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Exome and whole-genome analyses powered by next-generation sequencing (NGS) have become invaluable tools in identifying causal mutations responsible for Mendelian disorders. Given that individual exomes contain several thousand single nucleotide variants and insertions/deletions, it remains a challenge to analyze large numbers of variants from multiple exomes to identify causal alleles associated with inherited conditions. To this end, we have developed user-friendly software that analyzes variant calls from multiple individuals to facilitate identification of causal mutations. The software, termed exomeSuite, filters for putative causative variants of monogenic diseases inherited in one of three forms: dominant, recessive caused by a homozygous variant, or recessive caused by two compound heterozygous variants. In addition, exomeSuite can perform homozygosity mapping and analyze the variant data of multiple unrelated individuals. Here we demonstrate that filtering of variants with exomeSuite reduces datasets to a fraction of a percent of their original size. To the best of our knowledge this is the first freely available software developed to analyze variant data from multiple individuals that rapidly assimilates and filters large data sets based on pattern of inheritance.

Purpose: The purpose of this project was to determine the spectrum and frequency of mutations in the small nuclear riboprotein 200 kDa gene (SNRNP200) that cause autosomal dominant retinitis pigmentosa (adRP). Methods: A well-characterized adRP cohort of 2

Macular degeneration is a common cause of blindness in the elderly. To identify rare coding variants associated with a large increase in risk of age-related macular degeneration (AMD), we sequenced 2,335 cases and 789 controls in 10 candidate loci (57 gene

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., (© Published by Oxford University Press 2023.)

Purpose: The purpose of this study was to characterize the phenotypic spectrum of ophthalmic findings in patients with Alagille syndrome., Methods: We conducted a retrospective, observational, multicenter, study on 46 eyes of 23 subjects with Alagille syndrome. We reviewed systemic and ophthalmologic data extracted from medical records, color fundus photography, fundus autofluorescence, optical coherence tomography, visual fields, electrophysiological assessments, and molecular genetic findings., Results: Cardiovascular abnormalities were found in 83% of all cases (of those, 74% had cardiac murmur), whereas 61% had a positive history of hepatobiliary issues, and musculoskeletal anomalies were present in 61% of all patients. Dysmorphic facies were present in 16 patients, with a broad forehead being the most frequent feature. Ocular symptoms were found in 91%, with peripheral vision loss being the most frequent complaint. Median (range) Snellen visual acuity of all eyes was 20/25 (20/20 to hand motion [HM]). Anterior segment abnormalities were present in 74% of the patients; of those, posterior embryotoxon was the most frequent finding. Abnormalities of the optic disc were found in 52%, and peripheral retinal abnormalities were the most frequent ocular finding in this series, found in 96% of all patients. Fifteen JAG1 mutations were identified in 16 individuals; of those, 6 were novel., Conclusions: This study reports a cohort of patients with Alagille syndrome in which peripheral chorioretinal changes were more frequent than posterior embryotoxon, the most frequent ocular finding according to a number of previous studies. We propose that these peripheral chorioretinal changes are a new hallmark to help diagnose this syndrome.

