eyes (46 eyes), mean IOP at presentation was 21.8 ± 9.3 mmHg (median 20 mmHg, 4-45 mmHg) on 1.0 ± 1.6 glaucoma medications. Thirty-one eyes of 18 patients required glaucoma surgery with 2.2 ± 1.2 IOP-lowering surgeries per eye. Goniotomy (6 eyes) showed 43% success with 4.3 ± 3.9 years of IOP control. Trabeculotomy±trabeculectomy (6 eyes) had 17% success rate with 14.8 ± 12.7 years of IOP control. Trabeculectomy with anti-fibrotics (14 eyes) showed 57% success with 16.5 ± 13.5 years of IOP control. Ahmed© (FP7 or FP8) valve placement (8 eyes) had 25% success rate with 1.7 ± 1.9 years of IOP control. Baerveldt© (250 or 350) device placement (8 eyes) showed 70% success with 1.9 ± 2.3 years of IOP control. Cycloablation (4 eyes) had 33% success rate with 2.7 ± 3.5 years of IOP control. At final follow-up, mean IOP (12.6 ± 3.8 mmHg, median 11.8 mmHg, 7-19 mmHg) in glaucomatous eyes was significantly decreased (p < 0.0001), but there was no difference in number of glaucoma medications (1.6 ± 1.5, p = 0.1). CONCLUSIONS: In our series, greater than 70% of patients with ARS have secondary glaucoma that often requires multiple surgeries. Trabeculectomy with anti-fibrotics and Baerveldt glaucoma drainage devices showed the greatest success in obtaining IOP control.

Contribution of noncoding pathogenic variants to RPGRIP1-mediated inherited retinal degeneration.

PURPOSE: With the advent of gene therapies for inherited retinal degenerations (IRDs), genetic diagnostics will have an increasing role in clinical decision-making. Yet the genetic cause of disease cannot be identified using exon-based sequencing for a significant portion of patients. We hypothesized that noncoding pathogenic variants contribute significantly to the genetic causality of IRDs and evaluated patients with single coding pathogenic variants in RPGRIP1 to test this hypothesis. METHODS: IRD families underwent targeted panel sequencing. Unsolved cases were explored by exome and genome sequencing looking for additional pathogenic variants. Candidate pathogenic variants were then validated by Sanger sequencing, quantitative polymerase chain reaction, and in vitro splicing assays in two cell lines analyzed through amplicon sequencing. RESULTS: Among 1722 families, 3 had biallelic loss-of-function pathogenic variants in RPGRIP1 while 7 had a single disruptive coding pathogenic variants. Exome and genome sequencing revealed potential noncoding pathogenic variants in these 7 families. In 6, the noncoding pathogenic variants were shown to lead to loss of function in vitro. CONCLUSION: Noncoding pathogenic variants were identified in 6 of 7 families with single coding pathogenic variants in RPGRIP1. The results suggest that noncoding pathogenic variants contribute significantly to the genetic causality of IRDs and RPGRIP1-mediated IRDs are more common than previously thought.

Mutations in the gene PDE6C encoding the catalytic subunit of the cone photoreceptor phosphodiesterase in patients with achromatopsia.

Biallelic PDE6C mutations are a known cause for rod monochromacy, better known as autosomal recessive achromatopsia (ACHM), and early-onset cone photoreceptor dysfunction. PDE6C encodes the catalytic α'-subunit of the cone photoreceptor phosphodiesterase, thereby constituting an essential part of the phototransduction cascade. Here, we present the results of a study comprising 176 genetically preselected patients who remained unsolved after Sanger sequencing of the most frequent genes accounting for ACHM, and were subsequently screened for exonic and splice site variants in PDE6C applying a targeted next generation sequencing approach. We were able to identify potentially pathogenic biallelic variants in 15 index cases. The mutation spectrum comprises 18 different alleles, 15 of which are novel. Our study significantly contributes to the mutation spectrum of PDE6C and allows for a realistic estimate of the prevalence of PDE6C mutations in ACHM since our entire ACHM cohort comprises 1,074 independent families.

IFT88 mutations identified in individuals with non-syndromic recessive retinal degeneration result in abnormal ciliogenesis.

