Pathogenic Intronic Splice-Affecting Variants in MYBPC3 in Three Patients with Hypertrophic Cardiomyopathy

From MDPI via Jisc Publications RouterHistory: accepted 2021-05-25, pub-electronic 2021-06-02Publication status: PublishedFunder: Medical Research Council; Grant(s): 1916606, IS-BRC-1215-20007, BB/N000258/1Genetic variants in MYBPC3 are one of the most common causes of hypertrophic cardiomyopathy (HCM). While variants in MYBPC3 affecting canonical splice site dinucleotides are a well-characterised cause of HCM, only recently has work begun to investigate the pathogenicity of more deeply intronic variants. Here, we present three patients with HCM and intronic splice-affecting MYBPC3 variants and analyse the impact of variants on splicing using in vitro minigene assays. We show that the three variants, a novel c.927-8G>A variant and the previously reported c.1624+4A>T and c.3815-10T>G variants, result in MYBPC3 splicing errors. Analysis of blood-derived patient RNA for the c.3815-10T>G variant revealed only wild type spliced product, indicating that mis-spliced transcripts from the mutant allele are degraded. These data indicate that the c.927-8G>A variant of uncertain significance and likely benign c.3815-10T>G should be reclassified as likely pathogenic. Furthermore, we find shortcomings in commonly applied bioinformatics strategies to prioritise variants impacting MYBPC3 splicing and re-emphasise the need for functional assessment of variants of uncertain significance in diagnostic testing

Assessment of the incorporation of CNV surveillance into gene panel next-generation sequencing testing for inherited retinal diseases.

BACKGROUND: Diagnostic use of gene panel next-generation sequencing (NGS) techniques is commonplace for individuals with inherited retinal dystrophies (IRDs), a highly genetically heterogeneous group of disorders. However, these techniques have often failed to capture the complete spectrum of genomic variation causing IRD, including CNVs. This study assessed the applicability of introducing CNV surveillance into first-tier diagnostic gene panel NGS services for IRD. METHODS: Three read-depth algorithms were applied to gene panel NGS data sets for 550 referred individuals, and informatics strategies used for quality assurance and CNV filtering. CNV events were confirmed and reported to referring clinicians through an accredited diagnostic laboratory. RESULTS: We confirmed the presence of 33 deletions and 11 duplications, determining these findings to contribute to the confirmed or provisional molecular diagnosis of IRD for 25 individuals. We show that at least 7% of individuals referred for diagnostic testing for IRD have a CNV within genes relevant to their clinical diagnosis, and determined a positive predictive value of 79% for the employed CNV filtering techniques. CONCLUSION: Incorporation of CNV analysis increases diagnostic yield of gene panel NGS diagnostic tests for IRD, increases clarity in diagnostic reporting and expands the spectrum of known disease-causing mutations

Expanding the genotypic spectrum of TXNL4A variants in Burn‐McKeown syndrome

From Wiley via Jisc Publications RouterHistory: received 2021-09-06, rev-recd 2021-10-21, accepted 2021-10-23, pub-electronic 2021-11-05Article version: VoRPublication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; Id: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/N000358/1Funder: Health Education England Genomics Education ProgrammeFunder: Medical Research Council; Id: http://dx.doi.org/10.13039/501100007155; Grant(s): 1916606Funder: National Institute for Health Manchester Biomedical Research Centre; Grant(s): IS‐BRC‐1215‐20007Abstract: The developmental disorder Burn‐McKeown Syndrome (BMKS) is characterised by choanal atresia and specific craniofacial features. BMKS is caused by biallelic variants in the pre‐messenger RNA splicing factor TXNL4A. Most patients have a loss‐of‐function variant in trans with a 34‐base pair (bp) deletion (type 1 Δ34) in the promoter region. Here, we identified two patients with BMKS. One individual has a TXNL4A c.93_94delCC, p.His32Argfs *21 variant combined with a type 1 Δ34 promoter deletion. The other has an intronic TXNL4A splice site variant (c.258‐3C>G) and a type 1 Δ34 promoter deletion. We show the c.258‐3C>G variant and a previously reported c.258‐2A>G variant, cause skipping of the final exon of TXNL4A in a minigene splicing assay. Furthermore, we identify putative transcription factor binding sites within the 56 bp of the TXNL4A promoter affected by the type 1 and type 2 Δ34 and use dual luciferase assays to identify a 22 bp repeated motif essential for TXNL4A expression within this promoter region. We propose that additional variants affecting critical transcription factor binding nucleotides within the 22 bp repeated motif could be relevant to BMKS aetiology. Finally, our data emphasises the need to analyse the non‐coding sequence in individuals where a single likely pathogenic coding variant is identified in an autosomal recessive disorder consistent with the clinical presentation

