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To identify the genetic basis of an unusual pediatric cortical cataract demonstrating autosomal dominant inheritance in a large European-Australian pedigree.DNA from four affected individuals were exome sequenced utilizing a NimbleGen SeqCap EZ Exome V3 kit and HiSeq 2500. DNA from 12 affected and four unaffected individuals were genotyped using Human OmniExpress-24 BeadChips. Multipoint linkage and haplotyping were performed (Superlink-Online SNP). DNA from one affected individual and his unaffected father were whole-genome sequenced on a HiSeq X Ten system. Rare small insertions/deletions and single-nucleotide variants (SNVs) were identified in the disease-linked region (Golden Helix SVS). Combined Annotation Dependent Depletion (CADD) analysis predicted variant deleteriousness. Putative enhancer function and variant effects were determined using the Dual-Glo Luciferase Assay system.Linkage mapping identified a 6.23-centimorgan support interval at chromosome 7q36. A co-segregating haplotype refined the critical region to 6.03 Mbp containing 21 protein-coding genes. Whole-genome sequencing uncovered 114 noncoding variants from which CADD predicted one was highly deleterious, a novel substitution within intron-1 of the sonic hedgehog signaling molecule (SHH) gene. ENCODE data suggested this site was a putative enhancer, subsequently confirmed by luciferase reporter assays with variant-associated gene overexpression.In a large pedigree, we have identified a SHH intron variant that co-segregates with an unusual pediatric cortical cataract phenotype. SHH is important for lens formation, and mutations in its receptor (PTCH1) cause syndromic cataract. Our data implicate increased function of an enhancer important for SHH expression primarily within developing eye tissues.

Purpose Affecting children by age 3, primary congenital glaucoma (PCG) can cause debilitating vision loss by the developmental impairment of aqueous drainage resulting in high intraocular pressure (IOP), globe enlargement, and optic neuropathy. TEK haploinsufficiency accounts for 5% of PCG in diverse populations, with low penetrance explained by variable dysgenesis of Schlemm's canal (SC) in mice. We report eight families with TEK-related PCG, and provide evidence for SVEP1 as a disease modifier in family 8 with a higher penetrance and severity. Methods Exome sequencing identified coding/splice site variants with an allele frequency less than 0.0001 (gnomAD). TEK variant effects were assayed in construct-transfected HEK293 cells via detection of autophosphorylated (active) TEK protein. An enucleated eye from an affected member of family 8 was examined via histology. SVEP1 expression in developing outflow tissues was detected by immunofluorescent staining of 7-day mouse anterior segments. SVEP1 stimulation of TEK expression in human umbilical vascular endothelial cells (HUVECs) was measured by TaqMan quantitative PCR. Results Heterozygous TEK loss-of-function alleles were identified in eight PCG families, with parent-child disease transmission observed in two pedigrees. Family 8 exhibited greater disease penetrance and severity, histology revealed absence of SC in one eye, and SVEP1:p.R997C was identified in four of the five affected individuals. During SC development, SVEP1 is secreted by surrounding tissues. SVEP1:p.R997C abrogates stimulation of TEK expression by HUVECs. Conclusions We provide further evidence for PCG caused by TEK haploinsufficiency, affirm autosomal dominant inheritance in two pedigrees, and propose SVEP1 as a modifier of TEK expression during SC development, affecting disease penetrance and severity.

Primary congenital glaucoma (PCG) is a leading cause of blindness in children worldwide and is caused by developmental defects in 2 aqueous humor outflow structures, Schlemm’s canal (SC) and the trabecular meshwork. We previously identified loss-of-function mutations in the angiopoietin (ANGPT) receptor TEK in families with PCG and showed that ANGPT/TEK signaling is essential for SC development. Here, we describe roles for the major ANGPT ligands in the development of the aqueous outflow pathway. We determined that ANGPT1 is essential for SC development, and that Angpt1-knockout mice form a severely hypomorphic canal with elevated intraocular pressure. By contrast, ANGPT2 was dispensable, although mice deficient in both Angpt1 and Angpt2 completely lacked SC, indicating that ANGPT2 compensates for the loss of ANGPT1. In addition, we identified 3 human subjects with rare ANGPT1 variants within an international cohort of 284 PCG patients. Loss of function in 2 of the 3 patient alleles was observed by functional analysis of ANGPT1 variants in a combined in silico, in vitro, and in vivo approach, supporting a causative role for ANGPT1 in disease. By linking ANGPT1 with PCG, these results highlight the importance of ANGPT/TEK signaling in glaucoma pathogenesis and identify a candidate target for therapeutic development.

