Search

To delineate the genetic bases of primary congenital glaucoma (PCG), we ascertained a large cohort consisting of 48 consanguineous families. Of these, we previously reported 26 families with mutations in CYP1B1 and six families with LTBP2, whereas the genetic bases responsible for PCG in 16 families remained elusive. We employed next-generation whole exome sequencing to delineate the genetic basis of PCG in four of these 16 familial cases. Exclusion of linkage to reported PCG loci was established followed by next-generation whole exome sequencing, which was performed on 10 affected individuals manifesting cardinal systems of PCG belonging to four unresolved families along with four control samples consisting of genomic DNAs of individuals harboring mutations in CYP1B1 and LTBP2. The analyses of sequencing datasets failed to identify potential causal alleles in the 10 exomes whereas c.1169G > A (p. Arg390His) in CYP1B1 and c.3427delC (p.Gln1143Argfs*35) in LTBP2 were identified in the control samples. Taken together, next-generation whole exome sequencing failed to delineate the genetic basis of PCG in familial cases excluded from mutations in CYP1B1 and LTBP2. These data strengthen the notion that compound heterozygous coding variants or non-coding variants might contribute to PCG.

We previously identified a homozygous G178R mutation in human ASRGL1 (hASRGL1) through whole-exome analysis responsible for early onset retinal degeneration (RD) in patients with cone–rod dystrophy. The mutant G178R ASRGL1 expressed in Cos-7 cells showed altered localization, while the mutant ASRGL1 in E. coli lacked the autocatalytic activity needed to generate the active protein. To evaluate the effect of impaired ASRGL1 function on the retina in vivo, we generated a mouse model with c.578_579insAGAAA (NM_001083926.2) mutation (Asrgl1mut/mut) through the CRISPR/Cas9 methodology. The expression of ASGRL1 and its asparaginase activity were undetectable in the retina of Asrgl1mut/mut mice. The ophthalmic evaluation of Asrgl1mut/mut mice showed a significant and progressive decrease in scotopic electroretinographic (ERG) response observed at an early age of 3 months followed by a decrease in photopic response around 5 months compared with age-matched wildtype mice. Immunostaining and RT-PCR analyses with rod and cone cell markers revealed a loss of cone outer segments and a significant decrease in the expression of Rhodopsin, Opn1sw, and Opn1mw at 3 months in Asrgl1mut/mut mice compared with age-matched wildtype mice. Importantly, the retinal phenotype of Asrgl1mut/mut mice is consistent with the phenotype observed in patients harboring the G178R mutation in ASRGL1 confirming a critical role of ASRGL1 in the retina and the contribution of ASRGL1 mutations in retinal degeneration.

We previously identified a homozygous G178R mutation in human

FYCO1 (FYVE and coiled-coil domain containing 1) is an adaptor protein, expressed ubiquitously and required for microtubule-dependent, plus-end-directed transport of macroautophagic/autophagic vesicles. We have previously shown that loss-of-function mutations in FYCO1 cause cataracts with no other ocular and/or extra-ocular phenotype. Here, we show fyco1 homozygous knockout (fyco1(−/−)) mice recapitulate the cataract phenotype consistent with a critical role of FYCO1 and autophagy in lens morphogenesis. Transcriptome coupled with proteome and metabolome profiling identified many autophagy-associated genes, proteins, and lipids respectively perturbed in fyco1(−/−) mice lenses. Flow cytometry of FYCO1 (c.2206C>T) knock-in (KI) human lens epithelial cells revealed a decrease in autophagic flux and autophagic vesicles resulting from the loss of FYCO1. Transmission electron microscopy showed cellular organelles accumulated in FYCO1 (c.2206C>T) KI lens-like organoid structures and in fyco1(−/−) mice lenses. In summary, our data confirm the loss of FYCO1 function results in a diminished autophagic flux, impaired organelle removal, and cataractogenesis. Abbreviations: CC: congenital cataracts; DE: differentially expressed; ER: endoplasmic reticulum; FYCO1: FYVE and coiled-coil domain containing 1; hESC: human embryonic stem cell; KI: knock-in; OFZ: organelle-free zone; qRT-PCR: quantitative real-time PCR; PE: phosphatidylethanolamine; RNA-Seq: RNA sequencing; SD: standard deviation; sgRNA: single guide RNA; shRNA: shorthairpin RNA; TEM: transmission electron microscopy; WT: wild type

Purpose Retinal pigment epithelium (RPE) cell proliferation is precisely regulated to maintain retinal homoeostasis. Microphthalmia-associated transcription factor (MITF), a critical transcription factor in RPE cells, has two alternatively spliced isoforms: (+)MITF and (-)MITF. Previous work has shown that (-)MITF but not (+)MITF inhibits RPE cell proliferation. This study aims to investigate the role of long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) in regulating MITF splicing and hence proliferation of RPE cells. Methods Mouse RPE, primary cultured mouse RPE cells, and different proliferative human embryonic stem cell (hESC)-RPE cells were used to evaluate the expression of (+)MITF, (-)MITF, and NEAT1 by reverse-transcription PCR (RT-PCR) or quantitative RT-PCR. NEAT1 was knocked down using specific small interfering RNAs (siRNAs). Splicing factor proline- and glutamine-rich (SFPQ) was overexpressed with the use of lentivirus infection. Cell proliferation was analyzed by cell number counting and Ki67 immunostaining. RNA immunoprecipitation (RIP) was used to analyze the co-binding between the SFPQ and MITF or NEAT1. Results NEAT1 was highly expressed in proliferative RPE cells, which had low expression of (-)MITF. Knockdown of NEAT1 in RPE cells switched the MITF splicing pattern to produce higher levels of (-)MITF and inhibited cell proliferation. Mechanistically, NEAT1 recruited SFPQ to bind directly with MITF mRNA to regulate its alternative splicing. Overexpression of SFPQ in ARPE-19 cells enhanced the binding enrichment of SFPQ to MITF and increased the splicing efficiency of (+)MITF. The binding affinity between SFPQ and MITF was decreased after NEAT1 knockdown. Conclusions NEAT1 acts as a scaffold to recruit SFPQ to MITF mRNA and promote its binding affinity, which plays an important role in regulating the alternative splicing of MITF and RPE cell proliferation.

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., Author summary The study was performed to identify the underlying cause of inherited retinal degeneration (IRD) in 409 individuals from 108 families. Primarily, these families were recruited from three different geographic regions: Mexico, Pakistan and European Americans from the United States. Blood samples were collected from all individuals for genome analysis. This analysis detected causative variants in 61 out of the 108 pedigrees. A total of 93 gene variants were found in the 61 families. Among these, 54 were previously reported as causative variants and the remaining 39 have not been reported in IRD pedigrees. Interestingly, 54% of these novel variants were not listed in gnomAD. In addition to these findings, complex causative genotypes were observed in 20% of pedigrees. Overall, causative variants were detected in 63% Mexican, 60% Pakistani and 45% European American pedigrees. This study revealed the distribution of IRD causative variants in pedigrees with diverse ethnic and geographic backgrounds.

