Comprehensive Genetic Analysis Unraveled the Missing Heritability and a Founder Variant of BEST1 in a Chinese Cohort With Autosomal Recessive Bestrophinopathy.

Purpose: To describe the genetic landscape of BEST1 for a large Chinese cohort with autosomal recessive bestrophinopathy (ARB), identify the missing heritability, and report a common Chinese founder variant. Methods: We recruited 65 patients from 63 families with a clinical diagnosis of ARB. All patients underwent ophthalmic examinations and comprehensive genetic analyses, including Sanger DNA sequencing of BEST1 and whole genome sequencing (WGS). The effects of deep intronic variants (DIVs) on splicing were assessed using in vitro splicing assays in HEK293T cells and patient-derived peripheral blood mononuclear cells. Haplotype mapping was performed for 17 unrelated patients harboring variant c.867+97G>A. Results: We identified 54 distinct disease-causing variants of BEST1 in 63 pedigrees, 62 probands with biallelic variants, and one family with monoallelic variants. Sanger DNA sequencing of BEST1 initially detected 51 variants in 61 pedigrees, including 19 probands with one heterozygous variant. Subsequent WGS, combined with supplementary Sanger sequencing, revealed three missing DIVs (c.1101-491A>G, c.867+97G>A, and c.867+97G>T) in 20 families. The novel DIV c.1101-491A>G caused an abnormal splicing resulting in a 204-nt pseudoexon (PE) insertion, whereas c.867+97G>A/T relatively strengthened an alternative donor site, resulting in a 203-nt intron retention (IR). The PE and IR generated a premature termination codon downstream. Haplotype analysis identified c.867+97G>A as a common founder variant with an allele frequency of 16%. Conclusions: Our results expand the pathogenic variant spectrum of BEST1, and DIVs can explain almost all of the missing heritability. The c.867+97G>A DIV is a common founder variant for Chinese patients with ARB.

Unraveling the genetic cause of hereditary ophthalmic disorders in Arab societies from Israel and the Palestinian Authority.

Visual impairment due to inherited ophthalmic disorders is amongst the most common disabilities observed in populations practicing consanguineous marriages. Here we investigated the molecular genetic basis of an unselected broad range of ophthalmic disorders in 20 consanguineous families from Arab villages of Israel and the Palestinian Authority. Most patients had little or very poor prior clinical workup and were recruited in a field study. Homozygosity mapping followed by candidate gene sequencing applying conventional Sanger sequencing or targeted next generation sequencing was performed in six families. In the remaining 14 families, one affected subject per family was chosen for whole exome sequencing. We discovered likely disease-causing variants, all homozygous, in 19 of 20 independent families (95%) including a previously reported novel disease gene for congenital nystagmus associated with foveal hypoplasia. Moreover, we found a family in which disease-causing variants for two collagenopathies - Stickler and Knobloch syndrome - segregate within a large sibship. Nine of the 19 distinct variants observed in this study were novel. Our study demonstrated a very high molecular diagnostic yield for a highly diverse spectrum of rare ophthalmic disorders in Arab patients from Israel and the Palestinian Authority, even with very limited prior clinical investigation. We conclude that 'genetic testing first' may be an economic way to direct clinical care and to support proper genetic counseling and risk assessment in these families.

CUTICULAR DRUSEN IN AN INDIGENOUS AUSTRALIAN.

BACKGROUND/PURPOSE: To report a case of cuticular drusen in an indigenous Australian. METHODS: A 37-year-old indigenous (aboriginal) Australian woman from a remote Western Australian town presented with a 2-month history of vision loss. Clinical history, examination, and multimodal retinal imaging data from spectral domain optical coherence tomography, fundus autofluorescence, fluorescein angiography, and indocyanine green angiography were analyzed. RESULTS: Multimodal imaging confirmed cuticular drusen complicated by a right choroidal neovascularization with pigment epithelial detachment and a left foveal vitelliform lesion. An unusual "negative-staining" pattern was noted on late phase indocyanine green angiography and this spared the regions affected by cuticular drusen. CONCLUSION: To the best of our knowledge, this is the first published report of cuticular drusen in an indigenous Australian. We hypothesize that this may be secondary to ancestral mixing of the gene pool. An unusual staining pattern witnessed on indocyanine green angiography suggests widespread disturbance of lipoprotein deposition in the Bruch membrane.

A novel frameshift mutation in the PITX2 gene in a family with Axenfeld-Rieger syndrome using targeted exome sequencing.