Major advances in the study of inherited retinal diseases (IRDs) have placed efforts to develop treatments for these blinding conditions at the forefront of the emerging field of precision medicine. As a result, the growth of clinical trials for IRDs has increased rapidly over the past decade and is expected to further accelerate as more therapeutic possibilities emerge and qualified participants are identified. Although guided by established principles, these specialized trials, requiring analysis of novel outcome measures and endpoints in small patient populations, present multiple challenges relative to study design and ethical considerations. This position paper reviews recent accomplishments and existing challenges in clinical trials for IRDs and presents a set of recommendations aimed at rapidly advancing future progress. The goal is to stimulate discussions among researchers, funding agencies, industry, and policy makers that will further the design, conduct, and analysis of clinical trials needed to accelerate the approval of effective treatments for IRDs, while promoting advocacy and ensuring patient safety., Competing Interests: Disclosure: D.A. Thompson, MeiraGTx (F), filed patent on gene therapy (P); A. Iannaccone, Editas Medicine (C), Astellas (C), Roivant Pharma (C), IQVIA (C), Gyroscope/Orbit Biomedical (C), Rhythm Pharmaceuticals (C), Applied Genetic Technologies Corp (F), Allergan (F), Acucela (F), ProQR Therapeutics (F), Foundation Fighting Blindness Clinical Research Institute (F), Alia Therapeutics (S); R.R. Ali, MeiraGTx (S), filed patents on gene and cell therapies (P); V.Y. Arshavsky, None; I. Audo, None; J.W.B. Bainbridge, MeiraGTx (S), filed patents on gene and cell therapies (P); C.G. Besirli, MeiraGTx (F), Spark Therapeutics (F); D.G. Birch, Nightstar Therapeutics/Biogen (C), Applied Genetic Technologies Corp (S), Nacuity Pharmaceuticals (C), Editas Medicine (C), Acucela (S), Foundation Fighting Blindness (C), ProQR Therapeutics (C); K.E. Branham, ProQR Therapeutics (C); A.V. Cideciyan, Patents on gene therapies for RPGR, RHO, and NPHP5-associated IRDs (P); ProQR Therapeutics (F), IVERIC bio (F); S.P. Daiger, Applied Genetic Technologies Corp (S); D. Dalkara, Patent on AAV virions with variant capsid and methods of use (P); J.L. Duncan, 4D Therapeutics (C), Applied Genetic Technologies Corp (C), Editas Medicine (C), Gyroscope (C), Horama (C), Nightstar Therapeutics/Biogen (C), ProQR Therapeutics (C), SparingVision (S), Spark Therapeutics (C), Vedere Bio (C), Acucela (F), Allergan (F), Neurotech (F), Second Sight Medical Products (F); A.T. Fahim, None; J.G. Flannery, None; R. Gattegna, None; J.R. Heckenlively, None; E. Heon, None; K.T. Jayasundera, Editas Medicine (C); N.W. Khan, None; H. Klassen, jCyte (S, I); B.P. Leroy, Bayer (C), Nightstar Therapeutics/Biogen (C), IVERIC bio (C), Novartis Pharma (C), RegenX Bio (C), Vedere Bio (C); Novartis (R), Spark Therapeutics (R); GenSight Biologics (F), ProQR Therapeutics (F); R.S. Molday, Applied Genetic Technologies Corp (S); D.C. Musch, Belite Bio (S), Mactel Group NTMT-03 trial (S), NEI intramural branch clinical trials (S); M.E. Pennesi, Adverum (C), Allergan (C), Astellas (C), Pharmaceuticals (C), Biogen (C), IVERIC bio (C), Novartis (C), Regenix Bio (C), Spark Therapeutics (C), Wave Biosciences (C), Eyevensys (S), Horama (S), Nayan (S), Nacuity Pharmaceuticals (S, I), Ocugen (S, I), Verede (S), ProQR Therapeutics (S), Gensight (S), Applied Genetic Technologies Corp (F), Sanofi (F), Foundation Fighting Blindness (F), ProQR Therapeutics (F), Allergan (F); S.M. Petersen-Jones, None; E.A. Pierce, Astellas (C), Merck (C), Sanofi-Genzyme (C), Wave Therapeutics (C); GenSight Biologics (S), Odylia Therapeutics (S), Opsis Therapeutics (S), Sana Biotechnology (S); CRISPR Therapeutics (F), Spark Therapeutics (F); Editas Medicine (F), MeiraGTx (F), ProQR Therapeutics (F), filed patents on gene and genetic therapies (P); R.C. Rao, None; T.A. Reh, Nayan Pharmaceuticals (C, I); J.A. Sahel, Pixium Vision (S, I), GenSight Biologics (S, I), SparingVision (S, I); Prophesee (I), Chronolife (I); D. Sharon, None; P.A. Sieving, None; E. Strettoi, None; P. Yang, Astellas (C), Applied Genetic Technologies Corp (C); D.N. Zacks, ONL Therapeutics (C, I, P), (Copyright 2020 The Authors.)

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.

Purpose: To identify the causes of autosomal dominant retinitis pigmentosa (adRP) in a cohort of families without mutations in known adRP genes and consequently to characterize a novel dominant-acting missense mutation in SAG., Methods: Patients underwent ophthalmologic testing and were screened for mutations using targeted-capture and whole-exome next-generation sequencing. Confirmation and additional screening were done by Sanger sequencing. Haplotypes segregating with the mutation were determined using short tandem repeat and single nucleotide variant polymorphisms. Genealogies were established by interviews of family members., Results: Eight families in a cohort of 300 adRP families, and four additional families, were found to have a novel heterozygous mutation in the SAG gene, c.440G>T; p.Cys147Phe. Patients exhibited symptoms of retinitis pigmentosa and none showed symptoms characteristic of Oguchi disease. All families are of Hispanic descent and most were ascertained in Texas or California. A single haplotype including the SAG mutation was identified in all families. The mutation dramatically alters a conserved amino acid, is extremely rare in global databases, and was not found in 4000+ exomes from Hispanic controls. Molecular modeling based on the crystal structure of bovine arrestin-1 predicts protein misfolding/instability., Conclusions: This is the first dominant-acting mutation identified in SAG, a founder mutation possibly originating in Mexico several centuries ago. The phenotype is clearly adRP and is distinct from the previously reported phenotypes of recessive null mutations, that is, Oguchi disease and recessive RP. The mutation accounts for 3% of the 300 families in the adRP Cohort and 36% of Hispanic families in this cohort.

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.

Although rare in the general population, retinal dystrophies occupy a central position in current efforts to develop innovative therapies for blinding diseases. This status derives, in part, from the unique biology, accessibility, and function of the retina, as well as from the synergy between molecular discoveries and transformative advances in functional assessment and retinal imaging. The combination of these factors has fueled remarkable progress in the field, while at the same time creating complex challenges for organizing collective efforts aimed at advancing translational research. The present position paper outlines recent progress in gene therapy and cell therapy for this group of disorders, and presents a set of recommendations for addressing the challenges remaining for the coming decade. It is hoped that the formulation of these recommendations will stimulate discussions among researchers, funding agencies, industry, and policy makers that will accelerate the development of safe and effective treatments for retinal dystrophies and related diseases., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Age-related macular degeneration (AMD) is a major cause of blindness in the western world. While genetic studies have linked both common and rare variants in genes involved in regulation of the complement system to increased risk of development of AMD, environmental factors, such as smoking and nutrition, can also significantly affect the risk of developing the disease and the rate of disease progression. Since epigenetics has been implicated in mediating, in part, the disease risk associated with some environmental factors, we investigated a possible epigenetic contribution to AMD. We performed genome-wide DNA methylation profiling of blood from AMD patients and controls. No differential methylation site reached genome-wide significance; however, when epigenetic changes in and around known GWAS-defined AMD risk loci were explored, we found small but significant DNA methylation differences in the blood of neovascular AMD patients near age-related maculopathy susceptibility 2 (ARMS2), a top-ranked GWAS locus preferentially associated with neovascular AMD. The methylation level of one of the CpG sites significantly correlated with the genotype of the risk SNP rs10490924, suggesting a possible epigenetic mechanism of risk. Integrating genome-wide DNA methylation analysis of retina samples with and without AMD together with blood samples, we further identified a consistent, replicable change in DNA methylation in the promoter region of protease serine 50 (PRSS50). These methylation changes may identify sites in novel genes that are susceptible to non-genetic factors known to contribute to AMD development and progression.

