Novel mutations in CACNA1F and NYX in Dutch families with X-linked congenital stationary night blindness

PURPOSE: To describe the clinical features and genetic analysis of eight X-linked congenital stationary night blindness (XLCSNB) Dutch patients. METHODS: Electroretinogram (ERG) measurements were assessed in Dutch patients. Molecular genetic testing by denaturing high performance liquid chromatography (DHPLC), single stranded conformation polymorphism (SSCP) analysis, and direct sequencing of the CACNA1F and NYX genes were performed in the patients possessing a negative Schubert Bornschein ERG. RESULTS: Molecular genetic testing of CACNA1F and NYX revealed three novel and two known CACNA1F sequence variants as well as two novel sequence alterations in the NYX gene. While one of the CACNA1F sequence variants (5756G>A, R1919H) has been previously described as a common polymorphism in Japanese families, we did not found this transition in 100 European control alleles. CONCLUSIONS: In a pool of eight diagnosed XLCSNB patients, five showed a sequence variation in the CACNA1F and two in the NYX gene. In only one of the eight patients no sequence alteration could be detected. This might be explained by a mutation in other, as yet unidentified coding or regulatory sequences of NYX or CACNA1F or additional genes

A nonsense mutation in S-antigen (p.Glu306*) causes Oguchi disease

Contains fulltext : 110974.pdf (publisher's version ) (Open Access)PURPOSE: Genetic studies were performed to identify the causative mutation in a 15-year-old girl diagnosed with congenital stationary night blindness (CSNB) presenting Mizuo-Nakamura phenomenon, a typical Oguchi disease symptom. The patient also had dural sinus thrombosis (DST), thrombocytopenia, and systemic lupus erythematosus (SLE). METHODS: Mutation analysis was done by sequencing two candidate genes, S-antigen (SAG; arrestin 1), associated with Oguchi type 1, and rhodopsin kinase (GRK1), associated with Oguchi type 2. In addition, the C677T variation in the methylenetetrahydrofolate reductase (MTHFR) gene was also screened in the family, to determine its probable association with hyperhomocysteinemia in the patient. RESULTS: Sequencing of the SAG and GRK1 resulted in identifying a novel homozygous nonsense mutation (c.916G>T; p.Glu306*) in SAG, which in unaffected siblings either was present in a heterozygous state or absent. The C677T heterozygous allele in the MTHFR gene was found to be associated with hyperhomocysteinemia in the patient and other family members. CONCLUSIONS: This is the first report of Oguchi type 1 in a Pakistani patient due to a nonsense mutation (c.916G>T; p.Glu306*) in SAG. The neurologic and hematological abnormalities likely are not associated with the SAG variant

Identification of recurrent and novel mutations in TULP1 in Pakistani families with early-onset retinitis pigmentosa

Contains fulltext : 108208.pdf (publisher's version ) (Open Access)PURPOSE: To identify the genetic defects underlying retinitis pigmentosa (RP) in Pakistani families. METHODS: Genome-wide high-density single-nucleotide-polymorphism microarray analysis was performed using the DNA of nine affected individuals from two large families with multiple consanguineous marriages. Data were analyzed to identify homozygous regions that are shared by affected sibs in each family. Sanger sequencing was performed for genes previously implicated in autosomal recessive RP and allied retinal dystrophies that resided in the identified homozygous regions. Probands from both families underwent fundus examination and electroretinogram measurements. RESULTS: The tubby-like protein 1 gene (TULP1) was present in the largest homozygous region in both families. Sequence analysis identified a previously reported mutation (c.1138A>G; p.Thr380Ala) in one family and a novel pathogenic variant (c.1445G>A; p.Arg482Gln) in the other family. Both variants were found to be present in a homozygous state in all affected individuals, were heterozygous present in the unaffected parents, and heterozygous present or absent in normal individuals. Affected individuals of both families showed an early-onset form of RP. CONCLUSIONS: Homozygosity mapping, combined with candidate-gene analysis, successfully identified genetic defects in TULP1 in two large Pakistani families with early-onset retinitis pigmentosa

A homozygous p.Glu150Lys mutation in the opsin gene of two Pakistani families with autosomal recessive retinitis pigmentosa

