In Cystic Fibrosis (CF), correction of splicing defects represents an interesting therapeutic approach to restore normal CFTR function. In this study, we focused on ten common mutations/variants, 711+3A>G/C, 711+5G>A, 1863C>T, 1898+3A>G, 2789+5G>A, TG13T3, TG13T5, TG12T5 and 3120G>A that induce skipping of the corresponding CFTR exons 5, 9, 13, 16 and 18. To rescue the splicing defects we tested, in a minigene assay, a panel of modified U1 snRNAs, named Exon Specific U1s (ExSpeU1) that were engineered to bind to intronic sequences downstream of each defective exon. Using this approach, we show that all ten splicing mutations analysed are efficiently corrected by specific ExSpeU1s. Using cDNA-splicing competent minigenes, we also show that the ExspeU1-mediated splicing correction at the RNA level recovered the full-length CFTR protein for 1863C>T, 1898+3A>G, 2789+5G>A variants. In addition, detailed mutagenesis experiments performed on exon 13 led us to identify a novel intronic regulatory element involved in the ExSpeU1-mediated splicing rescue. These results provide a common strategy based on modified U1 snRNAs to correct exon skipping in a group of disease-causing CFTR mutations.
Background: CHEK2 variants are associated with intermediate breast cancer risk among other cancers. We aimed to comprehensively describe CHEK2 variants in a Spanish hereditary cancer (HC) cohort and adjust American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines for their classification. Methods: First, three CHEK2 frequent variants were screened in a retrospective Hereditary Breast and Ovarian Cancer cohort of 516 patients. After, the whole CHEK2 coding region was analyzed by next-generation sequencing in 1,848 prospective patients with HC suspicion. We refined ACMGAMP criteria and applied different combinatorial rules to classify CHEK2 variants and define risk alleles. Results: We identified 10 CHEK2 null variants, 6 missense variants with discordant interpretation in ClinVar database, and 35 additional variants of unknown significance. Twelve variants were classified as (likely)-pathogenic; 2 can also be considered “established risk-alleles” and one as “likely risk-allele”. The prevalence of (likely)-pathogenic variants in the HC cohort was 0.8% (1.3% in breast cancer patients and 1.0% in hereditary non-polyposis colorectal cancer patients). Conclusions: Here we provide ACMG adjustment guidelines to classify CHEK2 variants. We hope that this work would be useful for variant classification of other genes with low effect variants
Full genome analysis of a young girl with deafness, dystonia, central hypomyelination, refractory seizure, and fluctuating liver function impairment revealed a heterozygous, de novo variant in the BCAP31 gene on chromosome X28q (NC_000023.11(BCAP31_v001):c.92G>A), mutations of which caused the X-linked recessive severe neurologic disorder DDCH (Deafness, Dystonia, and Cerebral Hypomyelination, OMIM#300475). Reverse transcription-PCR (RT-PCR) of the patient’s white blood cells showed the absence of wild-type BCAP31 mRNA but the presence of two novel BCAP31 mRNAs. The major alternatively-spliced mRNA is due to exon 2 skipping and the utilization of a new initiation site in exon 3 that leads to a frameshift and truncated transcript while the minor novel mRNA has a 110 nucleotide insertion to exon 2. Phasing studies showed that the de novo variant arose in the paternal X chromosome. X chromosome inactivation assay was done and confirmed that the patient’s maternal X chromosome was preferentially inactivated, providing evidence that the mutated BCAP31 gene was the predominantly expressed. According to the ACMG guideline, this variant is deemed “pathogenic” (PS2, PS3, PM2, PP3, PP4) and deleterious. This is the first reported female patient in BCAP31-related syndrome resulted from skewed X-inactivation and a de novo mutation in the active X chromosome.
In hemophilia A and B, analysis of the F8 and F9 variants has become standard over recent decades, giving information on the severity of hemophilia, inhibitor formation and allowing counseling for the families. The PedNet Registry collects data on hemophilia in children and has more than 2000 children listed. Genetic reports are collected uniformly and re-evaluated following international guidelines. We report 90 novel variants in the F8 and F9 gene, respectively, causing hemophilia with detailed information on severity, factor level and inhibitor formation. This will lead to further guidance for genetic laboratories and the treating physician. These findings can be implemented in hemophilia variant databases. The study highlights the need to re-evaluate and update earlier genetic reports in hemophilia both locally but also in variant databases in the light of changed nomenclature, the use of in silico prediction and new sequencing techniques.