Whole genome sequencing (WGS) was performed to identify the variants responsible for inherited retinal degeneration (IRD) in a Caucasian family. Segregation analysis of selected rare variants with pathogenic potential identified a set of compound heterozygous changes p.Arg266*:c.796C>T and p.Ala568Thr:c.1702G>A in the intraflagellar transport protein-88 (IFT88) gene segregating with IRD. Expression of IFT88 with the p.Arg266* and p.Ala568Thr mutations in mIMDC3 cells by transient transfection and in HeLa cells by introducing the mutations using CRISPR-cas9 system suggested that both mutations result in the formation of abnormal ciliary structures. The introduction of the IFT88 p.Arg266* variant in the homozygous state in HeLa cells by CRISPR-Cas9 genome-editing revealed that the mutant transcript undergoes nonsense-mediated decay leading to a significant depletion of IFT88 transcript. Additionally, abnormal ciliogenesis was observed in these cells. These observations suggest that the rare and unique combination of IFT88 alleles observed in this study provide insight into the physiological role of IFT88 in humans and the likely mechanism underlying retinal pathology in the pedigree with IRD.

Contrast sensitivity deficits in patients with mutation-proven inherited retinal degenerations.

BACKGROUND: Patients with retinal diseases frequently complain of poor visual function even when visual acuity is relatively unaffected. This clinical finding has been attributed to deficits in contrast sensitivity (CS). The purpose of our study was to evaluate the CS in patients with clinical and genetic diagnosis of inherited retinal degeneration (IRD) and relatively preserved visual acuity. METHODS: Seventeen patients (30 eyes) with IRD and visual acuity of 20/40 or better, and 18 controls (18 eyes) without any ocular condition underwent slit lamp examination, visual acuity testing via standard Snellen chart testing, CS testing via the Quick Contrast Sensitivity Function (QCSF), and Spectral Domain Optical Coherence Tomography (SD-OCT). CS were measured at 1.0, 1.5, 3.0, 6.0, 12.0, and 18.0 cycles per degree (cpd). T tests with general estimated equations were used to compare CS between groups. Wald chi square followed by pairwise comparisons was used to compare CS between multiple groups. RESULTS: We included 12 patients with rod-cone dystrophy (RCD), 3 patients with Stargardt disease (STGD) and 2 patients with Best disease. Patients with IRD had significantly worse CS than controls (p < 0.001) in all spatial frequencies. Patients with STGD had more marked deficits in CS than patients with Best disease (p < 0.001) and RCD (p < 0.001) despite having similar visual acuities. CONCLUSION: Patients with IRD, especially patients with STGD with relatively preserved visual acuity have marked deficits in CS when measured across a range of spatial frequencies. We recommend that clinical trials for STGD incorporate CS measured over a range of spatial frequencies as a secondary clinical endpoint for monitoring visual function. CS may provide an explanation for complaints of visual dysfunction when visual acuity is not significantly altered.

Molecular Findings in Families with an Initial Diagnose of Autosomal Dominant Retinitis Pigmentosa (adRP).

Genetic testing of probands in families with an initial diagnosis of autosomal dominant retinitis pigmentosa (adRP) usually confirms the diagnosis, but there are exceptions. We report results of genetic testing in a large cohort of adRP families with an emphasis on exceptional cases including X-linked RP with affected females; homozygous affected individuals in families with heterozygous, dominant disease; and independently segregating mutations in the same family. Genetic testing was conducted in more than 700 families with a provisional or probable diagnosis of adRP. Exceptions to the proposed mode of inheritance were extracted from our comprehensive patient and family database. In a subset of 300 well-characterized families with a probable diagnosis of adRP, 195 (70%) have dominant mutations in known adRP genes but 25 (8%) have X-linked mutations, 3 (1%) have multiple segregating mutations, and 3 (1%) have dominant-acting mutations in genes previously associated with recessive disease. It is currently possible to determine the underlying disease-causing gene and mutation in approximately 80% of families with an initial diagnosis of adRP, but 10% of "adRP" families have a variant mode of inheritance. Informed genetic diagnosis requires close collaboration between clinicians, genetic counselors, and laboratory scientists.

Identification of Novel Deletions as the Underlying Cause of Retinal Degeneration in Two Pedigrees.