Inherited retinal degenerations are a clinically and genetically heterogeneous group of blinding diseases characterized by progressive degeneration of the neuroretina and/or the retinal pigment epithelium. Currently, over 300 genes have been implicated in retinal degenerations (RetNet: https://sph.uth.edu/RetNet/). While mutations, or causative variants, in each of these genes are relatively rare, taken together they are a significant cause of blindness, especially in working-age individuals, which increases their economic and societal impact. Clinically, inherited retinal degenerations vary from retinitis pigmentosa and Leber congenital amaurosis, which are often manifest at birth and initially involve rod photoreceptors in the retinal periphery, to early-onset macular degeneration, which is a progressive degeneration affecting central vision (1). Inherited retinal degenerations can show autosomal-dominant, autosomal-recessive, and X-linked inheritance, as well as more complex or multifactorial inheritance patterns, especially for some of the later-onset progressive diseases. While mutations in the same gene usually show the same inheritance pattern, it is not uncommon for mutations in the same gene to cause two or more forms of retinal disease, complicating the nosology of this group of diseases (2). In addition, the presence of disease-associated alleles at one gene can affect the penetrance or expressivity of mutations at a second, resulting in complicated family histories and in some cases even digenic diallelic (3) or digenic triallelic (4) inheritance. In PNAS, Hanany et al. (5) address this intimidating set of related diseases by developing a system to identify disease alleles at each of the associated genes and, beyond that, to calculate the gene-specific carrier frequency and the prevalence of homozygosity for variants causing autosomal recessive retinal degenerations. While appearing somewhat mundane on the surface, this work actually has far-reaching implications for both the basic science of the retina and the clinical practice of ophthalmology. The genes associated with retinal degeneration and the changes … [↵][1]1To whom correspondence may be addressed. Email: hejtmancikj{at}nei.nih.gov. [1]: #xref-corresp-1-1

Cataract, the clinical correlate of opacity or light scattering in the eye lens, is usually caused by the presence of high-molecular-weight (HMW) protein aggregates or disruption of the lens microarchitecture. In general, genes involved in inherited cataracts reflect important processes and pathways in the lens including lens crystallins, connexins, growth factors, membrane proteins, intermediate filament proteins, and chaperones. Usually, mutations causing severe damage to proteins cause congenital cataracts, while milder variants increasing susceptibility to environmental insults are associated with age-related cataracts. These may have different pathogenic mechanisms: Congenital cataracts induce the unfolded protein response and apoptosis. By contrast, denatured crystallins in age-related cataracts are bound by α-crystallin and form light-scattering HMW aggregates. New therapeutic approaches to age-related cataracts use chemical chaperones to solubilize HMW aggregates, while attempts are being made to regenerate lenses using endogenous stem cells to treat congenital cataracts.

High hyperopia is a common and severe form of refractive error. Genetic factors play important roles in the development of high hyperopia but the exact gene responsible for this condition is mostly unknown. We identified a large Chinese family with autosomal dominant high hyperopia. A genome-wide linkage scan mapped the high hyperopia to chromosome 11p12–q13.3, with maximum log of the odds scores of 4.68 at theta = 0 for D11S987. Parallel whole-exome sequencing detected a novel c.3377delG (p.Gly1126Valfs*31) heterozygous mutation in the MYRF gene within the linkage interval. Whole-exome sequencing in other 121 probands with high hyperopia identified additional novel mutations in MYRF within two other families: a de novo c.3274_3275delAG (p.Leu1093Profs*22) heterozygous mutation and a c.3194+2T>C heterozygous mutation. All three mutations are located in the C-terminal region of MYRF and are predicted to result in truncation of that portion. Two patients from two of the three families developed angle-closure glaucoma. These three mutations were present in neither the ExAC database nor our in-house whole-exome sequencing data from 3280 individuals. No other truncation mutations in MYRF were detected in the 3280 individuals. Knockdown of myrf resulted in small eye size in zebrafish. These evidence all support that truncation mutations in the C-terminal region of MYRF are responsible for autosomal dominant high hyperopia in these families. Our results may provide useful clues for further understanding the functional role of the C-terminal region of this critical myelin regulatory factor, as well as the molecular pathogenesis of high hyperopia and its associated angle-closure glaucoma. Electronic supplementary material The online version of this article (10.1007/s00439-019-02039-z) contains supplementary material, which is available to authorized users.

Here we report whole genome sequencing of four individuals (H3, H4, H5, and H6) from a family of Pakistani descent. Whole genome sequencing yielded 1084.92, 894.73, 1068.62, and 1005.77 million mapped reads corresponding to 162.73, 134.21, 160.29, and 150.86 Gb sequence data and 52.49x, 43.29x, 51.70x, and 48.66x average coverage for H3, H4, H5, and H6, respectively. We identified 3,529,659, 3,478,495, 3,407,895, and 3,426,862 variants in the genomes of H3, H4, H5, and H6, respectively, including 1,668,024 variants common in the four genomes. Further, we identified 42,422, 39,824, 28,599, and 35,206 novel variants in the genomes of H3, H4, H5, and H6, respectively. A major fraction of the variants identified in the four genomes reside within the intergenic regions of the genome. Single nucleotide polymorphism (SNP) genotype based comparative analysis with ethnic populations of 1000 Genomes database linked the ancestry of all four genomes with the South Asian populations, which was further supported by mitochondria based haplogroup analysis. In conclusion, we report whole genome sequencing of four individuals of Pakistani descent., Measurement(s) SNV • genome Technology Type(s) whole genome sequencing • DNA sequencing Factor Type(s) individual Sample Characteristic - Organism Homo sapiens Sample Characteristic - Location Pakistan Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12642761

Autosomal dominant optic atrophy (ADOA) is an important cause of irreversible visual impairment in children and adolescents. About 60-90% of ADOA is caused by the pathogenic variants of OPA1 gene. By evaluating the pathogenicity of OPA1 variants and summarizing the relationship between the genotype and phenotype, this study aimed to provide a reference for clinical genetic test involving OPA1. Variants in OPA1 were selected from the exome sequencing results in 7092 cases of hereditary eye diseases and control groups from our in-house data. At the same time, the urine cells of some optic atrophy patients with OPA1 variants as well as their family members were collected and oxygen consumption rates (OCR) were measured in these cells to evaluate the pathogenicity of variants. As a result, 97 variants were detected, including 94 rare variants and 3 polymorphisms. And the 94 rare variants were classified into three groups: pathogenic (33), variants of uncertain significance (19), and likely benign (42). Our results indicated that the frameshift variants at the 3' terminus might be pathogenic, while the variants in exon 7 and intron 4 might be benign. The penetrance of the missense variants was higher than that of truncation variants. The OCR of cells with pathogenic OPA1 variants were significantly lower than those without pathogenic variants. In conclusion, some variants might be benign although predicted pathogenic in previous studies while some might have unknown pathogenesis. Measuring the OCR in urine cells could be used as a method to evaluate the pathogenicity of some OPA1 variants.

Peroxisomes, single-membrane intracellular organelles, play an important role in various metabolic pathways. The translocation of proteins from the cytosol to peroxisomes depends on peroxisome import receptor proteins and defects in peroxisome transport result in a wide spectrum of peroxisomal disorders. Here, we report a large consanguineous family with autosomal recessive congenital cataracts and developmental defects. Genome-wide linkage analysis localized the critical interval to chromosome 12p with a maximum two-point LOD score of 4.2 (θ = 0). Next-generation exome sequencing identified a novel homozygous missense variant (c.653 T > C; p.F218S) in peroxisomal biogenesis factor 5 (PEX5), a peroxisome import receptor protein. This missense mutation was confirmed by bidirectional Sanger sequencing. It segregated with the disease phenotype in the family and was absent in ethnically matched control chromosomes. The lens-specific knockout mice of Pex5 recapitulated the cataractous phenotype. In vitro import assays revealed a normal capacity of the mutant PEX5 to enter the peroxisomal Docking/Translocation Module (DTM) in the presence of peroxisome targeting signal 1 (PTS1) cargo protein, be monoubiquitinated and exported back into the cytosol. Importantly, the mutant PEX5 protein was unable to form a stable trimeric complex with peroxisomal biogenesis factor 7 (PEX7) and a peroxisome targeting signal 2 (PTS2) cargo protein and, therefore, failed to promote the import of PTS2 cargo proteins into peroxisomes. In conclusion, we report a novel missense mutation in PEX5 responsible for the defective import of PTS2 cargo proteins into peroxisomes resulting in congenital cataracts and developmental defects.