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is an autosomal dominant genetic disorder that is characterized by specific abnormalities of the anterior segment of the eye. Heterozygous mutations in two developmental transcription factor genes PITX2 and FOXC1 have been identified within ARS patients, accounting for 40 to 70% of cases. Our purpose is to describe clinical and genetic findings in a Chinese family with ARS. METHODS: An ARS family with three affected members was recruited. The patients underwent a series of complete ophthalmologic examinations, general physical examination and dental radiography. DNA samples of proband II-1 were used for targeted exome sequencing of the FOXC1 and PITX2 genes. Sanger sequencing was used to validate the variation in PITX2. Quantitative real-time PCR was carried out to detect the expression of PITX2 in patients and normal controls. RESULTS: All affected members showed iris atrophy, corectopia, shallow anterior chamber, complete or partial angle closure, and advanced glaucoma. In addition, they revealed systemic anomalies, including microdontia, hypodontia, and redundant periumbilical skin. A novel heterozygous frameshift variation, c.515delA, in PITX2 was found in the proband, which might lead to a truncated PITX2 protein (p.Gln172ArgfsX36). Sanger sequencing validated that the variation completely cosegregated with the ARS phenotype among this family, but was absent in 100 unrelated controls. Quantitative real-time PCR analysis revealed that the mRNA expression of PITX2 was significantly decreased in patients compared with that in unrelated normal controls. CONCLUSIONS: PITX2 c.515delA (p.Gln172ArgfsX36) was the genetic etiology of our pedigree. The mutation led to decreased PITX2 gene expression and a truncated mRNA transcript.

Variable reduction in Norrin signaling activity caused by novel mutations in FZD4 identified in patients with familial exudative vitreoretinopathy.

Purpose: To identify novel mutations in FZD4 and to investigate their pathogenicity in a cohort of Chinese patients with familial exudative vitreoretinopathy (FEVR). Methods: Next-generation sequencing was performed in patients with a clinical diagnosis of FEVR. Wide-field angiography was performed in probands and family members if available. Clinical data were collected from patient charts. The effect of the mutations in FZD4 on its biologic activity in the Norrin/ß-catenin signaling pathway was analyzed with the luciferase reporter assay. Results: Four novel mutations in FZD4 (c.1188_1192del/p.F396fs, c.1220delC/p.A407Vfs*24, c.905G>A/p.C302Y, c.1325T>A/p.V442E) were identified in four unrelated families. The mutations were not detected in 200 healthy individuals. The variability of the ocular phenotypes was not only observed in the probands and parents harboring the same mutation but also between two eyes in one individual. All four novel mutations introduced reduction in luciferase activity. Compared with the wild-type, the FZD4 level of the four mutants also decreased variably. Conclusions: Four novel mutations in FZD4 were identified in Chinese patients with FEVR. No correlation in the reduced luciferase activity and the ocular phenotype was observed in this study. This study further emphasized the complexity of the FEVR-causing machinery.

Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases.

Central corneal thickness (CCT) is a highly heritable trait associated with complex eye diseases such as keratoconus and glaucoma. We perform a genome-wide association meta-analysis of CCT and identify 19 novel regions. In addition to adding support for known connective tissue-related pathways, pathway analyses uncover previously unreported gene sets. Remarkably, >20% of the CCT-loci are near or within Mendelian disorder genes. These included FBN1, ADAMTS2 and TGFB2 which associate with connective tissue disorders (Marfan, Ehlers-Danlos and Loeys-Dietz syndromes), and the LUM-DCN-KERA gene complex involved in myopia, corneal dystrophies and cornea plana. Using index CCT-increasing variants, we find a significant inverse correlation in effect sizes between CCT and keratoconus (r = -0.62, P = 5.30 × 10-5) but not between CCT and primary open-angle glaucoma (r = -0.17, P = 0.2). Our findings provide evidence for shared genetic influences between CCT and keratoconus, and implicate candidate genes acting in collagen and extracellular matrix regulation.

Genotypic spectrum and phenotype correlations of ABCA4-associated disease in patients of south Asian descent.