Neurodegenerative diseases affecting the macula constitute a major cause of incurable vision loss and exhibit considerable clinical and genetic heterogeneity, from early-onset monogenic disease to multifactorial late-onset age-related macular degeneration (AMD). As part of our continued efforts to define genetic causes of macular degeneration, we performed whole exome sequencing in four individuals of a two-generation family with autosomal dominant maculopathy and identified a rare variant p.Glu1144Lys in Fibrillin 2 (FBN2), a glycoprotein of the elastin-rich extracellular matrix (ECM). Sanger sequencing validated the segregation of this variant in the complete pedigree, including two additional affected and one unaffected individual. Sequencing of 192 maculopathy patients revealed additional rare variants, predicted to disrupt FBN2 function. We then undertook additional studies to explore the relationship of FBN2 to macular disease. We show that FBN2 localizes to Bruch's membrane and its expression appears to be reduced in aging and AMD eyes, prompting us to examine its relationship with AMD. We detect suggestive association of a common FBN2 non-synonymous variant, rs154001 (p.Val965Ile) with AMD in 10 337 cases and 11 174 controls (OR = 1.10; P-value = 3.79 × 10(-5)). Thus, it appears that rare and common variants in a single gene--FBN2--can contribute to Mendelian and complex forms of macular degeneration. Our studies provide genetic evidence for a key role of elastin microfibers and Bruch's membrane in maintaining blood-retina homeostasis and establish the importance of studying orphan diseases for understanding more common clinical phenotypes., (Published by Oxford University Press 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Next generation sequencing (NGS) technology, with the ability to sequence many genomic regions at once, can provide clinicians with increased information, in the form of more mutations detected. Discussions on broad testing technology have largely been focused on incidental findings, or unanticipated results related to diseases beyond the primary indication for testing. By examining multiple genes that could be responsible for the patient's presentation, however, there is also the possibility of unexpected results that are related to the reason genetic testing was ordered. We present a case study where multiple potentially causative mutations were detected using NGS technology. This case raises questions of scientific uncertainty, and has important implications for medical management and secondary studies. Clinicians and genetic counselors should be aware of the potential for increased information to affect one's understanding of genetic risk, and the pre- and post-testing counseling process.

Estimating individual ancestry is important in genetic association studies where population structure leads to false positive signals, although assigning ancestry remains challenging with targeted sequence data. We propose a new method for the accurate estimation of individual genetic ancestry, based on direct analysis of off-target sequence reads, and implement our method in the publicly available LASER software. We validate the method using simulated and empirical data and show that the method can accurately infer worldwide continental ancestry when used with sequencing data sets with whole-genome shotgun coverage as low as 0.001×. For estimates of fine-scale ancestry within Europe, the method performs well with coverage of 0.1×. On an even finer scale, the method improves discrimination between exome-sequenced study participants originating from different provinces within Finland. Finally, we show that our method can be used to improve case-control matching in genetic association studies and to reduce the risk of spurious findings due to population structure.

Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling.

Age-related macular degeneration (AMD) is a leading cause of visual impairment worldwide. Aberrant DNA methylation within the promoter of IL17RC in peripheral blood mononuclear cells has recently been reported in AMD. To validate this association, we examined DNA methylation of the IL17RC promoter in peripheral blood. First, we used Illumina Human Methylation450 Bead Arrays, a widely accepted platform for measuring global DNA methylation. Second, methylation status at multiple sites within the IL17RC promoter was determined by bisulfite pyrosequencing in two cohorts. Third, a methylation-sensitive quantitative PCR-based assay was performed on a subset of samples. In contrast to previous findings, we did not find evidence of differential methylation between AMD cases and age-matched controls. We conclude that hypomethylation within the IL17RC gene promoter in peripheral blood is not suitable for use as a clinical biomarker of AMD. This study highlights the need for considerable replication of epigenetic association studies prior to clinical application., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: The purpose of this project was to determine the spectrum and frequency of mutations in the small nuclear riboprotein 200 kDa gene (SNRNP200) that cause autosomal dominant retinitis pigmentosa (adRP)., Methods: A well-characterized adRP cohort of 251 families was tested for mutations in the exons and intron/exon junctions of SNRNP200 using fluorescent dideoxy sequencing. An additional 21 adRP families from the eyeGENE® Network were tested for possible mutations. Bioinformatic and segregation analysis was performed on novel variants., Results: SNRNP200 mutations were identified in seven of the families tested. Two previously reported mutations, p.Arg681Cys and p.Ser1087Leu, were found in two families each. One family had the previously reported p.Arg681His mutation. Two novel SNRNP200 variants, p.Pro682Ser and p.Ala542Val, were also identified in one family each. Bioinformatic and segregation analyses suggested that these novel variants are likely to be pathogenic. Clinical examination of patients with SNRNP200 mutations showed a wide range of clinical symptoms and severity, including one instance of non-penetrance., Conclusions: Mutations in SNRNP200 caused 1.6% of disease in our adRP cohort. Pathogenic mutations were found primarily in exons 16 and 25, but the novel p.Ala542Val mutation in exon 13 suggests that variation in other genetic regions is also responsible for causing dominant disease. SNRNP200 mutations were associated with a wide range of clinical symptoms similar to those of individuals with other splice-factor gene mutations.