To identify possible genetic variants influencing expression of EPHA2 (Ephrin-receptor Type-A2), a tyrosine kinase receptor that has been shown to be important for lens development and to contribute to both congenital and age related cataract when mutated, the extended promoter region of EPHA2 was screened for variants. SNP rs6603883 lies in a PAX2 binding site in the EPHA2 promoter region. The C (minor) allele decreased EPHA2 transcriptional activity relative to the T allele by reducing the binding affinity of PAX2. Knockdown of PAX2 in human lens epithelial (HLE) cells decreased endogenous expression of EPHA2. Whole RNA sequencing showed that extracellular matrix (ECM), MAPK-AKT signaling pathways and cytoskeleton related genes were dysregulated in EPHA2 knockdown HLE cells. Taken together, these results indicate a functional non-coding SNP in EPHA2 promoter affects PAX2 binding and reduces EPHA2 expression. They further suggest that decreasing EPHA2 levels alters MAPK, AKT signaling pathways and ECM and cytoskeletal genes in lens cells that could contribute to cataract. These results demonstrate a direct role for PAX2 in EPHA2 expression and help delineate the role of EPHA2 in development and homeostasis required for lens transparency.

The crystalline lens plays an important role in the refractive vision of vertebrates by facilitating variable fine focusing of light onto the retina. Loss of lens transparency, or cataract, is a frequently acquired cause of visual impairment in adults and may also present during childhood. Genetic studies have identified mutations in over 30 causative genes for congenital or other early-onset forms of cataract as well as several gene variants associated with age-related cataract. However, the pathogenic mechanisms resulting from genetic determinants of cataract are only just beginning to be understood. Here, we briefly summarize current concepts pointing to differences in the molecular mechanisms underlying congenital and age-related forms of cataract.

Author(s): Li, Lin; Chen, Yabin; Jiao, Xiaodong; Jin, Chongfei; Jiang, Dan; Tanwar, Mukesh; Ma, Zhiwei; Huang, Li; Ma, Xiaoyin; Sun, Wenmin; Chen, Jianjun; Ma, Yan; M'hamdi, Oussama; Govindarajan, Gowthaman; Cabrera, Patricia E; Li, Jiali; Gupta, Nikhil; Naeem, Muhammad Asif; Khan, Shaheen N; Riazuddin, Sheikh; Akram, Javed; Ayyagari, Radha; Sieving, Paul A; Riazuddin, S Amer; Hejtmancik, J Fielding | Abstract: PurposeThe Pakistan Punjab population has been a rich source for identifying genes causing or contributing to autosomal recessive retinal degenerations (arRD). This study was carried out to delineate the genetic architecture of arRD in the Pakistani population.MethodsThe genetic origin of arRD in a total of 144 families selected only for having consanguineous marriages and multiple members affected with arRD was examined. Of these, causative mutations had been identified in 62 families while only the locus had been identified for an additional 15. The remaining 67 families were subjected to homozygosity exclusion mapping by screening of closely flanking microsatellite markers at 180 known candidate genes/loci followed by sequencing of the candidate gene for pathogenic changes.ResultsOf these 67 families subjected to homozygosity mapping, 38 showed homozygosity for at least one of the 180 regions, and sequencing of the corresponding genes showed homozygous cosegregating mutations in 27 families. Overall, mutations were detected in approximately 61.8 % (89/144) of arRD families tested, with another 10.4% (15/144) being mapped to a locus but without a gene identified.ConclusionsThese results suggest the involvement of unmapped novel genes in the remaining 27.8% (40/144) of families. In addition, this study demonstrates that homozygosity mapping remains a powerful tool for identifying the genetic defect underlying genetically heterogeneous arRD disorders in consanguineous marriages for both research and clinical applications.

Purpose This study was designed to identify the pathogenic variants in three consanguineous families with congenital cataracts segregating as a recessive trait. Methods Consanguineous families with multiple individuals manifesting congenital cataracts were ascertained. All participating members underwent an ophthalmic examination. A small aliquot of the blood sample was collected from all participating individuals, and genomic DNAs were extracted. Homozygosity-based linkage analysis was performed using short tandem repeat (STR) markers. The haplotypes were constructed with alleles of the STR markers, and the two-point logarithm of odds (LOD) scores were calculated. The candidate gene was sequenced bidirectionally to identify the disease-causing mutations. Results Linkage analysis localized the disease interval to chromosome 3p in three families. Subsequently, bidirectional Sanger sequencing identified two novel mutations—a single base deletion resulting in a frameshift (c.3196delC; p.His1066IlefsTer10) mutation and a single base substitution resulting in a nonsense (c.4270C>T; p.Arg1424Ter) mutation—and a known missense (c.4127T>C, p.Leu1376Pro) mutation in FYCO1. All three mutations showed complete segregation with the disease phenotype and were absent in 96 ethnically matched control individuals. Conclusions We report two novel mutations and a previously reported mutation in FYCO1 in three large consanguineous families. Taken together, mutations in FYCO1 contribute nearly 15% to the total genetic load of autosomal recessive congenital cataracts in this cohort.

Congenital motor nystagmus (CMN) is characterized by early-onset bilateral ocular oscillations without other ocular deficits. To date, mutations in only one gene have been identified to be responsible for CMN, i.e., FRMD7 for X-linked CMN. Four loci for autosomal dominant CMN, including NYS7 (OMIM 614826), have been mapped but the causative genes have yet to be identified. NYS7 was mapped to 1q32.1 based on independent genome-wide linkage scan on two large families with CMN. In this study, mutations in all known protein-coding genes, both intronic sequence with predicted effect and coding sequence, in the linkage interval were excluded by whole-genome sequencing. Then, long-read genome sequencing based on the Nanopore platform was performed with a sample from each of the two families. Two deletions with an overlapping region of 775,699 bp, located in a region without any known protein-coding genes, were identified in the two families in the linkage region. The two deletions as well as their breakpoints were confirmed by Sanger sequencing and co-segregated with CMN in the two families. The 775,699 bp deleted region contains uncharacterized non-protein-coding expressed sequences and pseudogenes but no protein-coding genes. However, Hi-C data predicted that the deletions span two topologically associated domains and probably lead to a change in the 3D genomic architecture. These results provide novel evidence of a strong association between structural variations in non-coding genomic regions and human hereditary diseases like CMN with a potential mechanism involving changes in 3D genome architecture, which provides clues regarding the molecular pathogenicity of CMN. Electronic supplementary material The online version of this article (10.1007/s00439-020-02156-0) contains supplementary material, which is available to authorized users.

βγ-Crystallins, having a uniquely stable two domain four Greek key structure, are crucial for transparency of the eye lens,. Mutations in lens crystallins have been proposed to cause cataract formation by a variety of mechanisms most of which involve destabilization of the protein fold. The underlying molecular mechanism for autosomal dominant zonular cataracts with sutural opacities in an Indian family caused by a c.215+1G>A splice mutation in the βA3/A1-crystallin gene CRYBA1 was elucidated using three transgenic mice models. This mutation causes a splice defect in which the mutant mRNA escapes nonsense mediated decay by skipping both exons 3 and 4. Skipping these exons results in an in-frame deletion of the mRNA and synthesis of an unstable p.Ile33_Ala119del mutant βA3/A1-crystallin protein. Transgenic expression of mutant βA3/A1-crystallin but not the wild type protein results in toxicity and abnormalities in the maturation and orientation of differentiating lens fibers in c.97_357del CRYBA1 transgenic mice, leading to a small spherical lens, cataract, and often lens capsule rupture. On a cellular level, the lenses accumulated p.Ile33_Ala119del βA3/A1-crystallin with resultant activation of the stress signaling pathway - unfolded protein response (UPR) and inhibition of normal protein synthesis, culminating in apoptosis. This highlights the mechanistic contrast between mild mutations that destabilize crystallins and other proteins, resulting in their being bound by the α-crystallins that buffer lens cells against damage by denatured proteins, and severely misfolded proteins that are not bound by α-crystallin but accumulate and have a direct toxic effect on lens cells, resulting in early onset cataracts.