ObjectivesTo assess whether epigenetic mechanisms affecting gene expression may be involved in the pathogenesis of early-onset myopia, we performed genome-wide DNA methylation analyses of umbilical cord tissues, and assessed any associations between CpG site-specific methylation and the development of the disorder when the children were 3 years old.MethodsGenome-wide DNA methylation profiling of umbilical cord samples from 519 Singaporean infants involved in a prospective birth cohort 'Growing Up in Singapore Towards healthy Outcomes' (GUSTO) was performed using the Illumina Infinium HumanMethylation450K chip microarray. Multivariable logistic regression models were used to assess any associations between site-specific CpG methylation of umbilical cord tissue at birth and myopia risk in 3 year old children, adjusting for potential confounders. Gene expression of genes located near CpG sites that demonstrated statistically significant associations were measured in relevant ocular tissues using human and mouse fetal and adult eye samples.ResultsWe identified statistically significant associations between DNA methylation levels at five CpG sites and early-onset myopia risk after correcting for multiple comparisons using a false discovery rate of 5%. Two statistically significant CpG sites were identified in intergenic regions: 8p23(p = 1.70×10-7) and 12q23.2(p = 2.53×10-7). The remaining 3 statistically significant CpG sites were identified within the following genes: FGB (4q28, p = 3.60×10-7), PQLC1 (18q23, p = 8.9×10-7) and KRT12 (17q21.2, p = 1.2×10-6). Both PQLC1 and KRT12 were found to be significantly expressed in fetal and adult cornea and sclera tissues in both human and mouse.ConclusionsWe identified five CpG methylation sites that demonstrate a statistically significant association with increased risk of developing early-onset myopia. These findings suggest that variability in the neonatal cord epigenome may influence early-onset myopia risk in children. Further studies of the epigenetic influences on myopia risk in larger study populations, and the associations with adulthood myopia risk are warranted.

Purpose: To evaluate the roles of known myopia-associated genetic variants for development of myopic macular degeneration (MMD) in individuals with high myopia (HM), using case-control studies from the Consortium of Refractive Error and Myopia (CREAM). Methods: A candidate gene approach tested 50 myopia-associated loci for association with HM and MMD, using meta-analyses of case-control studies comprising subjects of European and Asian ancestry aged 30 to 80 years from 10 studies. Fifty loci with the strongest associations with myopia were chosen from a previous published GWAS study. Highly myopic (spherical equivalent [SE] ≤ -5.0 diopters [D]) cases with MMD (N = 348), and two sets of controls were enrolled: (1) the first set included 16, 275 emmetropes (SE ≤ -0.5 D); and (2) second set included 898 highly myopic subjects (SE ≤ -5.0 D) without MMD. MMD was classified based on the International photographic classification for pathologic myopia (META-PM). Results: In the first analysis, comprising highly myopic cases with MMD (N = 348) versus emmetropic controls without MMD (N = 16, 275), two SNPs were significantly associated with high myopia in adults with HM and MMD: (1) rs10824518 (P = 6.20E-07) in KCNMA1, which is highly expressed in human retinal and scleral tissues; and (2) rs524952 (P = 2.32E-16) near GJD2. In the second analysis, comprising highly myopic cases with MMD (N = 348) versus highly myopic controls without MMD (N = 898), none of the SNPs studied reached Bonferroni-corrected significance. Conclusions: Of the 50 myopia-associated loci, we did not find any variant specifically associated with MMD, but the KCNMA1 and GJD2 loci were significantly associated with HM in highly myopic subjects with MMD, compared to emmetropes., Correction--10 Oct 2019: Wong YL, Hysi P, Cheung G, Tedja M, Hoang QV, et al. (2019) Correction: Genetic variants linked to myopic macular degeneration in persons with high myopia: CREAM Consortium. PLOS ONE 14(10): e0223942. https://doi.org/10.1371/journal.pone.0223942