Recently, we demonstrated that the qualitative American College of Medical Genetics and Genomics/ Association for Medical Pathology (ACMG/AMP) guidelines for evaluation of Mendelian disease gene variants are fundamentally compatible with a quantitative Bayesian formulation. Here, we show that the underlying ACMG/AMP “strength of evidence categories” can be abstracted into a point system. These points are proportional to Log(odds), are additive, and produce a system that recapitulates the Bayesian formulation of the ACMG/AMP guidelines. Strengths of this system are its simplicity and that the connection between point values and odds of pathogenicity allows empirical calibration of strength of evidence for individual data types. Weaknesses include that a narrow range of prior probabilities is locked in, and that the Bayesian nature of the system is inapparent. We conclude that a points-based system has useful attributes of user friendliness and can be useful so long as the underlying Bayesian principles are acknowledged.
Purpose:There have been concerted efforts towards cataloging rare and deleterious variants in different world population using high throughput genotyping and sequencing based methods. The Indian populations are underrepresented or its information w.r.t. clinically relevant variants are sparse in public datasets. The aim of this study was to estimate the burden of monogenic disease causing variants in Indian populations. Towards this, we have assessed the frequency profile of monogenic phenotype associated ClinVar variants. Methods: The study utilized genotype dataset (global-screening-array, Illumina) from 2795 individuals (multiple in-house genomics cohorts) representing diverse ethnic and geographically distinct Indian populations. Results: Of the analyzed variants from GSA, ~12% were found to be informative and were either not known earlier or underrepresented in public databases in terms of their frequencies. These variants were linked to disorders, viz. Inborn-errors of Metabolism, Monogenic-diabetes, hereditary cancers and various other hereditary conditions. We have also shown that our study cohort is genetically better representatives of Indian populations than its representation in1000 genome project (South-Asians). Conclusion: We have created a database, ClinIndb [(http://clinindb.igib.res.in) and (https://databases.lovd.nl/shared/variants?search_owned_by_=%3D%22Mohamed%20Faruq%22)], to help clinicians and researchers in diagnosis, counseling and development of appropriate genetic screening tools relevant to the Indian populations and Indians living abroad.
Holoprosencephaly (HPE) is the most common congenital anomaly affecting the forebrain and face in humans and occurs as frequently as 1:250 conceptions or 1:10,000 livebirths. Sonic hedgehog (SHH) is one of the best characterized HPE genes that plays crucial roles in numerous developmental processes including midline neural patterning and craniofacial development. The Frizzled class G-Protein Coupled Receptor (GPCR) SMOOTHENED (SMO), whose signalling activity is tightly regulated, is the sole obligate transducer of hedgehog-related signals. However, except for previous reports of somatic oncogenic driver mutations in human cancers (or mosaic tumors in rare syndromes), any potential disease-related role of SMO genetic variation in humans is largely unknown. To our knowledge, ours is the first report of a human hypomorphic variant revealed by functional testing of seven distinct non-synonymous SMO variants derived from HPE molecular and clinical data. Here we describe several zebrafish bioassays developed and guided by a systems biology analysis. This analysis strategy, and detection of hypomorphic variation in human SMO, demonstrates the necessity of integrating the genomic variant findings in HPE probands with other components of the hedgehog gene regulatory network (GRN) in overall medical interpretations.
This letter is a response to the commentary by Jonson & Do (Johnson and Do 2020) on our paper, entitled “A Vietnamese human genetic variation database” (Vinh et al. 2019). The commentators concerned about two issues: Firstly, the relation of Southeast Asian (SEA) and East Asian (EA) groups to African and European groups; Secondly, the history of migration and settlement in Southeast Asia. Our responses will clarify both concerns from the commentators.
It is possible to estimate the prior probability of pathogenicity for germline disease gene variants based on bioinformatic prediction of variant effect/s. However, routinely used approaches have likely led to the underestimation and underreporting of variants located outside donor and acceptor splice site motifs that affect mRNA processing. This review presents information about hereditary cancer gene germline variants, outside native splice sites, with experimentally validated splicing effects. We list 81 exonic variants that impact splicing regulatory elements in BRCA1, BRCA2, MLH1, MSH2, MSH6 and PMS2. We utilized a pre-existing large-scale BRCA1 functional dataset to map functional splicing regulatory elements, assess the relative performance of different tools to predict effects of 283 variants on such elements, and develop a generic workflow to prioritize variants that may impact splicing regulatory elements. We also describe rare examples of intronic variants that impact branchpoint sites and create pseudoexons. We discuss the challenges in predicting variant effect on branchpoint site usage and pseudoexonization, and suggest strategies to improve the bioinformatic prioritization of such variants for experimental validation. Importantly, our review highlights the importance of considering impact of variants outside donor and acceptor motifs on mRNA splicing and disease causation.