We identified a homozygous missense alteration (c.75C>A, p.D25E) in CLCC1, encoding a presumptive intracellular chloride channel highly expressed in the retina, associated with autosomal recessive retinitis pigmentosa (arRP) in eight consanguineous families of Pakistani descent. The p.D25E alteration decreased CLCC1 channel function accompanied by accumulation of mutant protein in granules within the ER lumen, while siRNA knockdown of CLCC1 mRNA induced apoptosis in cultured ARPE-19 cells. TALEN KO in zebrafish was lethal 11 days post fertilization. The depressed electroretinogram (ERG) cone response and cone spectral sensitivity of 5 dpf KO zebrafish and reduced eye size, retinal thickness, and expression of rod and cone opsins could be rescued by injection of wild type CLCC1 mRNA. Clcc1+/-KO mice showed decreased ERGs and photoreceptor number. Together these results strongly suggest that intracellular chloride transport by CLCC1 is a critical process in maintaining retinal integrity, and CLCC1 is crucial for survival and function of retinal cells.

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.

To identify the molecular basis of inherited retinal degeneration (IRD) in a familial case of Pakistani origin using whole-exome sequencing.A thorough ophthalmic examination was completed, and genomic DNA was extracted using standard protocols. Whole exome(s) were captured with Agilent V5 + UTRs probes and sequenced on Illumina HiSeq genome analyzer. The exomeSuite software was used to filter variants, and the candidate causal variants were prioritized, examining their allele frequency and PolyPhen2, SIFT, and MutationTaster predictions. Sanger dideoxy sequencing was performed to confirm the segregation with disease phenotype and absence in ethnicity-matched control chromosomes.Ophthalmic examination confirmed retinal degeneration in all affected individuals that segregated as an autosomal recessive trait in the family. Whole-exome sequencing identified two homozygous missense variants: c.1304G A; p.Arg435Gln in ZNF408 (NM_024741) and c.902G A; p.Gly301Asp in C1QTNF4 (NM_031909). Both variants segregated with the retinal phenotype in the PKRD320 and were absent in ethnically matched control chromosomes.Whole-exome sequencing coupled with bioinformatics analysis identified potential novel variants that might be responsible for IRD.

Here we report next-generation based whole genome sequencing of two individuals (H1 and H2) from a family of Pakistani descent. The genomic DNA was used to prepare paired-end libraries for whole-genome sequencing. Deep sequencing yielded 706.49 and 778.12 million mapped reads corresponding to 70.64 and 77.81 Gb sequence data and 23× and 25× average coverage for H1 and H2, respectively. Notably, a total of 448,544 and 470,683 novel variants, not present in the single nucleotide polymorphism database (dbSNP), were identified in H1 and H2, respectively. Comparative analysis identified 2,415,852 variants common in both genomes including 240,181 variants absent in the dbSNP. Principal component analysis linked the ancestry of both genomes with South Asian populations. In conclusion, we report whole genome sequences of two individuals from a family of Pakistani descent.

Purpose Previously, a genome-wide association study (GWAS) identified rs13382811 (near ZFHX1B) and rs6469937 (near SNTB1) to be associated with high myopia. The present study evaluates the association of these two single nucleotide polymorphisms (SNPs) with moderate to high myopia in two Chinese cohorts and two cohorts of European populations. Methods Two Chinese university student cohorts, including one with 300 unrelated subjects with high myopia and 308 emmetropic controls from Guangzhou and a second with 96 unrelated individuals with moderate to high myopia and 96 emmetropic controls of Chaoshanese origin in Guangzhou, were enrolled in this study. Two SNPs, rs6469937 and rs13382811, were selected for genotyping based on their reported associations with severe myopia. The SNPs were genotyped via DNA sequencing. In addition, association analysis of both SNPs was performed using genotype data from the database of Genotypes and Phenotypes (dbGaP) involving a total of 2,423 samples in two independent cohorts of European-derived populations, as follows: Kooperative Gesundheitsforschung in der Region Augsburg (KORA) and TwinsUK. The allelic and genotypic distribution among cases and controls were analyzed using the Chi-square test. Logistic regression was used to evaluate the SNP–SNP interaction. Fisher’s exact test was used for two-SNP comparisons. Results In the Guangzhou cohort, SNP rs13382811 near ZFHX1B showed significant association with high myopia (pallelic = 0.0001, pgenotypic = 4.07 × 10−5), with the minor T allele showing an increased risk of high myopia (odds ratio [OR] = 1.68, 95% confidence interval [CI] = 1.28–2.20). SNP rs6469937 near SNTB1 showed nominal evidence of association (pallelic = 0.0085, pgenotypic = 0.0166), which did not withstand correction for multiple testing. No significant association was detected in the smaller Chaoshan cohort alone. The association of SNPs rs13382811 and rs6469937 remained significant when both Han Chinese cohorts were combined (pallelic = 0.0033 and 0.0016, respectively), and it was also significant under the genotypic test (pgenotypic = 0.0036 and 0.0053, respectively). When both SNPs were considered together under a recessive model, their significance increased (p = 8.37 × 10−4), as did their effect (OR = 4.09, 95%CI = 1.7–9.8). The association between either of these two SNPs alone and myopia did not replicate significantly in the combined cohorts of European descent, providing only suggestive results (pallelic = 0.0088 for rs13382811 and pallelic = 0.0319 for rs6469937). However, the effects of the combined SNPs showed significant association (p = 8.2 × 10−4; OR = 1.56, 95%CI = 1.2–2.0). While the risk for myopia increased with risk alleles from both SNPs, the increase was additive rather representing a multiplicative interaction in both populations. Conclusions Our study confirms that the two susceptibility loci ZFHX1B and SNTB1 are associated with moderate to high myopia in a Han Chinese population, as well as in a European population, when both SNPs are combined. These results confirm previous reports of their associations, extend these observations to a European population, and suggest that additional interactive and possibly population-specific genetic or environmental factors may affect their contribution to myopia.

