Whole-exome screening for primary congenital glaucoma in Lebanon (original) (raw)
Related papers
Frontiers in Genetics, 2021
High throughput sequencing technologies have revolutionized the identification of mutations responsible for a diverse set of Mendelian disorders, including inherited retinal disorders (IRDs). However, the causal mutations remain elusive for a significant proportion of patients. This may be partially due to pathogenic mutations located in non-coding regions, which are largely missed by capture sequencing targeting the coding regions. The advent of whole-genome sequencing (WGS) allows us to systematically detect non-coding variations. However, the interpretation of these variations remains a significant bottleneck. In this study, we investigated the contribution of deep-intronic splice variants to IRDs. WGS was performed for a cohort of 571 IRD patients who lack a confident molecular diagnosis, and potential deep intronic variants that affect proper splicing were identified using SpliceAI. A total of six deleterious deep intronic variants were identified in eight patients. An in vitro minigene system was applied to further validate the effect of these variants on the splicing pattern of the associated genes. The prediction scores assigned to splice-site disruption positively correlated with the impact of mutations on splicing, as those with lower prediction scores demonstrated partial splicing. Through this study, we estimated the contribution of deep-intronic splice mutations to unassigned IRD patients and leveraged in silico and in vitro methods to establish a framework for prioritizing deep intronic variant candidates for mechanistic and functional analyses.
2019
Purpose Intraocular pressure leading to glaucoma is a major cause of childhood blindness in developing countries. In this study, we sought to identify gene variants potentially associated with primary congenital glaucoma (PCG) in the Mauritanian population. Methods Using next-generation sequencing (NGS), a panel of PCG candidate genes was screened in a search for DNA mutations in four families with multiple occurrences of PCG. Results Targeted exome sequencing analysis revealed predicted pathogenic mutations in four genes: CYP1B1 (c.217_218delTC, p.Ser73Valfs*150), MYOC (878C>A, p.T293K), NTF4 (c.601T>G, p.Cys201Gly), and WDR36 (c.2078A>G, p.Asn693Ser), each carried by a different family. Conclusions Genetic variation associated with PCG in this study reflects the ethnic heterogeneity of the Mauritanian population. However, a larger cohort is needed to identify additional families carrying these mutations and confirm their biologic role.
Genes
Aims: We aimed to validate the pathogenicity of genetic variants identified in inherited retinal dystrophy (IRD) patients, which were located in non-canonical splice sites (NCSS). Methods: After next generation sequencing (NGS) analysis (target gene panels or whole exome sequencing (WES)), NCSS variants were prioritized according to in silico predictions. In vivo and in vitro functional tests were used to validate their pathogenicity. Results: Four novel NCSS variants have been identified. They are located in intron 33 and 34 of ABCA4 (c.4774-9G>A and c.4849-8C>G, respectively), intron 2 of POC1B (c.101-3T>G) and intron 3 of RP2 (c.884-14G>A). Functional analysis detected different aberrant splicing events, including intron retention, exon skipping and intronic nucleotide addition, whose molecular effect was either the disruption or the elongation of the open reading frame of the corresponding gene. Conclusions: Our data increase the genetic diagnostic yield of IRD patie...
Scientific Reports, 2017
Retinitis pigmentosa is the most frequent group of inherited retinal dystrophies. It is highly heterogeneous, with more than 80 disease-causing genes 27 of which are known to cause autosomal dominant RP (adRP), having been identified. In this study a total of 29 index cases were ascertained based on a family tree compatible with adRP. A custom panel of 31 adRP genes was analysed by targeted next-generation sequencing using the Ion PGM platform in combination with Sanger sequencing. This allowed us to detect putative disease-causing mutations in 14 out of the 29 (48.28%) families analysed. Remarkably, around 38% of all adRP cases analysed showed mutations affecting the splicing process, mainly due to mutations in genes coding for spliceosome factors (SNRNP200 and PRPF8) but also due to splice-site mutations in RHO. Twelve of the 14 mutations found had been reported previously and two were novel mutations found in PRPF8 in two unrelated patients. In conclusion, our results will lead to more accurate genetic counselling and will contribute to a better characterisation of the disease. In addition, they may have a therapeutic impact in the future given the large number of studies currently underway based on targeted RNA splicing for therapeutic purposes. Retinitis pigmentosa (RP; MIM# 268000) is the most frequent form of inherited retinal dystrophy (IRD), with a prevalence of 1 in 3000-4000 cases worldwide 1. It is characterised by a progressive dysfunction associated with the death of rods and/or cones, which leads to retinal atrophy and loss of vision. The mode of inheritance of RP is complex, with autosomal dominant (ad), autosomal recessive (ar), X-linked (xl) Mendelian cases and some cases of digenism or mitochondrial forms having been reported 1-3. From a genetic perspective, over 80 disease-causing genes are currently associated with RP, 27 of which have been associated with adRP (http://www.sph.uth.tmc. edu/retnet). However, to date, mutations in the known adRP genes account for only 50-75% of dominant cases, depending on the test and population used in the study 4. This percentage is increasing, mainly due to the implementation of Next Generation Sequencing (NGS)-based techniques 5-7 and the discovery of new RP genes 8-11. Most human genes harbour introns that are removed during pre-mRNA splicing post-transcriptional modification 12. The splicing reaction is catalysed by the spliceosome, a multisubunit complex comprising small noncoding nuclear RNAs (U1, U2, U4, U5, and U6) and several associated proteins 13. The spliceosome orchestrates the two transesterification reactions needed to remove introns and to join the adjacent exons, and operates by step-wise formation of sub-complexes that recognise regulatory sequences and promote efficient splicing 12-14. Mis-regulation of splicing is a common feature of many human diseases, including several retinal diseases 15-18. These disorders can be caused by mutations that disrupt the splicing of specific genes or by mutations in genes coding for splicing factors, both of which lead to a general loss of spliceosomal function. Thousands of splice-site mutations have been identified in patients with retinal dystrophies. Although most of these mutations disrupt a consensus splice-site sequence and cause exon skipping, some result in intron inclusion, novel exon inclusion,
Next-generation whole exome sequencing to delineate the genetic basis of primary congenital glaucoma
Scientific Reports
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 Alter Retina: Alternative Splicing of Retinal Genes in Health and Disease
International Journal of Molecular Sciences, 2021
Alternative splicing of mRNA is an essential mechanism to regulate and increase the diversity of the transcriptome and proteome. Alternative splicing frequently occurs in a tissue- or time-specific manner, contributing to differential gene expression between cell types during development. Neural tissues present extremely complex splicing programs and display the highest number of alternative splicing events. As an extension of the central nervous system, the retina constitutes an excellent system to illustrate the high diversity of neural transcripts. The retina expresses retinal specific splicing factors and produces a large number of alternative transcripts, including exclusive tissue-specific exons, which require an exquisite regulation. In fact, a current challenge in the genetic diagnosis of inherited retinal diseases stems from the lack of information regarding alternative splicing of retinal genes, as a considerable percentage of mutations alter splicing or the relative produ...
