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Background Leber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) are inherited retinal diseases that cause early onset severe visual impairment. An accurate molecular diagnosis can refine the clinical diagnosis and allow gene specific treatments. Methods We developed a capture panel that enriches the exonic DNA of 163 known retinal disease genes. Using this panel, we performed targeted next generation sequencing (NGS) for a large cohort of 179 unrelated and prescreened patients with the clinical diagnosis of LCA or juvenile RP. Systematic NGS data analysis, Sanger sequencing validation, and segregation analysis were utilised to identify the pathogenic mutations. Patients were revisited to examine the potential phenotypic ambiguity at the time of initial diagnosis. Results Pathogenic mutations for 72 patients (40%) were identified, including 45 novel mutations. Of these 72 patients, 58 carried mutations in known LCA or juvenile RP genes and exhibited corresponding phenotypes, while 14 carried mutations in retinal disease genes that were not consistent with their initial clinical diagnosis. We revisited patients in the latter case and found that homozygous mutations in PRPH2 can cause LCA/juvenile RP. Guided by the molecular diagnosis, we reclassified the clinical diagnosis in two patients. Conclusions We have identified a novel gene and a large number of novel mutations that are associated with LCA/juvenile RP. Our results highlight the importance of molecular diagnosis as an integral part of clinical diagnosis.

Animal models of human disease are an invaluable component of studies aimed at understanding disease pathogenesis and therapeutic possibilities. Mutations in the gene encoding retinitis pigmentosa GTPase regulator (RPGR) are the most common cause of X-linked retinitis pigmentosa (XLRP) and are estimated to cause 20% of all retinal dystrophy cases. A majority of RPGR mutations are present in ORF15, the purine-rich terminal exon of the predominant splice-variant expressed in retina. Here we describe the genetic and phenotypic characterization of the retinal degeneration 9 (Rd9) strain of mice, a naturally occurring animal model of XLRP. Rd9 mice were found to carry a 32-base-pair duplication within ORF15 that causes a shift in the reading frame that introduces a premature-stop codon. Rpgr ORF15 transcripts, but not protein, were detected in retinas from Rd9/Y male mice that exhibited retinal pathology, including pigment loss and slowly progressing decrease in outer nuclear layer thickness. The levels of rhodopsin and transducin in rod outer segments were also decreased, and M-cone opsin appeared mislocalized within cone photoreceptors. In addition, electroretinogram (ERG) a- and b-wave amplitudes of both Rd9/Y male and Rd9/Rd9 female mice showed moderate gradual reduction that continued to 24 months of age. The presence of multiple retinal features that correlate with findings in individuals with XLRP identifies Rd9 as a valuable model for use in gaining insight into ORF15-associated disease progression and pathogenesis, as well as accelerating the development and testing of therapeutic strategies for this common form of retinal dystrophy.

Iris Heid, Gonçalo Abecasis, Sudha Iyengar and colleagues report the results of a large genome-wide association meta-analysis of macular degeneration based on over 43,000 subjects. They identify 16 new risk loci, including some very rare coding variants. Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants ( P < 5 × 10 −8 ) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 × 10 −10 ). Very rare coding variants (frequency <0.1%) in CFH , CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8 . Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.

Gonçalo Abecasis and colleagues report a large-scale meta-analysis of genome-wide association studies for age-related macular degeneration (AMD), including over 17,100 advanced cases and 60,000 controls. They identify seven loci newly associated with AMD and report pathway analysis that shows enrichment in the complement system and atherosclerosis signaling. Age-related macular degeneration (AMD) is a common cause of blindness in older individuals. To accelerate the understanding of AMD biology and help design new therapies, we executed a collaborative genome-wide association study, including >17,100 advanced AMD cases and >60,000 controls of European and Asian ancestry. We identified 19 loci associated at P < 5 × 10 −8 . These loci show enrichment for genes involved in the regulation of complement activity, lipid metabolism, extracellular matrix remodeling and angiogenesis. Our results include seven loci with associations reaching P < 5 × 10 −8 for the first time, near the genes COL8A1 - FILIP1L , IER3 - DDR1 , SLC16A8 , TGFBR1 , RAD51B , ADAMTS9 and B3GALTL . A genetic risk score combining SNP genotypes from all loci showed similar ability to distinguish cases and controls in all samples examined. Our findings provide new directions for biological, genetic and therapeutic studies of AMD.