The aim of this work is to identify the molecular cause of autosomal recessive early onset retinal degeneration in a consanguineous pedigree. Seventeen members of a four-generation Pakistani family were recruited and underwent a detailed ophthalmic examination. Exomes of four affected and two unaffected individuals were sequenced. Variants were filtered using exomeSuite to identify rare potentially pathogenic variants in genes expressed in the retina and/or brain and consistent with the pattern of inheritance. Effect of the variant observed in the gene Intraflagellar Transport Protein 43 (IFT43) was studied by heterologous expression in mIMCD3 and MDCK cells. Expression and sub-cellular localization of IFT43 in the retina and transiently transfected cells was examined by RT-PCR, western blot analysis, and immunohistochemistry. Affected members were diagnosed with early onset non-syndromic progressive retinal degeneration and the presence of bone spicules distributed throughout the retina at younger ages while the older affected members showed severe central choroidal atrophy. Whole-exome sequencing analysis identified a novel homozygous c.100 G > A change in IFT43 segregating with retinal degeneration and not present in ethnicity-matched controls. Immunostaining showed IFT43 localized in the photoreceptors, and to the tip of the cilia in transfected mIMCD3 and MDCK cells. The cilia in mIMCD3 and MDCK cells expressing mutant IFT43 were found to be significantly shorter (P

Mutations in RP2 and RPGR genes are responsible for the X-linked retinitis pigmentosa (XLRP). In this study, we analyzed the RP2 and RPGR gene mutations in five Han Chinese families with XLRP. An approximately 17Kb large deletion including the exon 4 and exon 5 of RP2 gene was found in an XLRP family. In addition, four frameshift mutations including three novel mutations of c.1059 + 1 G > T, c.2002dupC and c.2236_2237del CT, as well as a previously reported mutation of c.2899delG were detected in the RPGR gene in the other four families. Our study further expands the mutation spectrum of RP2 and RPGR, and will be helpful for further study molecular pathogenesis of XLRP.

PurposeTo investigate the genetic basis of autosomal recessive congenital cataracts (arCC) in a large consanguineous Pakistani family.MethodsAll participating members of family, PKCC074 underwent an ophthalmic examination. Slit-lamp photographs were ascertained for affected individuals that have not been operated for the removal of the cataractous lens. A small aliquot of the blood sample was collected from all participating individuals and genomic DNAs were extracted. A genome-wide scan was performed with polymorphic short tandem repeat (STR) markers and the logarithm of odds (LOD) scores were calculated. All coding exons and exon-intron boundaries of HSF4 were sequenced and expression of Hsf4 in mouse ocular lens was investigated. The C-terminal FLAG-tagged wild-type and mutant HSF4b constructs were prepared to examine the nuclear localization pattern of the mutant protein.ResultsThe ophthalmological examinations suggested that nuclear cataracts are present in affected individuals. Genome-wide linkage analyses localized the critical interval to a 10.95 cM (14.17 Mb) interval on chromosome 16q with a maximum two-point LOD score of 4.51 at θ = 0. Sanger sequencing identified a novel missense mutation: c.433G>C (p.Ala145Pro) that segregated with the disease phenotype in the family and was not present in ethnically matched controls. Real-time PCR analysis identified the expression of HSF4 in mouse lens as early as embryonic day 15 with a steady level of expression thereafter. The immunofluorescence tracking confirmed that both wild-type and mutant HSF4 (p.Ala145Pro) proteins localized to the nucleus.ConclusionHere, we report a novel missense mutation in HSF4 associated with arCC in a familial case of Pakistani descent.

To identify known and novel CYP4V2 mutations in patients with Bietti crystalline cornea (BCD), expand the spectrum of CYP4V2 mutations, and characterize the population history of the c.802-8-810del17insGC mutation common in Asian populations, genomic DNA was isolated from peripheral blood samples from 58 unrelated patients with clinical diagnoses of BCD. Exons and flanking intronic regions of the CYP4V2 gene were dideoxy DNA sequenced. Nonpathogenic polymorphisms were excluded and known mutations were identified by sequencing 192 unaffected individuals from similar ethnic backgrounds and examination of online databases. The age of the c.802-8-810del17insGC mutation was estimated using three independent approaches. A total of 28 CYP4V2 mutations, 9 of which were novel, were detected in the 58 patients with BCD. These included 19 missense, 4 nonsense, 2 deletion, 2 splice site, and 1 insertion-deletion mutations. Two missense variants of uncertain significance were also detected. The age of the c.802-8-810del17insGC mutation was estimated to be 1040-8200 generations in the Chinese and 300-1100 generations in the Japanese populations. These results expand the mutation spectrum of CYP4V2, and provide insight into the origin of the c.802-8-810del17insGC mutation in the Chinese population and its transmission to the Japanese population. To identify known and novel CYP4V2 mutations in patients with Bietti crystalline cornea (BCD), expand the spectrum of CYP4V2 mutations, and characterize the population history of the c.802-8-810del17insGC mutation common in Asian populations, genomic DNA was isolated from peripheral blood samples from 58 unrelated patients with clinical diagnoses of BCD. Exons and flanking intronic regions of the CYP4V2 gene were dideoxy DNA sequenced. Nonpathogenic polymorphisms were excluded and known mutations were identified by sequencing 192 unaffected individuals from similar ethnic backgrounds and examination of online databases. The age of the c.802-8-810del17insGC mutation was estimated using three independent approaches. A total of 28 CYP4V2 mutations, 9 of which were novel, were detected in the 58 patients with BCD. These included 19 missense, 4 nonsense, 2 deletion, 2 splice site, and 1 insertion-deletion mutations. Two missense variants of uncertain significance were also detected. The age of the c.802-8-810del17insGC mutation was estimated to be 1040-8200 generations in the Chinese and 300-1100 generations in the Japanese populations. These results expand the mutation spectrum of CYP4V2, and provide insight into the origin of the c.802-8-810del17insGC mutation in the Chinese population and its transmission to the Japanese population.

FOXE3 is a lens-specific transcription factor that has been associated with anterior segment ocular dysgenesis. To determine the transcriptional target(s) of FOXE3 that are indispensable for the anterior segment development, we examined the transcriptome and the proteome of cells expressing truncated FOXE3 responsible for Peters anomaly identified through linkage-coupled next-generation whole-exome sequencing. We found that DNAJB1, an autophagy-associated protein, was the only candidate exhibiting differential expression in both screens. We confirmed the candidacy of DNAJB1 through chromatin immunoprecipitation and luciferase assays while knockdown of DNAJB1 in human lens epithelial cells resulted in a mitotic arrest. Subsequently, we targeted dnajb1a in zebrafish through injection of a splice-blocking morpholino. The dnajb1a morphants exhibited underdeveloped cataractous lenses with persistent apoptotic nuclei. In conclusion, here we report DNAJB1 is a transcriptional target of FOXE3 in a novel pathway that is crucial for the development of the anterior segment of the eye., Peter's Anomaly is a developmental disorder of the eye and has been linked to mutations in a range of genes, including the transcription factor FOXE3. Here the authors use next-generation RNA sequencing and mass spectrometry to identify an autophagy-associated protein, DNAJB1 as the transcriptional target of FOXE3.

Congenital cataracts (CCs), responsible for about one-third of blindness in infants, are a major cause of vision loss in children worldwide. Autosomal-recessive congenital cataracts (arCC) form a clinically diverse and genetically heterogeneous group of disorders of the crystalline lens. To identify the genetic cause of arCC in consanguineous Pakistani families, we performed genome-wide linkage analysis and fine mapping and identified linkage to 3p21-p22 with a summed LOD score of 33.42. Mutations in the gene encoding FYVE and coiled-coil domain containing 1 (FYCO1), a PI(3)P-binding protein family member that is associated with the exterior of autophagosomes and mediates microtubule plus-end-directed vesicle transport, were identified in 12 Pakistani families and one Arab Israeli family in which arCC had previously been mapped to the overlapping CATC2 region. Nine different mutations were identified, including c.3755 delC (p.Ala1252AspfsX71), c.3858_3862dupGGAAT (p.Leu1288TrpfsX37), c.1045 C>T (p.Gln349X), c.2206C>T (p.Gln736X), c.2761C>T (p.Arg921X), c.2830C>T (p.Arg944X), c.3150+1 G>T, c.4127T>C (p.Leu1376Pro), and c.1546C>T (p.Gln516X). Fyco1 is expressed in the mouse embryonic and adult lens and peaks at P12d. Expressed mutant proteins p.Leu1288TrpfsX37 and p.Gln736X are truncated on immunoblots. Wild-type and p.L1376P FYCO1, the only missense mutant identified, migrate at the expected molecular mass. Both wild-type and p. Leu1376Pro FYCO1 proteins expressed in human lens epithelial cells partially colocalize to microtubules and are found adjacent to Golgi, but they primarily colocalize to autophagosomes. Thus, FYCO1 is involved in lens development and transparency in humans, and mutations in this gene are one of the most common causes of arCC in the Pakistani population.