Molecular vision
To examine the possible role of alternate splicing leading to aggregation of myocilin in primary open-angle glaucoma. Several single nucleotide variations found in the myocilin (MYOC) genomic region were collected and examined for their possible role in causing splice-site alterations. A model for myocilin built using a knowledge-based consensus method was used to map the altered protein products. A total of 150 open-angle glaucoma patients and 50 normal age-matched control subjects were screened for the predicted polymorphisms, and clustering was performed. A total of 124 genomic variations were screened, and six polymorphisms that lead to altered protein products were detected as possible candidates for the alternative splicing mechanism. Five of these lay in the intronic regions, and the one that lay in the exon region corresponded to the previously identified polymorphism (Tyr347Tyr) implicated in primary open-angle glaucoma. Experimentally screening the intronic region of the M...
Mutation analysis of seven known glaucoma-associated genes in Chinese patients with glaucoma
Investigative ophthalmology & visual science, 2014
To evaluate mutations in the MYOC, WDR36, OPTN, OPA1, NTF4, CYP1B1, and LTBP2 genes in a cohort of Chinese patients with primary glaucoma. Genomic DNA was prepared from 683 unrelated patients, including 50 with primary congenital glaucoma, 104 with juvenile open-angle glaucoma (JOAG), 186 with primary open-angle glaucoma (POAG), and 343 with primary angle-closure glaucoma (PACG). Mutations in the seven genes in 257 patients (36 with JOAG, 89 with POAG, and 132 with PACG) were initially analyzed by exome sequencing and then confirmed by Sanger sequencing. In addition, Sanger sequencing was used to detect MYOC mutations in the remaining 426 patients. Exome sequencing identified 19 mutations (6 in MYOC, 9 in WDR36, 3 in OPA1, and 1 in OPTN) in 20 of 257 patients, including 4 patients with JOAG, 8 patients with POAG, and 8 patients with PACG. No mutation was detected in the other three genes. In addition, Sanger sequencing detected additional MYOC mutations in 5 of the remaining 426 pat...
Human mutation, 2017
The genetic heterogeneity of Mendelian disorders results in a significant proportion of patients that are unable to be assigned a confident molecular diagnosis after conventional exon sequencing and variant interpretation. Here we evaluated how many patients with an inherited retinal disease (IRD) have variants of uncertain significance (VUS's) that are disrupting splicing in a known IRD gene by means other than affecting the canonical dinucleotide splice site. Three in silico splice-affecting variant predictors were leveraged to annotate and prioritize variants for splicing functional validation. An in vitro minigene system was used to assay each variant's effect on splicing. Starting with 745 IRD patients lacking a confident molecular diagnosis we validated 23 VUS's as splicing variants that likely explain disease in 26 patients. Using our results we optimized in silico score cutoffs to guide future variant interpretation. Variants that alter base pairs other than the ...
Investigative Ophthalmology & Visual Science, 2004
To map the locus and identify the gene causing autosomal recessive congenital cataracts in a large consanguineous Tunisian family. METHODS. DNA was extracted from blood samples from a large Tunisian family with an autosomal recessive, congenital, total white cataract. A genome-wide scan was performed with microsatellite markers. All exons and the splice sites of the HSF4 gene were sequenced in all members of the Tunisian family and in control individuals. RT-PCR was used to detect different transcripts of the HSF4 gene in the human lens. The transcripts were cloned in a TA cloning vector and sequenced. RESULTS. Two-point linkage analyses showed linkage to markers on 16q22 with a maximum lod score of 17.78 at ϭ 0.01 with D16S3043. Haplotype analysis refined the critical region to a 1.8-cM (4.8-Mb) interval, flanked by D16S3031 and D16S3095. This region contains HSF4, some mutations of which cause the autosomal dominant Marner cataract. Sequencing of HSF4 showed a homozygous mutation in the 5Ј splice site of intron 12 (c.1327ϩ4A3 G), which causes the skipping of exon 12. A more detailed study of the transcripts resulting from alternative splicing of the HSF4 gene in the lens is also reported, showing the major transcript HSF4b. CONCLUSIONS. This is the first report describing association of an autosomal recessive cataract with the HSF4 locus on 16q21-q22.1 and the first description of HSF4 splice variants in the lens showing that HSF4b is the major transcript. (Invest Ophthalmol Vis Sci.