Macular degeneration is a common cause of blindness in the elderly. To identify rare coding variants associated with a large increase in risk of age-related macular degeneration (AMD), we sequenced 2,335 cases and 789 controls in 10 candidate loci (57 genes). To increase power, we augmented our control set with ancestry-matched exome-sequenced controls. An analysis of coding variation in 2,268 AMD cases and 2,268 ancestry-matched controls identified 2 large-effect rare variants: previously described p.Arg1210Cys encoded in the CFH gene (case frequency (fcase) = 0.51%; control frequency (fcontrol) = 0.02%; odds ratio (OR) = 23.11) and newly identified p.Lys155Gln encoded in the C3 gene (fcase = 1.06%; fcontrol = 0.39%; OR = 2.68). The variants suggest decreased inhibition of C3 by complement factor H, resulting in increased activation of the alternative complement pathway, as a key component of disease biology.

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.

Purpose: We determined the fraction of families in a well-characterized cohort with a provisional diagnosis of autosomal dominant retinitis pigmentosa (adRP) that have disease-causing mutations in the X-linked retinitis pigmentosa GTPase regulator (RPGR) gene or the retinitis pigmentosa 2 (RP2) gene., Methods: Families with a provisional clinical diagnosis of adRP, and a pedigree consistent with adRP but no male-to-male transmission were selected from a cohort of 258 families, and tested for mutations in the RPGR and RP2 genes with di-deoxy sequencing. To facilitate testing of RPGR in "adRP" families that had no male members available for testing, the repetitive and purine-rich ORF15 of RPGR was subcloned and sequenced in heterozygous female subjects from 16 unrelated families., Results: Direct sequencing of RPGR and RP2 allowed for identification of a disease-causing mutation in 21 families. Of these "adRP" families 19 had RPGR mutations, and two had RP2 mutations. Subcloning and sequencing of ORF15 of RPGR in female subjects identified one additional RPGR mutation. Of the 22 mutations identified, 15 have been reported previously., Conclusions: These data show that 8.5% (22 in 258) of families thought to have adRP truly have X-linked retinitis pigmentosa (XLRP). These results have substantive implications for calculation of recurrence risk, genetic counseling, and potential treatment options, and illustrate the importance of screening families with a provisional diagnosis of autosomal inheritance and no male-to-male transmission for mutations in X-linked genes. Mutations in RPGR are one of the most common causes of all forms of retinitis pigmentosa.

Molecular diagnostics for patients with retinitis pigmentosa (RP) has been hampered by extreme genetic and clinical heterogeneity, with 52 causative genes known to date. Here, we developed a comprehensive next-generation sequencing (NGS) approach for the clinical molecular diagnostics of RP. All known inherited retinal disease genes (n = 111) were captured and simultaneously analyzed using NGS in 100 RP patients without a molecular diagnosis. A systematic data analysis pipeline was developed and validated to prioritize and predict the pathogenicity of all genetic variants identified in each patient, which enabled us to reduce the number of potential pathogenic variants from approximately 1,200 to zero to nine per patient. Subsequent segregation analysis and in silico predictions of pathogenicity resulted in a molecular diagnosis in 36 RP patients, comprising 27 recessive, six dominant, and three X-linked cases. Intriguingly, De novo mutations were present in at least three out of 28 isolated cases with causative mutations. This study demonstrates the enormous potential and clinical utility of NGS in molecular diagnosis of genetically heterogeneous diseases such as RP. De novo dominant mutations appear to play a significant role in patients with isolated RP, having major implications for genetic counselling., (© 2012 Wiley Periodicals, Inc.)

Background: Variation in the complement factor H gene (CFH) is associated with risk of late age-related macular degeneration (AMD). Previous studies have been case-control studies in populations of European ancestry with little differentiation in AMD subtype, and insufficient power to confirm or refute effect modification by smoking., Methods: To precisely quantify the association of the single nucleotide polymorphism (SNP rs1061170, 'Y402H') with risk of AMD among studies with differing study designs, participant ancestry and AMD grade and to investigate effect modification by smoking, we report two unpublished genetic association studies (n = 2759) combined with data from 24 published studies (26 studies, 26,494 individuals, including 14,174 cases of AMD) of European ancestry, 10 of which provided individual-level data used to test gene-smoking interaction; and 16 published studies from non-European ancestry., Results: In individuals of European ancestry, there was a significant association between Y402H and late-AMD with a per-allele odds ratio (OR) of 2.27 [95% confidence interval (CI) 2.10-2.45; P = 1.1 x 10(-161)]. There was no evidence of effect modification by smoking (P = 0.75). The frequency of Y402H varied by ancestral origin and the association with AMD in non-Europeans was less clear, limited by paucity of studies., Conclusion: The Y402H variant confers a 2-fold higher risk of late-AMD per copy in individuals of European descent. This was stable to stratification by study design and AMD classification and not modified by smoking. The lack of association in non-Europeans requires further verification. These findings are of direct relevance for disease prediction. New research is needed to ascertain if differences in circulating levels, expression or activity of factor H protein explain the genetic association.