Objective To identify disease-causing mutations in two consanguineous Pakistani families with fundus albipunctatus. Methods Affected individuals in both families underwent a thorough clinical examination including funduscopy and electroretinography. Blood samples were collected from all participating members and genomic DNA was extracted. Exclusion analysis was completed with microsatellite short tandem repeat markers that span all reported loci for fundus albipunctatus. Two-point logarithm of odds (LOD) scores were calculated, and coding exons and exon–intron boundaries of RLBP1 were sequenced bi-directionally. Results The ophthalmic examination of affected patients in both families was consistent with fundus albipunctatus. The alleles of markers on chromosome 15q flanking RLBP1 segregated with the disease phenotype in both families and linkage was further confirmed by two-point LOD scores. Bi-directional sequencing of RLBP1 identified a nonsense mutation (R156X) and a missense mutation (G116R) that segregated with the disease phenotype in their respective families. Conclusions These results strongly suggest that mutations in RLBP1 are responsible for fundus albipunctatus in the affected individuals of these consanguineous Pakistani families.

Purpose: To describe clinical and functional features of a patient with Bietti crystalline dystrophy and atypical electroretinogram responses. Methods: The patient underwent a thorough medical anamnesis, genetic counseling, peripheral blood draw for CYP4V2 gene analysis and electron microscopy, and a complete ophthalmological assessment including optical coherence tomography, indocyanine green angiography, microperimetry, full-field electroretinogram and multifocal electroretinogram. Results: The most striking features of the retina were deposits of yellowish-white glistening crystals and focal lobular areas of choriocapillary atrophy at the posterior pole and midperiphery. The full-field electroretinogram was normal and the multifocal electroretinogram showed extinguished central recordings. Mutation analysis revealed a homozygous c. 332T>C p.I111T mutation in exon 3 of the CYP4V2 gene. Typical cytoplasmic inclusions containing crystalline-like structure and large degenerative lysosomes were seen on electron microscopy of peripheral leukocytes. Conclusion: Here we describe a patient with Bietti crystalline dystrophy with a CYP4V2 gene mutation and typical leukocyte inclusions who showed the classical retinal lesions but had a normal electroretinogram. This suggests the existence of less severe forms of BCD related to relatively mild CYP4V2 mutations.

Congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder that can be associated with impaired night vision. The last decade has witnessed huge progress in ophthalmic genetics, including the identification of three genes implicated in the pathogenicity of autosomal-recessive CSNB. However, not all patients studied could be associated with mutations in these genes and thus other genes certainly underlie this disorder. Here, we report a large multigeneration family with five affected individuals manifesting symptoms of night blindness. A genome-wide scan localized the disease interval to chromosome 15q, and recombination events in affected individuals refined the critical interval to a 10.41 cM (6.53 Mb) region that harbors SLC24A1, a member of the solute carrier protein superfamily. Sequencing of all the coding exons identified a 2 bp deletion in exon 2: c.1613_1614del, which is predicted to result in a frame shift that leads to premature termination of SLC24A1 (p.F538CfsX23) and segregates with the disorder under an autosomal-recessive model. Expression analysis using mouse ocular tissues shows that Slc24a1 is expressed in the retina around postnatal day 7. In situ and immunohistological studies localized both SLC24A1 and Slc24a1 to the inner segment, outer and inner nuclear layers, and ganglion cells of the retina, respectively. Our data expand the genetic basis of CSNB and highlight the indispensible function of SLC24A1 in retinal function and/or maintenance in humans.

Background Retinitis pigmentosa (RP) is one of the most common ophthalmic disorders affecting one in approximately 5000 people worldwide. A nuclear family was recruited from the Punjab province of Pakistan to study the genetic basis of autosomal recessive RP. Methods All affected individuals underwent a thorough ophthalmic examination and the disease was characterised based upon results for fundus photographs and electroretinogram recordings. Genomic DNA was extracted from peripheral leucocytes. Exclusion studies were performed with short tandem repeat (STR) markers flanking reported autosomal recessive RP loci. Haplotypes were constructed and results were statistically evaluated. Results The results of exclusion analyses suggested that family PKRP173 was linked to chromosome 2q harbouring mer tyrosine kinase protooncogene ( MERTK ), a gene previously associated with autosomal recessive RP. Additional STR markers refined the critical interval and placed it in a 13.4 cM (17 Mb) region flanked by D2S293 proximally and D2S347 distally. Significant logarithm of odds (LOD) scores of 3.2, 3.25 and 3.18 at θ=0 were obtained with markers D2S1896, D2S2269 and D2S160. Sequencing of the coding exons of MERTK identified a mutation, c.718G→T in exon 4, which results in a premature termination of p.E240X that segregates with the disease phenotype in the family. Conclusion Our results strongly suggest that the nonsense mutation in MERTK , leading to premature termination of the protein, is responsible for RP phenotype in the affected individuals of the Pakistani family.

Objective To identify a disease locus for autosomal recessive retinitis pigmentosa in a consanguineous Pakistani family. Design Prospective linkage study. Methods Blood samples were collected and genomic DNA was extracted. A genome-wide scan was performed using 382 polymorphic microsatellite markers on genomic DNA from 4 affected and 5 unaffected family members, and logarithm of odds scores were calculated. Results A maximum 2-point logarithm of odds score of 3.14 at θ = 0 was obtained for marker D2S165 during the genome-wide scan. Fine mapping markers identified a 20.92-cM (19.98-Mb) interval flanked by D2S149 and D2S367 that cosegregates with the disease phenotype. Haplotype analyses further refined the critical interval, distal to D2S220 in a 12.31-cM (13.35-Mb) region that does not harbor any genes that previously have been associated with retinitis pigmentosa. Conclusions Linkage analysis identified a new locus for autosomal recessive retinitis pigmentosa that maps to chromosome 2p22.3-p24.1 in a consanguineous Pakistani family.

Uveitis is a complex multifactorial autoimmune disease of the eye characterized by inflammation of the uvea and retina, degeneration of the retina, and blindness in genetically predisposed patients. Using the rat model of experimental autoimmune uveitis (EAU), we previously identified three quantitative trait loci (QTL) associated with EAU on rat chromosomes 4, 12, and 10 (Eau1, Eau2, and Eau3). The primary goal of the current study is to delineate additional non-MHC chromosomal regions that control susceptibility to EAU, and to identify any QTLs that overlap with the QTLs of other autoimmune diseases. Using a set of informative microsatellite markers and F2 generations of resistant and susceptible MHC class II-matched rat strains (F344 and LEW), we have identified several new significant or suggestive QTLs on rat chromosomes 2, 3, 7, 10, and 19 that control susceptibility to EAU. A protective allele was identified in the susceptible LEW strain in the Eau5 locus at D7Wox18, and epistatic interactions between QTLs were found to influence the severity of disease. The newly identified regions (Eau4 through Eau9) colocalize with the genetic determinants of other autoimmune disease models, and to disease-regulating syntenic regions identified in autoimmune patients on human chromosomes 4q21-31, 5q31-33, 16q22-24, 17p11-q12, 20q11-13, and 22q12-13. Our results suggest that uveitis shares some of the pathogenic mechanisms associated with other autoimmune diseases, and lends support to the “common gene, common pathway” hypothesis for autoimmune disorders.