Variants in the ABCA4 gene are the most common cause of juvenile-onset blindness affecting close to 1 in 10 000 people worldwide. Disease severity varies largely according to genotype, which can be specific to ethnic and racial groups. Here we investigate the spectrum of ABCA4 variation and its phenotypic expression in 38 patients of South Asian descent, notably from India, Pakistan, Bangladesh and Sri Lanka. Sequencing of all exons and flanking intronic sequences of ABCA4 revealed disease-causing variants in all patients: 3 in 2.6%, 2 in 81.6% and 1 in 15.8%. Altogether, 36 distinct variants were identified, including 9 previously not described. The most frequent variant c.5882G>A, p.(G1961E) was found in half the patients, the highest ever reported in a single study cohort. The South Asian founder variant c.859-9T>C was identified along with other founder variants ascribed to Danish, Chinese, Mexican and African patients. Patients carrying c.5882G>A, p.(G1961E) exhibited a consistently confined disease phenotype, normal quantitative autofluorescence (qAF) levels and preserved full-field ERG (ffERG) while c.859-9T>C resulted in widespread disease, significantly elevated qAF and reduced to non-detectable ffERG. South Asian patients present with a relatively unique ABCA4 profile comprised of various ethnic founder variants resulting in two or three major retinal phenotypes.

Screening for BEST1 gene mutations in Chinese patients with bestrophinopathy.

PURPOSE: The purpose of this study was to analyze BEST1 gene mutations in Chinese patients with bestrophinopathy and to describe the clinical features of these patients. METHODS: Thirteen patients from 12 unrelated Chinese families affected by bestrophinopathy were recruited and clinically evaluated with best-corrected visual acuity examination, slit-lamp biomicroscopy, fundus examination and photography, optical coherence tomography, fundus autofluorescence, electro-oculography, and electroretinography. Blood samples were collected for DNA extraction. Mutation analysis was performed by direct sequencing of the BEST1 gene. One hundred control chromosomes were also screened to exclude nonpathogenic polymorphisms. RESULTS: Seven patients showed clinical pictures of Best vitelliform macular dystrophy (BVMD) and harbored heterozygous mutations compatible with autosomal dominant inheritance. Two novel mutations (p.T4I and p.A291V) and three reported mutations (p.R218C, p.Q293H, and p.D301G) were identified. Six patients carried BEST1 mutations on both alleles compatible with autosomal recessive inheritance. Compound heterozygous mutations were detected in four patients who presented a BVMD phenotype, while homozygous mutations were detected in two patients with autosomal recessive bestrophinopathy. Mutation analysis revealed eight mutations. Four (p.Y33H, p.R130L, p.M163R, and c.519delA) were novel, and four (p.R13H, p.A195V, p.R255W, and p.W287*) had previously been reported. CONCLUSIONS: Patients with biallelic BEST1 mutations were common among Chinese patients with bestrophinopathy, and the phenotypes varied. The features and combinations of different BEST1 mutations as well as epistatic effects may influence phenotype expression. Our results expand the BEST1 mutation spectrum.

Incidence of fovea plana in normal children.

PURPOSE: To characterize the prevalence and features of subclinical foveal hypoplasia detected by optical coherence tomography (OCT) in children. METHODS: Fast macular OCT scans were performed on normal children with normal vision for the development of a normative OCT-3 database; from this data, eyes with no discernable foveal depression were identified. When possible, the ocular imaging was repeated 3 years later using both OCT-3 and spectral domain OCT (SD-OCT). SD-OCT results were compared to age-matched controls. RESULTS: Of the 286 normal children (mean age, 8.6 ± 3.1 years) scanned, 9 (mean age, 8 ± 2.9 years; 6 males) were found to have bilateral shallow foveal depression on OCT-3 imaging, including 8 of 154 white children (5.4%) and 1 child of mixed ethnicity (white/black). Children with shallow foveas (n = 9) had larger average foveal thickness (FT) compared to the cohort of controls (n = 277) with a defined fovea (FT = 231.4 ± 8.8 vs 188.8 ± 25.0, resp. [P < 0.0001]). Mean macular volume did not differ from that of controls. SD-OCT performed 3 years later on 5 of the 9 children with shallow foveal depression showed persistence of the inner macular layers over the foveal center, corresponding to grades 1 or 2 of foveal hypoplasia. The FT was increased compared to 5 age-matched controls with a defined fovea (FT = 294.5 ± 5.1 vs 219.75 ± 5.68 µm, resp. [P = 0.029]). CONCLUSIONS: Up to 3% of children with clinically normal eyes had an anatomically underdeveloped foveal pit bilaterally on OCT.

Clinical and hereditary features of familial vitreous amyloidosis in Chinese.