PURPOSE: To identify the gene mutations responsible for autosomal recessive retinitis pigmentosa (arRP) in Pakistani families. METHODS: A cohort of consanguineous families with typical RP phenotype in patients was screened by homozygosity mapping using microsatellite markers that mapped close to 21 known arRP genes and five arRP loci. Mutation analysis was performed by direct sequencing of the candidate gene. RESULTS: In two families, RP21 and RP53, homozygosity mapping suggested RHO, the gene encoding rhodopsin, as a candidate disease gene on chromosome 3q21. In six out of seven affected members from the two families, direct sequencing of RHO identified a homozygous c.448G>A mutation resulting in the p.Glu150Lys amino acid change. This variant was first reported in PMK197, an Indian arRP family. Single nucleotide polymorphism analysis in RP21, RP53, and PMK197 showed a common disease-associated haplotype in the three families. CONCLUSIONS: In two consanguineous Pakistani families with typical arRP phenotype in the patients, we identified a disease-causing mutation (p.Glu150Lys) in the RHO gene. Single nucleotide polymorphism analysis suggests that the previously reported Indian family (PMK197) and the two Pakistani families studied here share the RHO p.Glu150Lys mutation due to a common ancestry

Asymmetric Inter-Eye Progression in Stargardt Disease

PURPOSE. Asymmetry in disease progression between left and right eyes can occur in Stargardt disease (STGD1), and this needs to be considered in novel therapeutic trials with a fellow-eye paired controlled design. This study investigated the inter-eye discordance of best-corrected visual acuity (BCVA) and progression of RPE atrophy in STGD1. METHODS. We performed a retrospective cohort study collecting 68 STGD1 patients (136 eyes) with ‡1 ABCA4 variants and ‡0.5-year follow-up on BCVA and fundus autofluorescence. We compared inter-eye correlations of RPE atrophy progression between early-onset ( 10 years), intermediate-onset , and late-onset ( ‡45) STGD1 and ABCA4 variant combinations by v 2 tests. We identified associations of discordant baseline BCVA and RPE atrophy with discordant RPE atrophy progression by odds ratios (OR). We defined discordance by differences >1.5 interquartile ranges 6 first/third interquartiles. RESULTS. Progression of RPE atrophy correlated moderately between eyes (q ¼ 0.766), which decreased with later onset (P ¼ 9.8 3 10 À7 ) and lower pathogenicity of ABCA4 combinations (P ¼ 0.007). Twelve patients (17.6%) had discordant inter-eye RPE atrophy progression, associated with baseline discordance of RPE atrophy (OR,.34]), but not BCVA (OR, ]). CONCLUSIONS. Lower inter-eye correlations are more likely found in late-onset STGD1 and patients carrying low pathogenic ABCA4 combinations. To achieve the highest power in a therapeutic trial, early-phase studies should minimize inter-eye discordance by selecting earlyonset STGD1 patients carrying severe ABCA4 variants without evidence of asymmetry at baseline

Late-Onset Stargardt Disease Due to Mild, Deep-Intronic ABCA4 Alleles

PURPOSE. To investigate the role of two deep-intronic ABCA4 variants, that showed a mild splice defect in vitro and can occur on the same allele as the low penetrant c.5603A>T, in Stargardt disease (STGD1). METHODS. Ophthalmic data were assessed of 18 STGD1 patients who harbored c.769-784C>T or c.4253+43G>A in combination with a severe ABCA4 variant. Subjects carrying c.[769784C>T; 5603A>T] were clinically compared with a STGD1 cohort previously published carrying c.5603A>T noncomplex. We calculated the penetrances of the intronic variants using ABCA4 allele frequency data of the general population and investigated the effect of c.769-784C>T on splicing in photoreceptor progenitor cells (PPCs). RESULTS. Mostly, late-onset, foveal-sparing STGD1 was observed among subjects harboring c.769-784C>T or c.4253+43G>A (median age of onset, 54.5 and 52.0 years, respectively). However, ages of onset, phenotypes in fundo, and visual acuity courses varied widely. No significant clinical differences were observed between the c.[769-784C>T; 5603A>T] cohort and the c.4253+43G>A or the c.5603A>T cohort. The penetrances of c.769-784C>T (20.5%-39.6%) and c.4253+43G>A (35.8%-43.1%) were reduced, when not considering the effect of yet unidentified or known factors in cis, such as c.5603A>T (identified in 7/7 probands with c.769-784C>T; 1/8 probands with c.4253+43G>A). Variant c.769-784C>T resulted in a pseudo-exon insertion in 15% of the total mRNA (i.e., similar to 30% of the c.769-784C>T allele alone). CONCLUSIONS. Two mild intronic ABCA4 variants could further explain missing heritability in late-onset STGD1, distinguishing it from AMD. The observed clinical variability and calculated reduced penetrance urge research into modifiers within and outside of the ABCA4 gene

Identification of splice defects due to noncanonical splice site or deep‐intronic variants in ABCA4