Mutation in the PROM1 gene previously has been identified in one family with retinal degeneration for which neither ERG recordings nor detailed information about visual impairment is available. A large family with multiple individuals affected by retinal degeneration was ascertained in the Punjab province of Pakistan. The visual acuity of all affected patients in the family was severely compromised beginning in early childhood. The retinal disease in this family is a severe form of retinitis pigmentosa (RP) accompanied by macular degeneration. Fundus changes advanced with age. Choriocapillaris atrophy and posterior RPE atrophy were obvious allowing visualization of the large choroidal vessels in patients over 40 years of age. Rod and cone responses on ERG recordings were extinguished in patient's teens. A genome-wide scan mapped the disease to a 34.7 cM region of chromosome 4p14-p16 between D4S1599 and D4S405. A maximum lod score of 3.96 with D4S403 and D4S391 is seen at theta = 0. Sequence analysis of PROM1 located in the linkage interval identified a c.1726CT homozygous transition in exon 15: resulting in p.Gln576X in the translated protein. This mutation is found in a homozygous state in all six affected individuals and was heterozygous in five of the six unaffected family members examined. The mutation was not detected in 192 chromosomes of unrelated control individuals of the same ethnicity and from the same region. This delineates the phenotypic characteristics of retinopathy caused by mutations in PROM1.

Purpose This study was undertaken to identify causal mutations responsible for autosomal recessive retinitis pigmentosa (arRP) in consanguineous families. Methods Large consanguineous families were ascertained from the Punjab province of Pakistan. An ophthalmic examination consisting of a fundus evaluation and electroretinography (ERG) was completed, and small aliquots of blood were collected from all participating individuals. Genomic DNA was extracted from white blood cells, and a genome-wide linkage or a locus-specific exclusion analysis was completed with polymorphic short tandem repeats (STRs). Two-point logarithm of odds (LOD) scores were calculated, and all coding exons and exon–intron boundaries of RP1 were sequenced to identify the causal mutation. Results The ophthalmic examination showed that affected individuals in all families manifest cardinal symptoms of RP. Genome-wide scans localized the disease phenotype to chromosome 8q, a region harboring RP1, a gene previously implicated in the pathogenesis of RP. Sanger sequencing identified a homozygous single base deletion in exon 4: c.3697delT (p.S1233Pfs22*), a single base substitution in intron 3: c.787+1G>A (p.I263Nfs8*), a 2 bp duplication in exon 2: c.551_552dupTA (p.Q185Yfs4*) and an 11,117 bp deletion that removes all three coding exons of RP1. These variations segregated with the disease phenotype within the respective families and were not present in ethnically matched control samples. Conclusions These results strongly suggest that these mutations in RP1 are responsible for the retinal phenotype in affected individuals of all four consanguineous families.

Background Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a rare autosomal dominant disease. Mutations in the forkhead box L2 (FOXL2) gene cause two types of BPES distinguished by the presence (type I) and absence (type II) of premature ovarian failure (POF). The purpose of this study was to identify possible mutations in FOXL2 in two Chinese families with BPES. Methods Two large autosomal dominant Chinese BPES families were enrolled in this study. Genomic DNA was obtained from the leukocytes in peripheral venous blood. Four overlapping sets of primers were used to amplify the entire coding region and nearby intron sequences of the FOXL2 gene for mutations detection using polymerase chain reaction (PCR) and sequencing analyses. The sequencing results were analyzed using DNAstar software. Results All patients of the two families demonstrated typical features of BPES type II, including small palpebral fissures, ptosis, telecanthus, and epicanthus inversus without female infertility (POF). A novel FOXL2 heterozygous indel mutation c.675_690delinsT, including a 16-bp deletion and a 1-bp(T) insertion (p.Ala226_Ala230del), which would result in deletion of 5 alanine residues of a poly-alanine (poly-Ala) tract in the protein, was identified in all affected members of family A. A novel heterozygous missense mutation (c.223C > T, p.Leu75Phe) was identified in family B. Conclusions Two novel FOXL2 mutations were identified in Chinese families with BPES. Our results expand the spectrum of FOXL2 mutations and provide additional structure-function insights into the FOXL2 protein.

This introduction provides an overview of the retina, in which we survey the fundus, layers of the retina, retinal cell types, visual transduction cascade, vitamin A cycle, neuronal wiring of the retina, and blood supply of the retina.

PURPOSE:To define the molecular basis of retinal degeneration in consanguineous Pakistani pedigrees with early onset retinal degeneration. METHODS:A cohort of 277 individuals representing 26 pedigrees from the Punjab province of Pakistan was analyzed. Exomes were captured with commercial kits and sequenced on an Illumina HiSeq 2500. Candidate variants were identified using standard tools and analyzed using exomeSuite to detect all potentially pathogenic changes in genes implicated in retinal degeneration. Segregation analysis was performed by dideoxy sequencing and novel variants were additionally investigated for their presence in ethnicity-matched controls. RESULTS:We identified a total of nine causal mutations, including six novel variants in RPE65, LCA5, USH2A, CNGB1, FAM161A, CERKL and GUCY2D as the underlying cause of inherited retinal degenerations in 13 of 26 pedigrees. In addition to the causal variants, a total of 200 variants each observed in five or more unrelated pedigrees investigated in this study that were absent from the dbSNP, HapMap, 1000 Genomes, NHLBI ESP6500, and ExAC databases were identified, suggesting that they are common in, and unique to the Pakistani population. CONCLUSIONS:We identified causal mutations associated with retinal degeneration in nearly half of the pedigrees investigated in this study through next generation whole exome sequencing. All novel variants detected in this study through exome sequencing have been cataloged providing a reference database of variants common in, and unique to the Pakistani population.

Purpose This study was initiated to identify causal mutations responsible for autosomal recessive congenital cataracts in consanguineous familial cases. Methods Affected individuals underwent a detailed ophthalmological and clinical examination, and slit-lamp photographs were ascertained for affected individuals who have not yet been operated for the removal of the cataractous lens. Blood samples were obtained, and genomic DNA was extracted from white blood cells. A genome-wide scan was completed with short tandem repeat (STR) markers, and the logarithm of odds (LOD) scores were calculated. Protein coding exons of CRYAB were sequenced, bi-directionally. Evolutionary conservation was investigated by aligning CRYAB orthologues, and the expression of Cryab in embryonic and postnatal mice lens was investigated with TaqMan probe. Results The clinical and ophthalmological examinations suggested that all affected individuals had nuclear cataracts. Genome-wide linkage analysis suggested a potential region on chromosome 11q23 harboring CRYAB. DNA sequencing identified a missense variation: c.34C>T (p.R12C) in CRYAB that segregated with the disease phenotype in the family. Subsequent interrogation of our entire cohort of familial cases identified a second familial case localized to chromosome 11q23 harboring a c.31C>T (p.R11C) mutation. In silico analyses suggested that the mutations identified in familial cases, p.R11C and p.R12C will not be tolerated by the three-dimensional structure of CRYAB. Real-time PCR analysis identified the expression of Cryab in mouse lens as early as embryonic day 15 (E15) that increased significantly until postnatal day 6 (P6) with steady level of expression thereafter. Conclusion Here, we report two novel missense mutations, p.R11C and p.R12C, in CRYAB associated with autosomal recessive congenital nuclear cataracts.

In order to accomplish its function of transmitting and focusing light, the crystalline lens of the vertebrate eye has evolved a unique cellular structure and protein complement. These distinct adaptations have provided a rich source of scientific discovery ranging from biochemistry and genetics to optics and physics. In addition, because of these adaptations, lens cells persist for the lifetime of an organism, providing an excellent model of the aging process. The chapters dealing with the lens will demonstrate how the different aspects of lens biology and biochemistry combine in this singular refractive organ to accomplish its critical role in the visual system.