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 dipalmitoylphosphatidylcholine (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.

Objectives: To assess the phenotype of patients with X-linked retinitis pigmentosa (XLRP) with RP2 mutations and to correlate the findings with their genotype., Methods: Six hundred eleven patients with RP were screened for RP2 mutations. From this screen, 18 patients with RP2 mutations were evaluated clinically with standardized electroretinography, Goldmann visual fields, and ocular examinations. In addition, 7 well-documented cases from the literature were used to augment genotype-phenotype correlations., Results: Of 11 boys younger than 12 years, 10 (91%) had macular involvement and 9 (82%) had best-corrected visual acuity worse than 20/50. Two boys from different families (aged 8 and 12 years) displayed a choroideremia-like fundus, and 9 boys (82%) were myopic (mean error, -7.97 diopters [D]). Of 10 patients with electroretinography data, 9 demonstrated severe rod-cone dysfunction. All 3 female carriers had macular atrophy in 1 or both eyes and were myopic (mean, -6.23 D). All 9 nonsense and frameshift and 5 of 7 missense mutations (71%) resulted in severe clinical presentations., Conclusions: Screening of the RP2 gene should be prioritized in patients younger than 16 years characterized by X-linked inheritance, decreased best-corrected visual acuity (eg, >20/40), high myopia, and early-onset macular atrophy. Patients exhibiting a choroideremia-like fundus without choroideremia gene mutations should also be screened for RP2 mutations., Clinical Relevance: An identifiable phenotype for RP2-XLRP aids in clinical diagnosis and targeted genetic screening.

We executed a genome-wide association scan for age-related macular degeneration (AMD) in 2,157 cases and 1,150 controls. Our results validate AMD susceptibility loci near CFH (P < 10(-75)), ARMS2 (P < 10(-59)), C2/CFB (P < 10(-20)), C3 (P < 10(-9)), and CFI (P < 10(-6)). We compared our top findings with the Tufts/Massachusetts General Hospital genome-wide association study of advanced AMD (821 cases, 1,709 controls) and genotyped 30 promising markers in additional individuals (up to 7,749 cases and 4,625 controls). With these data, we identified a susceptibility locus near TIMP3 (overall P = 1.1 x 10(-11)), a metalloproteinase involved in degradation of the extracellular matrix and previously implicated in early-onset maculopathy. In addition, our data revealed strong association signals with alleles at two loci (LIPC, P = 1.3 x 10(-7); CETP, P = 7.4 x 10(-7)) that were previously associated with high-density lipoprotein cholesterol (HDL-c) levels in blood. Consistent with the hypothesis that HDL metabolism is associated with AMD pathogenesis, we also observed association with AMD of HDL-c-associated alleles near LPL (P = 3.0 x 10(-3)) and ABCA1 (P = 5.6 x 10(-4)). Multilocus analysis including all susceptibility loci showed that 329 of 331 individuals (99%) with the highest-risk genotypes were cases, and 85% of these had advanced AMD. Our studies extend the catalog of AMD associated loci, help identify individuals at high risk of disease, and provide clues about underlying cellular pathways that should eventually lead to new therapies.

Retinitis pigmentosa (RP) refers to a genetically heterogeneous group of progressive neurodegenerative diseases that result in dysfunction and/or death of rod and cone photoreceptors in the retina. So far, 18 genes have been identified for autosomal-dominant (ad) RP. Here, we describe an adRP locus (RP42) at chromosome 7p15 through linkage analysis in a six-generation Scandinavian family and identify a disease-causing mutation, c.449G-->A (p.S150N), in exon 6 of the KLHL7 gene. Mutation screening of KLHL7 in 502 retinopathy probands has revealed three different missense mutations in six independent families. KLHL7 is widely expressed, including expression in rod photoreceptors, and encodes a 75 kDa protein of the BTB-Kelch subfamily within the BTB superfamily. BTB-Kelch proteins have been implicated in ubiquitination through Cullin E3 ligases. Notably, all three putative disease-causing KLHL7 mutations are within a conserved BACK domain; homology modeling suggests that mutant amino acid side chains can potentially fill the cleft between two helices, thereby affecting the ubiquitination complexes. Mutations in an identical region of another BTB-Kelch protein, gigaxonin, have previously been associated with giant axonal neuropathy. Our studies suggest an additional role of the ubiquitin-proteasome protein-degradation pathway in maintaining neuronal health and in disease.

Objective: To report discordant phenotypes, resulting from the same mutation in exon ORF15 (GenBank AF286472) of the retinitis pigmentosa GTPase regulator gene (RPGR) (GenBank U57629), in 2 presumed dizygotic twin brothers with X-linked retinal disease., Methods: The 2 brothers underwent complete ophthalmic examination that included best-corrected visual acuity, slitlamp biomicroscopy, and detailed fundus examination. Visual field recording using Goldmann kinetic perimetry and a full-field electroretinogram were also obtained in both patients. Mutational screening was performed for RPGR because of an X-linked pattern of inheritance indicated by pedigree analysis., Results: One brother had a phenotypic expression of cone-rod dystrophy, while the other exhibited X-linked retinitis pigmentosa. A 1-nucleotide deletion was identified in the 3' region of exon ORF15 of RPGR (ORF15 + 1339delA)., Conclusions: An identical mutation in RPGR-ORF15 manifested distinct clinical phenotypes in individuals of the same family. Our data provide strong evidence in support of additional modifier genes that can produce diverse disease phenotypes in patients with RPGR mutations., Clinical Relevance: The clinical observation of different retinal phenotypes in a family with the same mutation in exon ORF15 of RPGR implicates the potential importance of modifier genes for the phenotypic expression of this form of X-linked retinal disease.