Retinal dystrophies are a phenotypically and genetically complex group of conditions. Because of this complexity, it can be challenging in many families to determine the inheritance based on pedigree analysis alone. Clinical examinations were performed and blood samples were collected from a North American (M1186) and a consanguineous Pakistani (PKRD168) pedigree affected with two different retinal dystrophies (RD). Based on the structure of the pedigrees, inheritance patterns in the families were difficult to determine. In one family, linkage analysis was performed with markers on X-chromosome. In the second family, whole-exome sequencing (WES) was performed. Subsequent Sanger sequencing of genes of interest was performed. Linkage and haplotype analysis localized the disease interval to a 70 Mb region on the X chromosome that encompassed RP2 and RPGR in M1186 . The disease haplotype segregated with RD in all individuals except for an unaffected man (IV:3) and his affected son (V:1) in this pedigree. Subsequent analysis identified a novel RPGR mutation (p. Lys857Glu fs221X) in all affected members of M1186 except V:1. This information suggests that there is an unidentified second cause of retinitis pigmentosa (RP) within the family. A novel two-base-pair deletion (p. Tyr565Ter fsX) in CHM (choroideremia) was found to segregate with RD in PKRD168. This paper highlights the challenges of interpreting family history in families with RD and reports on the identification of novel mutations in two RD families.

Genetic analysis of 10 pedigrees with inherited retinal degeneration by exome sequencing and phenotype-genotype association.

Our purpose was to identify causative mutations and characterize the phenotype associated with the genotype in 10 unrelated families with autosomal recessive retinal degeneration. Ophthalmic evaluation and DNA isolation were carried out in 10 pedigrees with inherited retinal degenerations (IRD). Exomes of probands from eight pedigrees were captured using Nimblegen V2/V3 or Agilent V5+UTR kits, and sequencing was performed on Illumina HiSeq. The DHDDS gene was screened for mutations in the remaining two pedigrees with Ashkenazi Jewish ancestry. Exome variants were filtered to detect candidate causal variants using exomeSuite software. Segregation and ethnicity-matched control sample analysis were performed by dideoxy sequencing. Retinal histology of a patient with DHDDS mutation was studied by microscopy. Genetic analysis identified six known mutations in ABCA4 (p.Gly1961Glu, p.Ala1773Val, c.5461-10T>C), RPE65 (p.Tyr249Cys, p.Gly484Asp), PDE6B (p.Lys706Ter) and DHDDS (p.Lys42Glu) and ten novel potentially pathogenic variants in CERKL (p.Met323Val fsX20), RPE65 (p.Phe252Ser, Thr454Leu fsX31), ARL6 (p.Arg121His), USH2A (p.Gly3142Ter, p.Cys3294Trp), PDE6B (p.Gln652Ter), and DHDDS (p.Thr206Ala) genes. Among these, variants/mutations in two separate genes were observed to segregate with IRD in two pedigrees. Retinal histopathology of a patient with a DHDDS mutation showed severe degeneration of retinal layers with relative preservation of the retinal pigment epithelium. Analysis of exome variants in ten pedigrees revealed nine novel potential disease-causing variants and nine previously reported homozygous or compound heterozygous mutations in the CERKL, ABCA4, RPE65, ARL6, USH2A, PDE6B, and DHDDS genes. Mutations that could be sufficient to cause pathology were observed in more than one gene in one pedigree.

Establishing the involvement of the novel gene AGBL5 in retinitis pigmentosa by whole genome sequencing.

While more than 250 genes are known to cause inherited retinal degenerations (IRD), nearly 40-50% of families have the genetic basis for their disease unknown. In this study we sought to identify the underlying cause of IRD in a family by whole genome sequence (WGS) analysis. Clinical characterization including standard ophthalmic examination, fundus photography, visual field testing, electroretinography, and review of medical and family history was performed. WGS was performed on affected and unaffected family members using Illumina HiSeq X10. Sequence reads were aligned to hg19 using BWA-MEM and variant calling was performed with Genome Analysis Toolkit. The called variants were annotated with SnpEff v4.11, PolyPhen v2.2.2, and CADD v1.3. Copy number variations were called using Genome STRiP (svtoolkit 2.00.1611) and SpeedSeq software. Variants were filtered to detect rare potentially deleterious variants segregating with disease. Candidate variants were validated by dideoxy sequencing. Clinical evaluation revealed typical adolescent-onset recessive retinitis pigmentosa (arRP) in affected members. WGS identified about 4 million variants in each individual. Two rare and potentially deleterious compound heterozygous variants p.Arg281Cys and p.Arg487* were identified in the gene ATP/GTP binding protein like 5 (AGBL5) as likely causal variants. No additional variants in IRD genes that segregated with disease were identified. Mutation analysis confirmed the segregation of these variants with the IRD in the pedigree. Homology models indicated destabilization of AGBL5 due to the p.Arg281Cys change. Our findings establish the involvement of mutations in AGBL5 in RP and validate the WGS variant filtering pipeline we designed.