The primary function of the lens resides in its transparency and ability to focus light on the retina. These require both that the lens cells contain high concentrations of densely packed lens crystallins to maintain a refractive index constant over distances approximating the wavelength of the light to be transmitted, and a specific arrangement of anterior epithelial cells and arcuate fiber cells lacking organelles in the nucleus to avoid blocking transmission of light. Because cells in the lens nucleus have shed their organelles, lens crystallins have to last for the lifetime of the organism, and are specifically adapted to this function. The lens crystallins comprise two major families: the βγ-crystallins are among the most stable proteins known and the α-crystallins, which have a chaperone-like function. Other proteins and metabolic activities of the lens are primarily organized to protect the crystallins from damage over time and to maintain homeostasis of the lens cells. Membrane protein channels maintain osmotic and ionic balance across the lens, while the lens cytoskeleton provides for the specific shape of the lens cells, especially the fiber cells of the nucleus. Perhaps most importantly, a large part of the metabolic activity in the lens is directed toward maintaining a reduced state, which shelters the lens crystallins and other cellular components from damage from UV light and oxidative stress. Finally, the energy requirements of the lens are met largely by glycolysis and the pentose phosphate pathway, perhaps in response to the avascular nature of the lens. Together, all these systems cooperate to maintain lens transparency over time.

Item does not contain fulltext OBJECTIVE: To determine the refractive error in patients with autosomal recessive retinitis pigmentosa (arRP) caused by RP1 mutations and to compare it with that of other genetic subtypes of RP. METHODS: Twenty-six individuals had arRP with RP1 mutations, 25 had autosomal dominant RP (adRP) with RP1 mutation, 8 and 33 had X-linked RP (xlRP) with RP2 and RPGR mutations, respectively, 198 and 93 had Usher syndrome and arRP without RP1 mutations, respectively. The median of the spherical equivalent (SE) and the IQR (Q25-Q75) was determined and multiple comparisons were performed. RESULTS: arRP patients with RP1 mutations had SE median at -4.0 dioptres (D) OD (Ocula Dextra); -3.88 D OS (Ocula Sinistra), whereas arRP patients without RP1 mutations (-0.50 D OD; -0.75 D OS) and Usher syndrome patients (-0.50 D OD; -0.38 D OS) were significantly less myopic (p

Purpose To identify the genes causing autosomal recessive retinal dystrophy in Indian families and to characterize the associated phenotypes. Design Experimental and observational. Methods Families with autosomal recessive nonsyndromic retinal dystrophies were recruited. Complete ophthalmic evaluation, including visual acuity, visual fields, fundus examinations, and electroretinography, was performed on all members. Genotyping of 14 families for two or more microsatellite markers flanking each of 21 different genes causing retinal dystrophy was done by standard methods to screen for the presence of homozygosity by descent. Mutational screening of the ABCA4 gene was carried out on 18 members (five affected) of two families by amplification and direct automated sequencing of exons and flanking sequences. Sequence alterations identified were tested for cosegregation with disease in the families and for presence in 100 unrelated normal controls. Results Two of 14 families showed homozygosity shared by affected individuals for markers flanking the ABCA4 locus. A homozygous nonsense mutation in the ABCA4 gene of Arg2030Stop was found in one family and a homozygous single base deletion leading to frameshift at Arg409 was found in the second family. Both of these mutations were found to cosegregate with disease. Five affected individuals from the two families had early-onset visual loss, diminished rod and cone electroretinographic responses, and widespread atrophy of the retinal pigment epithelium. Conclusion Homozygous null mutations in ABCA4 produced a severe widespread retinal degeneration that showed marked central retinal involvement.

A severe form of autosomal recessive retinitis pigmentosa (arRP) was identified in a large Pakistani family ascertained in the Punjab province of Pakistan. All affected individuals in the family had night blindness in early childhood, early complete loss of useful vision, and typical RP fundus changes plus macular degeneration. After exclusion of known arRP loci, a genome-wide scan was performed using microsatellite markers at about 10 cM intervals and calculating two-point lod scores. PCR cycle dideoxynucleotide sequencing was used to sequence candidate genes inside the linked region for mutations. RP in this family shows linkage to markers in a 10.5 cM (8.9 Mbp) region of chromosome 1p13.3-p21.2 between D1S2896 and D1S457. D1S485 yields the highest lod score of 6.54 at theta=0. Sequencing the exons and intron-exon boundaries of five candidate genes and six ESTs in this region, OLFM3, GNAI3, LOC126987, FLJ25070, DKFZp586G0123, AV729694, BU662869, BU656110, BU171991, BQ953690, and CA397743, did not identify any causative mutations. This novel locus lies approximately 4.9 cM (7.1 Mbp) from ABCA4, which is excluded from the linked region. Identification and study of this gene may help to elucidate the phenotypic diversity of arRP mapping to this region.

Usher syndrome type I (USH1), the most severe form of this syndrome, is characterized by profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. At least seven USH1 loci, USH1A-G, have been mapped to the chromosome regions 14q32, 11q13.5, 11p15, 10q21-q22, 21q21, 10q21-q22, and 17q24-25, respectively. Mutations in five genes, including MYO7A, USH1C, CDH23, PCDH15 and SANS, have been shown to be the cause of Usher syndrome type 1B, type 1C, type 1D, type 1F and type 1G, respectively. In the present study, we carried out a systematic mutation screening of these genes in USH1 patients from USA and from UK. We identified a total of 27 different mutations; of these, 19 are novel, including nine missense, two nonsense, four deletions, one insertion and three splicing defects. Approximatelly 35–39% of the observed mutations involved the USH1B and USH1D genes, followed by 11% for USH1F and 7% for USH1C in non-Acadian alleles and 7% for USH1G. Two of the 12 MYO7A mutations, R666X and IVS40-1G>T accounted for 38% of the mutations at that locus. A 193delC mutation accounted for 26% of CDH23 (USH1D) mutations, confirming its high frequency. The most common PCDH15 (USH1F) mutation in this study, 5601-5603delAAC, accounts for 33% of mutant alleles. Interestingly, a novel SANS mutation, W38X, was observed only in the USA cohort. The present study suggests that mutations in MYO7A and CDH23 are the two major components of causes for USH1, while PCDH15, USH1C, and SANS are less frequent causes.

Advances in molecular biological and genetic technology have greatly accelerated elucidation of the genetic contribution to age-related cataract. Epidemiological studies have documented tendencies for cataracts to occur more frequently in relatives of cataract patients than in the general population, genetic studies have demonstrated contributory roles of some specific genes in age related cataract in small populations, and molecular studies have shown changes in expression of specific genes in cataractous lenses. Together, these studies are beginning to provide a conceptual framework for understanding age-related cataracts.

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive retinal dystrophy characterized by multiple glistening intraretinal crystals scattered over the fundus, a characteristic degeneration of the retina, and sclerosis of the choroidal vessels, ultimately resulting in progressive night blindness and constriction of the visual field. The BCD region of chromosome 4q35.1 was refined to an interval flanked centromerically by D4S2924 by linkage and haplotype analysis; mutations were found in the novel CYP450 family member CYP4V2 in 23 of 25 unrelated patients with BCD tested. The CYP4V2 gene, transcribed from 11 exons spanning 19 kb, is expressed widely. Homology to other CYP450 proteins suggests that CYP4V2 may have a role in fatty acid and steroid metabolism, consistent with biochemical studies of patients with BCD.