Pathogenic variants in the ATP-binding cassette transporter A4 (ABCA4) gene cause a continuum of retinal disease phenotypes, including Stargardt disease. Noncanonical splice site (NCSS) and deep-intronic variants constitute a large fraction of disease-causing alleles, defining the functional consequences of which remains a challenge. We aimed to determine the effect on splicing of nine previously reported or unpublished NCSS variants, one near exon splice variant and nine deep-intronic variants in ABCA4, using in vitro splice assays in human embryonic kidney 293T cells. Reverse transcription-polymerase chain reaction and Sanger sequence analysis revealed splicing defects for 12 out of 19 variants. Four deep-intronic variants create pseudoexons or elongate the upstream exon. Furthermore, eight NCSS variants cause a partial deletion or skipping of one or more exons in messenger RNAs. Among the 12 variants, nine lead to premature stop codons and predicted truncated ABCA4 proteins. At least two deep-intronic variants affect splice enhancer and silencer motifs and, therefore, these conserved sequences should be carefully evaluated when predicting the outcome of NCSS and deep-intronic variants

LONGITUDINAL STUDY OFRPE65-ASSOCIATED INHERITED RETINAL DEGENERATIONS

Purpose: To study the disease course ofRPE65-associated inherited retinal degenerations (IRDs) as a function of the genotype, define a critical age for blindness, and identify potential modifiers. Methods: Forty-five patients with IRD from 33 families with biallelicRPE65mutations, 28 stemming from a genetic isolate. We collected retrospective data from medical charts. Coexisting variants in 108 IRD-associated genes were identified with Molecular Inversion Probe analysis. Results: Most patients were diagnosed within the first years of life. Daytime visual function ranged from near-normal to blindness in the first four decades and met WHO criteria for blindness for visual acuity and visual field in the fifth decade. p.(Thr368His) was the most common variant (54%). Intrafamilial variability and interfamilial variability in disease severity and progression were observed. Molecular Inversion Probe analysis confirmed allRPE65variants and identified one additional variant inLRATand one inEYSin two separate patients. Conclusion: All patients withRPE65-associated IRDs developed symptoms within the first year of life. Visual function in childhood and adolescence varied but deteriorated inevitably toward blindness after age 40. In this study, genotype was not predictive of clinical course. The variance in severity of disease could not be explained by double hits in other IRD genes

Cost-effective sequence analysis of 113 genes in 1,192 probands with retinitis pigmentosa and Leber congenital amaurosis

Introduction: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies. Methods: Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases. Results and discussion: After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.This study received funding from Novartis. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. This work was supported by grants from Foundation Fighting Blindness Career Development Award CDGE-0621-0809-RAD (SR), Foundation Fighting Blindness project program award PPA-0123-0841-UCL (SR and SdB), Retinitis Pigmentosa Fighting Blindness, Fight for Sight UK (RP Genome Project GR586), Ghent University Special Research Fund (BOF20/GOA/023) (EDB and BL); EJP RD Solve-RET EJPRD19-234 (EDB, BL, SB, CR, FC, and SR). EDB (1802220N) and BL (1803816N) are FWO Senior Clinical Investigators of the Research Foundation Flanders (FWO). EDB, BL, SB, FC, and SR are members of ERN-EYE (Framework Partnership Agreement No. 739534)

CDK19 is disrupted in a female patient with bilateral congenital retinal folds, microcephaly and mild mental retardation

Microcephaly, mental retardation and congenital retinal folds along with other systemic features have previously been reported as a separate clinical entity. The sporadic nature of the syndrome and lack of clear inheritance patterns pointed to a genetic heterogeneity. Here, we report a genetic analysis of a female patient with microcephaly, congenital bilateral falciform retinal folds, nystagmus, and mental retardation. Karyotyping revealed a de novo pericentric inversion in chromosome 6 with breakpoints in 6p12.1 and 6q21. Fluorescence in situ hybridization analysis narrowed down the region around the breakpoints, and the breakpoint at 6q21 was found to disrupt the CDK19 gene. CDK19 was found to be expressed in a diverse range of tissues including fetal eye and fetal brain. Quantitative PCR of the CDK19 transcript from Epstein–Barr virus-transformed lymphoblastoid cell lines of the patient revealed ~50% reduction in the transcript (p = 0.02), suggesting haploinsufficiency of the gene. cdk8, the closest orthologue of human CDK19 in Drosophila has been shown to play a major role in eye development. Conditional knock-down of Drosophila cdk8 in multiple dendrite (md) neurons resulted in 35% reduced dendritic branching and altered morphology of the dendritic arbour, which appeared to be due in part to a loss of small higher order branches. In addition, Cdk8 mutant md neurons showed diminished dendritic fields revealing an important role of the CDK19 orthologue in the developing nervous system of Drosophila. This is the first time the CDK19 gene, a component of the mediator co-activator complex, has been linked to a human disease