Age-related macular degeneration (AMD) is a progressive neurodegenerative disease, which affects quality of life for millions of elderly individuals worldwide. AMD is associated with a diverse spectrum of clinical phenotypes, all of which include the death of photoreceptors in the central part of the human retina (called the macula). Tremendous progress has been made in identifying genetic susceptibility variants for AMD. Variants at chromosome 1q32 (in the region of CFH) and 10q26 (LOC387715/ARMS2) account for a large part of the genetic risk to AMD and have been validated in numerous studies. In addition, susceptibility variants at other loci, several as yet unidentified, make substantial cumulative contribution to genetic risk for AMD; among these, multiple studies support the role of variants in APOE and C2/BF genes. Genome-wide association and re-sequencing projects, together with gene-environment interaction studies, are expected to further define the causal relationships that connect genetic variants to AMD pathogenesis and should assist in better design of prevention and intervention.

Genetic variants at chromosomes 1q31-32 and 10q26 are strongly associated with susceptibility to age-related macular degeneration (AMD), a common blinding disease of the elderly. We demonstrate, by evaluating 45 tag SNPs spanning HTRA1, PLEKHA1, and predicted gene LOC387715/ARMS2, that rs10490924 SNP alone, or a variant in strong linkage disequilibrium, can explain the bulk of association between the 10q26 chromosomal region and AMD. A previously suggested causal SNP, rs11200638, and other examined SNPs in the region are only indirectly associated with the disease. Contrary to previous reports, we show that rs11200638 SNP has no significant impact on HTRA1 promoter activity in three different cell lines, and HTRA1 mRNA expression exhibits no significant change between control and AMD retinas. However, SNP rs10490924 shows the strongest association with AMD (P = 5.3 x 10(-30)), revealing an estimated relative risk of 2.66 for GT heterozygotes and 7.05 for TT homozygotes. The rs10490924 SNP results in nonsynonymous A69S alteration in the predicted protein LOC387715/ARMS2, which has a highly conserved ortholog in chimpanzee, but not in other vertebrate sequences. We demonstrate that LOC387715/ARMS2 mRNA is detected in the human retina and various cell lines and encodes a 12-kDa protein, which localizes to the mitochondrial outer membrane when expressed in mammalian cells. We propose that rs10490924 represents a major susceptibility variant for AMD at 10q26. A likely biological mechanism is that the A69S change in the LOC387715/ARMS2 protein affects its presumptive function in mitochondria.

Unlabelled: The Madeline 2.0 Pedigree Drawing Engine (PDE) is a pedigree drawing program for use in linkage and family-based association studies. The program is designed to handle large and complex pedigrees with an emphasis on readability and aesthetics. For complex pedigrees, we use a hybrid algorithm in which consanguinous loops are drawn as cyclic graphs whenever possible, but we resort to acyclic graphs when matings can no longer be connected without line crossings. A similar hybrid approach is used to avoid line crossings for matings between distant descendants of different founding groups. Written in object-oriented C++ and released under the GNU General Public License (GPL), Madeline 2.0 PDE reads input files specified on the command line and generates pedigree drawings without user interaction. Pedigree output in scalable vector graphics (SVG) format can be viewed in browsers with native SVG rendering support or in vector graphics editors. We provide an easy-to-use public web service, which is experimental and still under development., Availability: http://kellogg.umich.edu/madeline.

Purpose: To investigate macular photoreceptor structure in patients with inherited retinal degeneration using high-resolution images and to correlate the findings with clinical phenotypes and genetic mutations., Methods: Adaptive optics scanning laser ophthalmoscopy (AOSLO) images of photoreceptors were obtained in 16 eyes: five with retinitis pigmentosa (RP), three with cone-rod dystrophy (CRD), and eight without retinal disease. A quadratic model was used to illustrate cone spacing as a function of retinal eccentricity. Cone spacing at 1 degrees eccentricity was compared with standard measures of central visual function, including best-corrected visual acuity (BCVA), foveal threshold, and multifocal electroretinogram (mfERG) amplitude and timing. Intervisit variations were studied in one patient with RP and one patient with CRD. Screening of candidate disease genes identified mutations in two patients, one with RP (a rhodopsin mutation) and the other with CRD (a novel RPGR-ORF15 mutation)., Results: Cone spacing values were significantly different from normal for patients with RP (P = 0.01) and CRD (P < 0.0001) and demonstrated a statistically significant correlation with foveal threshold (P = 0.0003), BCVA (P = 0.01), and mfERG amplitude (P = 0.008). Although many RP patients showed normal cone spacing within 1 degrees of fixation, cones could not be unambiguously identified in several retinal regions. Cone spacing increased in all CRD patients, even those with early disease. Little variation was observed in cone spacing measured during two sessions fewer than 8 days apart., Conclusions: AOSLO images can be used to study macular cones with high resolution in patients with retinal degeneration. The authors present the first report of cone structure in vivo in patients with mutations in rhodopsin and RPGR-ORF15 and show that macular cones display distinct characteristics, depending on the underlying disease. AOSLO imaging, therefore, can provide new insight into possible mechanisms of cone vision loss in patients with retinal degeneration.