Retinal degenerative diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are a leading cause of untreatable blindness with substantive impact on the quality of life of affected individuals and their families. Mouse mutants with retinal dystrophies have provided a valuable resource to discover human disease genes and helped uncover pathways critical for photoreceptor function. Here we show that the rd11 mouse mutant and its allelic strain, B6-JR2845, exhibit rapid photoreceptor dysfunction, followed by degeneration of both rods and cones. Using linkage analysis, we mapped the rd11 locus to mouse chromosome 13. We then identified a one-nucleotide insertion (c.420–421insG) in exon 3 of the Lpcat1 gene. Subsequent screening of this gene in the B6-JR2845 strain revealed a seven-nucleotide deletion (c.14–20delGCCGCGG) in exon 1. Both sequence changes are predicted to result in a frame-shift, leading to premature truncation of the lysophosphatidylcholine acyltransferase-1 (LPCAT1) protein. LPCAT1 (also called AYTL2) is a phospholipid biosynthesis/remodeling enzyme that facilitates the conversion of palmitoyl-lysophosphatidylcholine to dipalmitoyl-phosphatidylcholine (DPPC). The analysis of retinal lipids from rd11 and B6-JR2845 mice showed substantially reduced DPPC levels compared with C57BL/6J control mice, suggesting a causal link to photoreceptor dysfunction. A follow-up screening of LPCAT1 in retinitis pigmentosa and Leber congenital amaurosis patients did not reveal any obvious disease-causing mutations. Previously, LPCAT1 has been suggested to be critical for the production of lung surfactant phospholipids and biosynthesis of platelet-activating factor in noninflammatory remodeling pathway. Our studies add another dimension to an essential role for LPCAT1 in retinal photoreceptor homeostasis.

Mutations in the Crumbs homologue 1 (CRB1) gene have been reported in patients with a variety of autosomal recessive retinal dystrophies, including retinitis pigmentosa (RP) with preserved paraarteriolar retinal pigment epithelium (PPRPE), RP with Coats‐like exudative vasculopathy, early onset RP without PPRPE, and Leber congenital amaurosis (LCA). We extended our investigations of CRB1 in these retinal dystrophies, and identified nine novel CRB1 sequence variants. In addition, we screened patients with “classic” RP and classic Coats disease (without RP), but no pathologic sequence variants were found in the CRB1 gene. In total, 71 different sequence variants have been identified on 184 CRB1 alleles of patients with retinal dystrophies, including amino acid substitutions, frameshift, nonsense, and splice site mutations, in‐frame deletions, and large insertions. Recent studies in two animal models, mouse and Drosophila, and in vivo high‐resolution microscopy in patients with LCA, have shed light on the role of CRB1 in the pathogenesis of retinal dystrophies and its function in the photoreceptors. In this article, we provide an overview of the currently known CRB1 sequence variants, predict their effect, and propose a genotype–phenotype correlation model for CRB1 mutations. Hum Mutat 24:355–369, 2004. © 2004 Wiley‐Liss, Inc.

Inherited retinopathies are a genetically and phenotypically heterogeneous group of diseases affecting approximately one in 2000 individuals worldwide. For the past 10 years, the Laboratory for Molecular Diagnosis of Inherited Eye Diseases (LMDIED) at the University of Texas‐Houston Health Science Center has screened subjects ascertained in the United States and Canada for mutations in genes causing dominant and recessive autosomal retinopathies. A combination of single strand conformational analysis (SSCA) and direct sequencing of five genes (rhodopsin, peripherin/RDS, RP1, CRX, and AIPL1) identified the disease‐causing mutation in approximately one‐third of subjects with autosomal dominant retinitis pigmentosa (adRP) or with autosomal dominant cone‐rod dystrophy (adCORD). In addition, the causative mutation was identified in 15% of subjects with Leber congenital amaurosis (LCA). Overall, we report identification of the causative mutation in 105 of 506 (21%) of unrelated subjects (probands) tested; we report five previously unreported mutations in rhodopsin, two in peripherin/RDS, and one previously unreported mutation in the cone‐rod homeobox gene, CRX. Based on this large survey, the prevalence of disease‐causing mutations in each of these genes within specific disease categories is estimated. These data are useful in estimating the frequency of specific mutations and in selecting individuals and families for mutation‐specific studies. Hum Mutat 17:42–51, 2001. © 2001 Wiley‐Liss, Inc.

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.

In this genomewide association study of age-related corneal dystrophy, there was a significant association with variants of TCF4, which encodes the E2-2 transcription factor. The risk variant of the most strongly associated genetic marker had an odds ratio of approximately 5. Fuchs's corneal dystrophy (FCD) is a progressive, bilateral condition characterized by dysfunction of the corneal endothelium, leading to reduced vision. The prevalence of FCD has been estimated at about 5% among persons over the age of 40 years in the United States. 1 Corneal edema that is associated with FCD may progress after cataract surgery and is the most common indication for the 42,000 corneal transplantations that take place each year in the United States. Enthusiasm for refractive surgery is decreased among patients who are at high risk for FCD. 2 Thus, the ability to diagnose FCD before symptoms develop and knowledge . . .

Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes.