Purpose To devise a comprehensive multi-platform genetic testing strategy for inherited retinal disease and describe its performance in 1,000 consecutive families seen by a single clinician. Methods The clinical records of all patients seen by a single retina specialist between January 2010 and June 2016 were reviewed and all patients who met the clinical criteria for a diagnosis of inherited retinal disease were included in the study. Each patient was assigned to one of 62 diagnostic categories and this clinical diagnosis was used to define the scope and order of the molecular investigations that were performed. The number of nucleotides evaluated in a given subject ranged from two (a multiplex allele-specific assay for the most common mutations in BBS1 and BBS10) to nearly 900,000 (the coding sequences, and splice junctions of 305 genes known to cause inherited retinal disease). Results Disease-causing genotypes were identified in 760 families (76%). These genotypes were distributed across 104 different genes. More than 70% of these 104 genes have coding sequences small enough to be efficiently packaged into an adeno-associated virus. Mutations in ABCA4 were the most common cause of disease in this cohort (173 families) while mutations in 80 genes caused disease in five or fewer families (i.e., 0.5% or less). Disease-causing genotypes were identified in 576 of the families without next generation sequencing (NGS). This included 23 families with mutations in the repetitive region of RPGR exon 15 that would have been missed by NGS. Whole exome sequencing of the remaining 424 families revealed mutations in an additional 182, and whole genome sequencing of four of the remaining 242 families revealed two additional genotypes that were invisible by the other methods. Performing the testing in a clinically-focused tiered fashion would be 6.1% more sensitive, 17.7% less expensive and have a significantly lower average false genotype rate than using whole exome sequencing to assess more than 300 genes in all patients (7.1 vs. 128%; p<0.001). Conclusions Genetic testing for inherited retinal disease is now more than 75% sensitive. A clinically-directed tiered testing strategy can increase sensitivity and improve statistical significance without increasing cost.
Purpose To identify specific mutations causing North Carolina Macular Dystrophy (NCMD). Study Design Whole genome sequencing coupled with RT-PCR analysis of gene expression in human retinal cells. Subjects 141 members of 12 families with NCMD and 261 unrelated control individuals. Methods Genome sequencing was performed on eight affected individuals from three families affected with chromosome-6-linked NCMD (MCDR1) and two individuals affected with chromosome-5-linked NCMD (MCDR3). Variants observed in the MCDR1 locus with frequencies of less than 1% in published databases were confirmed using Sanger sequencing. Confirmed variants absent from all published databases were sought in affected individuals from 8 additional MCDR1 families and the 261 controls. RT-PCR analysis of selected genes was performed in stem-cell-derived human retinal cells. Main Outcome Measure Cosegregation of rare genetic variants with disease phenotype. Results Five sequenced individuals with MCDR1-linked NCMD shared a haplotype of 14 rare variants that spanned one megabase of the disease-causing allele. One of these variants (V1) was absent from all published databases and all 261 controls, but was found in five additional NCMD kindreds. This variant lies in a DNase 1 hypersensitivity site (DHS) upstream of both the PRDM13 and CCNC genes. Sanger sequencing of 1000 base pairs centered on V1 was performed in the remaining four NCMD probands and two additional novel single nucleotide variants (V2 in three families and V3 in a single family) were identified in the DHS within 134 base pairs of the location of V1. A complete duplication of the PRDM13 gene was also discovered in a single family (V4). RT-PCR analysis of PRDM13 expression in developing retinal cells revealed marked developmental regulation. Next generation sequencing of two individuals affected with chromosome-5-linked NCMD revealed a 900kb duplication that included the entire IRX1 gene (V5). The five mutations V1–V5 segregated perfectly in the 102 affected and 39 unaffected members of the 12 NCMD families. Conclusion We have identified five rare mutations that are each capable of arresting the development of the human macula. Four of these strongly implicate the involvement of the gene PRDM13 in macular development, while the pathophysiologic mechanism of the fifth remains unknown but may involve the developmental dysregulation of IRX1.
Age-related macular degeneration (AMD) is a common and devastating disease that can result in severe visual dysfunction. Over the last decade, great progress has been made in identifying genetic variants that contribute to AMD, many of which lie in genes involved in the complement cascade. In this review we discuss the significance of complement activation in AMD, particularly with respect to the formation of the membrane attack complex in the aging choriocapillaris. We review the clinical, histological and biochemical data that indicate that vascular loss in the choroid occurs very early in the pathogenesis of AMD, and discuss the potential impact of vascular dropout on the retinal pigment epithelium, Bruch's membrane and the photoreceptor cells. Finally, we present a hypothesis for the pathogenesis of early AMD and consider the implications of this model on the development of new therapies.
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