In developed countries, age-related macular degeneration is a common cause of blindness in the elderly. A common polymorphism, encoding the sequence variation Y402H in complement factor H (CFH), has been strongly associated with disease susceptibility. Here, we examined 84 polymorphisms in and around CFH in 726 affected individuals (including 544 unrelated individuals) and 268 unrelated controls. In this sample, 20 of these polymorphisms showed stronger association with disease susceptibility than the Y402H variant. Further, no single polymorphism could account for the contribution of the CFH locus to disease susceptibility. Instead, multiple polymorphisms defined a set of four common haplotypes (of which two were associated with disease susceptibility and two seemed to be protective) and multiple rare haplotypes (associated with increased susceptibility in aggregate). Our results suggest that there are multiple disease susceptibility alleles in the region and that noncoding CFH variants play a role in disease susceptibility.

Using a large sample of cases and controls from a single center, we show that a T-->C substitution in exon 9 (Y402H) of the complement factor H gene is strongly associated with susceptibility to age-related macular degeneration, the most common cause of blindness in the elderly. Frequency of the C allele was 0.61 in cases, versus 0.34 in age-matched controls (P<1x10(-24)). Genotype frequencies also differ markedly between cases and controls (chi2=112.68 [2 degrees of freedom]; P<1x10(-24)). A multiplicative model fits the data well, and we estimate the population frequency of the high-risk C allele to be 0.39 (95% confidence interval 0.36-0.42) and the genotype relative risk to be 2.44 (95% confidence interval 2.08-2.83) for TC heterozygotes and 5.93 (95% confidence interval 4.33-8.02) for CC homozygotes.

Age-related macular degeneration (AMD) is a genetically heterogeneous disease that leads to progressive and irreversible vision loss among the elderly. Inflammation, oxidative damage, cholesterol metabolism and/or impaired function of retinal pigment epithelium (RPE) have been implicated in AMD pathogenesis. We examined toll-like receptor 4 (TLR4) as a candidate gene for AMD susceptibility because: (i) the TLR4 gene is located on chromosome 9q32-33, a region exhibiting evidence of linkage to AMD in three independent reports; (ii) the TLR4-D299G variant is associated with reduced risk of atherosclerosis, a chronic inflammatory disease with subendothelial accumulation; (iii) the TLR4 is not only a key mediator of proinflammatory signaling pathways but also linked to regulation of cholesterol efflux and (iv) the TLR4 participates in phagocytosis of photoreceptor outer segments by the RPE. We examined D299G and T399I variants of TLR4 in a sample of 667 unrelated AMD patients and 439 unrelated controls, all of Caucasian ancestry. Multiple logistic regression demonstrated an increased risk of AMD in carriers of the G allele at TLR4 residue 299 (odds ratio=2.65, P=0.025), but lack of an independent effect by T399I variant. TLR4-D299G showed an additive effect on AMD risk (odds ratio=4.13, P=0.002) with allelic variants of apolipoprotein E (APOE) and ATP-binding cassette transporter-1 (ABCA1), two genes involved in cholesterol efflux. Interestingly, the effect of TLR4, APOE and ABCA1 variants on AMD susceptibility was opposite to that of association with atherosclerosis risk. Our data provide evidence of a link between multiple diverse mechanisms underlying AMD pathogenesis.

Purpose: To examine the effect of apolipoprotein E (APOE) alleles on age-related macular degeneration (AMD) risk and on age at diagnosis of AMD in a large patient cohort recruited from a single center., Methods: The frequency of APOE alleles was analyzed in 632 unrelated AMD patients and 206 unrelated controls, all of whom were of white ancestry. The presence or absence of disease symptoms in all patients and controls was based on clinical examination and/or ophthalmic records. The association with APOE was explored in the context of AMD subtypes, family history status, possible interaction with smoking, and distribution of age at diagnosis of AMD., Results: The frequency of the epsilon4 allele was significantly reduced in patients compared with controls (0.10 vs. 0.14, P < or = 0.02). Gender- and age-adjusted odds ratios indicated that epsilon4-carriers have significantly lower risk of developing AMD compared to epsilon3epsilon3 subjects (OR = 0.55, 95% CI: 0.37-0.82, P = 0.004). In the cohort, AMD patients with a positive family history exhibited a significant 3.5 years earlier age at diagnosis (P = 0.001); however, APOE alleles did not appear to modulate the age at diagnosis of AMD., Conclusions: The association between the APOE-epsilon4 allele and a reduced risk of AMD was established in a large cohort with sufficient statistical power. How distinct APOE alleles affect AMD susceptibility warrants further investigation.