BACKGROUND: In a four-generation Caucasian family variably diagnosed with autosomal dominant (AD) Stickler or Wagner disease, commercial gene screening failed to identify a mutation in COL2A1 or VCAN. We utilized linkage mapping and exome sequencing to identify the causal variant. MATERIALS AND METHODS: Genomic DNA samples collected from 40 family members were analyzed. A whole-genome linkage scan was performed using Illumina HumanLinkage-24 BeadChip followed by two-point and multipoint linkage analyses using FASTLINK and MERLIN. Exome sequencing was performed on two affected individuals, followed by co-segregation analysis. RESULTS: Parametric multipoint linkage analysis using an AD inheritance model demonstrated HLOD scores > 2.00 at chromosomes 1p36.13–1p36.11 and 12q12–12q14.1. SIMWALK multipoint analysis replicated the peak in chromosome 12q (peak LOD = 1.975). FASTLINK two-point analysis highlighted several clustered chromosome 12q SNPs with HLOD > 1.0. Exome sequencing revealed a novel nonsense mutation (c.115C>T, p.Gln39*) in exon 2 of COL2A1 that is expected to result in nonsense-mediated decay of the RNA transcript. This mutation co-segregated with all clinically affected individuals and seven individuals who were clinically unaffected. CONCLUSIONS: The utility of combining traditional linkage mapping and exome sequencing is highlighted to identify gene mutations in large families displaying a Mendelian inheritance of disease. Historically, nonsense mutations in exon 2 of COL2A1 have been reported to cause a fully penetrant ocular-only Stickler phenotype with few or no systemic manifestations. We report a novel nonsense mutation in exon 2 of COL2A1 that displays incomplete penetrance and/or variable age of onset with extraocular manifestations.

Purpose To identify causal gene mutations in 14 families with autosomal dominant (AD) high myopia using exome sequencing. Methods Select individuals from 14 large Caucasian families with high myopia were exome sequenced. Gene variants were filtered to identify potential pathogenic changes. Sanger sequencing was used to confirm variants in original DNA, and to test for disease cosegregation in additional family members. Candidate genes and chromosomal loci previously associated with myopic refractive error and its endophenotypes were comprehensively screened. Results In 14 high myopia families, we identified 73 rare and 31 novel gene variants as candidates for pathogenicity. In seven of these families, two of the novel and eight of the rare variants were within known myopia loci. A total of 104 heterozygous nonsynonymous rare variants in 104 genes were identified in 10 out of 14 probands. Each variant cosegregated with affection status. No rare variants were identified in genes known to cause myopia or in genes closest to published genome-wide association study association signals for refractive error or its endophenotypes. Conclusions Whole exome sequencing was performed to determine gene variants implicated in the pathogenesis of AD high myopia. This study provides new genes for consideration in the pathogenesis of high myopia, and may aid in the development of genetic profiling of those at greatest risk for attendant ocular morbidities of this disorder.

Primary congenital glaucoma (PCG) is a devastating eye disease and an important cause of childhood blindness worldwide. In PCG, defects in the anterior chamber aqueous humor outflow structures of the eye result in elevated intraocular pressure (IOP); however, the genes and molecular mechanisms involved in the etiology of these defects have not been fully characterized. Previously, we observed PCG-like phenotypes in transgenic mice that lack functional angiopoietin-TEK signaling. Herein, we identified rare TEK variants in 10 of 189 unrelated PCG families and demonstrated that each mutation results in haploinsufficiency due to protein loss of function. Multiple cellular mechanisms were responsible for the loss of protein function resulting from individual TEK variants, including an absence of normal protein production, protein aggregate formation, enhanced proteasomal degradation, altered subcellular localization, and reduced responsiveness to ligand stimulation. Further, in mice, hemizygosity for Tek led to the formation of severely hypomorphic Schlemm’s canal and trabecular meshwork, as well as elevated IOP, demonstrating that anterior chamber vascular development is sensitive to Tek gene dosage and the resulting decrease in angiopoietin-TEK signaling. Collectively, these results identify TEK mutations in patients with PCG that likely underlie disease and are transmitted in an autosomal dominant pattern with variable expressivity. Published version