PURPOSE: To investigate the clinical and hereditary features of a Chinese Han pedigree with familial vitreous amyloidosis. METHODS: The hereditary features of familial traits were detected by drawing genealogy, and the clinical manifestations were observed. RESULTS: This family with 4 generations of 32 family members had the characteristics of euchromosome dominant inheritance. The age of onset in heterozygotes was over 40 years old in male and over 55 in female. All affected individuals had curly hair.Among the 23 family members of the first 3 generations, 7 had the final diagnosis . Four of the cases treated by vitrectomy was found to have open angle glaucoma during the follow-up. CONCLUSION: We reported a Chinese Han pedigree with familial vitreous amyloidosis which is a rare condition in Chinese and described the clinical and hereditary features. The genetic sequencing and animal model are undergoing.

Retinal and optic disc atrophy associated with a CACNA1F mutation in a Japanese family.

OBJECTIVE: To describe retinal and optic disc atrophy and a progressive decrease of visual function in 2 Japanese brothers. Both had a mutation in the CACNA1F gene, the causative gene of incomplete congenital stationary night blindness (CSNB). METHODS: We studied observational case reports and performed comprehensive ophthalmologic examinations including best-corrected visual acuity, biomicroscopy, ophthalmoscopy, fundus photography, and electroretinography. Genomic DNA was extracted from the peripheral blood, and all 48 exons of the CACNA1F gene were directly sequenced. RESULTS: The 2 brothers had retinal and optic disc atrophy and a progressive reduction of visual acuity with increasing age. Although these clinical features are not typical of previous patients with incomplete CSNB, both patients had an in-frame mutation with deletion and insertion in exon 4 of the CACNA1F gene. In both patients, the bright-flash, mixed rod-cone electroretinogram had a negative configuration, a characteristic of incomplete CSNB. However, the full-field scotopic and photopic electroretinograms were nonrecordable, indicating severe, diffuse retinal malfunction, which is not typical in incomplete CSNB. CONCLUSION: These findings indicate that a mutation of the CACNA1F gene may be associated with retinal and optic disc atrophy with a progressive decline of visual function. Clinical Relevance In patients with retinal and optic disc atrophy associated with negative-type electroretinograms, a CACNA1F gene mutation should be considered.

A nonsense mutation (W9X) in CRYAA causes autosomal recessive cataract in an inbred Jewish Persian family.

PURPOSE: To identify the genetic defect causing autosomal recessive cataract in two inbred families. METHODS: Linkage analysis was performed with polymorphic markers close to 14 loci previously shown to be involved in autosomal dominant congenital cataract. In one of the families a gene segregating with the disease was analyzed by single-strand conformation polymorphism (SSCP) and eventually sequenced. RESULTS: Three polymorphic markers close to the CRYAA gene located on chromosome 21q segregated with the disease phenotype in one of the families, but not in the other. Sequencing of the CRYAA in this Jewish Persian family revealed a G-to-A substitution, resulting in the formation of a premature stop codon (W9X). CONCLUSIONS: A nonsense mutation in the CRYAA gene causes autosomal recessive cataract in one family. This constitutes the first description of the molecular defect underlying nonsyndromic autosomal recessive congenital cataract. That there was no linkage to this locus in another family provides evidence for genetic heterogeneity.

A frequent 1085delC/insGAAG mutation in the RDH5 gene in Japanese patients with fundus albipunctatus.

PURPOSE: To identify the frequency of a mutation of the RDH5 gene in Japanese patients with hereditary retinal degeneration and to characterize clinical findings for the patients associated with a 1085delC/insGAAG mutation in the RDH5 gene. METHODS: Mutation screening by single-strand conformation polymorphism was performed on 6 patients with fundus albipunctatus and 150 patients with autosomal recessive retinitis pigmentosa. The DNA fragment that showed abnormal mobility on SSCP was then sequenced. Clinical features were characterized by visual acuity, slit-lamp biomicroscopy, electroretinography, fluorescein angiography, kinetic visual field testing, and dark adaptometry. RESULTS: A novel 1085delC/insGAAG mutation in the RDH5 gene was identified in all 6 patients, from 4 unrelated families with fundus albipunctatus. The ophthalmic findings of each affected member were very similar, which may provide the natural course of the phenotype produced by the 1085delC/insGAAG mutation. CONCLUSIONS: A homozygous1085delC/insGAAG mutation in the RDH5 gene produces fundus albipunctatus in Japanese patients. These findings suggest that this mutation was a founder effect in Japanese patients with fundus albipunctatus.