Age-related macular degeneration (AMD) is a complex multifactorial disease that affects the central region of the retina. AMD is clinically heterogeneous, leading to geographic atrophy (GA) and/or choroidal neovascularization (CNV) at advanced stages. Considerable data exists in support of a genetic predisposition for AMD. Recent linkage studies have provided evidence in favor of several AMD susceptibility loci. We have performed a high-resolution (5-cM) genome scan of 412 affected relative pairs that were enriched for late-stage disease (GA and/or CNV). Nonparametric linkage analysis was performed using two different diagnostic criteria and also by dividing the affected individuals according to GA or CNV phenotype. Our results demonstrate evidence of linkage in regions that were suggested in at least one previous study at chromosomes 1q (236-240 cM in the Marshfield genetic map), 5p (40-50 cM), and 9q (111 cM). Multipoint analysis of affected relatives with CNV provided evidence of additional susceptibility loci on chromosomes 2p (10 cM) and 22q (25 cM). A recently identified Gln5345Arg change in HEMICENTIN-1 on chromosome 1q25 was not detected in 274 affected members in the restricted group with AMD, 346 additional patients with AMD, and 237 unaffected controls. Our results consolidate the chromosomal locations of several AMD susceptibility loci and, together with previous reports, should facilitate the search for disease-associated sequence variants.

Poly(lactide-co-ethylphosphate)s, a new class of linear phosphorus-containing copolymers made by chain-extending low-molecular-weight polylactide prepolymers with ethyl dichlorophosphate, were investigated for their in vitro and in vivo degradation mechanism and kinetics. Microspheres made from poly(lactide-co-ethylphosphate) were studied under both accelerated and normal in vitro degradation conditions. Gel permeation chromatography (GPC), 1H- and 31P-NMR, weight loss measurements, and differential scanning calorimetry (DSC) techniques were used to characterize the change of molecular weight (M(w)), chemical composition, and glass transition temperature (T(g)) of the degrading polymers. The results indicated that the copolymers degraded in a two-stage fashion, with cleavage of the phosphate-lactide linkages contributing mostly to the initial more rapid degradation phase and cleavage of the lactide-lactide bonds being responsible for the slower latter stage degradation. The decrease in the copolymer M(w) was accompanied by a continuous mass loss. Results from the accelerated degradation studies confirmed that the copolymers degraded into various monomers of the copolymers, which were non-toxic and biocompatible. A two-stage hydrolysis pathway was thus proposed to explain the degradation behavior of the copolymers. In vivo degradation studies performed in mice demonstrated a good in vitro and in vivo correlation for the degradation rates. In vivo clearance of the polymer was faster and without any lag phase. These copolymers are potentially advantageous for drug delivery and other biomedical applications where rapid clearance of the polymer carrier and repeated dosing capability are essential to the success of the treatment.

10-Formyl-7,8-dihydrofolic acid (10-HCO-H2folate) was prepared by controlled air oxidation of 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H4folate). The UV spectra of the 10-HCO-H2folate preparation has lambda max. 234, 333 nm and lambda min. 301 nm at pH 7.4, and lambda max. 257, 328 nm and lambda min. 229, 307 nm at pH 1. 1H-NMR spectroscopy of 10-HCO-H2folate (in 2H2O; 300 MHz) suggested a pure compound and gave resonances for one formyl group proton, two protons on C-7 and C-9, and no evidence for a C-6 proton, which is consistent with the structure proposed. The spectral properties indicated that the 10-HCO-H2folate preparation is not appreciably contaminated with 10-HCO-H4folate, 5,10-methenyltetrahydrofolic acid (5,10-CH = H4folate) or 10-formylfolic acid (10-HCO-folate). The above data establish that the 10-HCO-H2folate prepared here is authentic. In contrast, a folate with a UV spectrum having lambda max. 272 nm and lambda min. 256 nm at pH 7, which was prepared by 2,6-dichloro-indophenol oxidation of 10-HCO-H4folate and reported to be 97% pure [Baram, Chabner, Drake, Fitzhugh, Sholar and Allegra (1988) J. Biol. Chem. 263, 7105-7111], is apparently not 10-HCO-H2folate. 10-HCO-H2folate is utilized by Jurkat-cell (human T-cell leukaemia) and chicken liver aminoimidazolecarboxamide ribonucleotide transformylase (AICAR T'ase; EC 2.1.2.3) in the presence of excess 5-amino-imidazole-4-carboxamide ribotide (AICAR) resulting in the appearance of approximately 1 mol of H2folate product for each mol of AICAR formylated. The present 10-HCO-H2folate preparation had a kinetic advantage over 10-HCO-H4folate resulting from a difference of approx. 5-fold in K(m) values when both folates were used as cofactors for Jurkat-cell and rat bone marrow AICAR T'ase. No substantial kinetic advantage was observed using chicken liver AICAR T'ase. 10-HCO-H2folate had little or no activity with Jurkat-cell or chicken liver glycinamide ribonucleotide transformylase (GAR T'ase, EC 2.1.2.2). The existence in vivo of 10-HCO-H2folate is suggested in mammals by several reports of detectable amounts of radiolabelled 10-HCO-folate in bile and urine after administration of radiolabelled folic acid.

Angiotensin-converting enzyme from human lung was purified to apparent homogeneity using a five-step purification procedure consisting of ammonium sulfate precipitation, ion-exchange chromatography on DEAE Sephadex A-50, gel permeation on Sephadex G-200, chromatofocusing on a polybuffer exchange (PBE 94) column and high-performance liquid chromatographic gel permeation on a Bio-Sil TSK-250 column. This procedure gave an approximately 700-fold purification with a 20% yield compared to a 550-fold purification and a 1% yield with an affinity chromatography-based procedure. The 20-fold greater yield of the five-step procedure offers a major advantage for preparative use in the structural characterization of angiotensin-converting enzyme.