EyeG2P: an automated variant filtering approach improves efficiency of diagnostic genomic testing for inherited ophthalmic disorders

BACKGROUND: Genomic variant prioritisation is one of the most significant bottlenecks to mainstream genomic testing in healthcare. Tools to improve precision while ensuring high recall are critical to successful mainstream clinical genomic testing, in particular for whole genome sequencing where millions of variants must be considered for each patient. METHODS: We developed EyeG2P, a publicly available database and web application using the Ensembl Variant Effect Predictor. EyeG2P is tailored for efficient variant prioritisation for individuals with inherited ophthalmic conditions. We assessed the sensitivity of EyeG2P in 1234 individuals with a broad range of eye conditions who had previously received a confirmed molecular diagnosis through routine genomic diagnostic approaches. For a prospective cohort of 83 individuals, we assessed the precision of EyeG2P in comparison with routine diagnostic approaches. For 10 additional individuals, we assessed the utility of EyeG2P for whole genome analysis. RESULTS: EyeG2P had 99.5% sensitivity for genomic variants previously identified as clinically relevant through routine diagnostic analysis (n=1234 individuals). Prospectively, EyeG2P enabled a significant increase in precision (35% on average) in comparison with routine testing strategies (p<0.001). We demonstrate that incorporation of EyeG2P into whole genome sequencing analysis strategies can reduce the number of variants for analysis to six variants, on average, while maintaining high diagnostic yield. CONCLUSION: Automated filtering of genomic variants through EyeG2P can increase the efficiency of diagnostic testing for individuals with a broad range of inherited ophthalmic disorders

Gene Panel Testing for Breast Cancer Reveals Differential Effect of Prior BRCA1/2 Probability

From MDPI via Jisc Publications RouterHistory: accepted 2021-08-16, pub-electronic 2021-08-18Publication status: PublishedFunder: Manchester Biomedical Research Centre; Grant(s): IS-BRC-1215-20007Funder: Prevent breast cancer; Grant(s): GA19-002Whilst panel testing of an extended group of genes including BRCA1/2 is commonplace, these studies have not been subdivided by histiotype or by a priori BRCA1/2 probability. Patients with a breast cancer diagnosis undergoing extended panel testing were assessed for frequency of actionable variants in breast cancer genes other than BRCA1/2 by histiotype and Manchester score (MS) to reflect a priori BRCA1/2 likelihood. Rates were adjusted by prior testing for BRCA1/2 in an extended series. 95/1398 (6.3%) who underwent panel testing were found to be positive for actionable non-BRCA1/2 breast/ovarian cancer genes (ATM, BARD1, CDH1, CHEK2, PALB2, PTEN, RAD51C, RAD51D, TP53). As expected, PALB2, CHEK2 and ATM were predominant with 80-(5.3%). The highest rate occurred in Grade-3 ER+/HER2− breast cancers-(9.6%). Rates of non-BRCA actionable genes was fairly constant over all likelihoods of BRCA1/2 but adjusted rates were three times higher with MS 9 (BRCA1/2 = 1.5%, other = 4.7%), but was only 1.6% compared to 79.3% with MS ≥ 40. Although rates of detection of non-BRCA actionable genes are relatively constant across BRCA1/2 likelihoods this disguises an overall adjusted low frequency in high-likelihood families which have been heavily pre-tested for BRCA1/2. Any loss of detection sensitivity for BRCA1/2 actionable variants in breast cancer panels should lead to bespoke BRCA1/2 testing being conducted first

Multi-disciplinary team directed analysis of whole genome sequencing reveals pathogenic non-coding variants in molecularly undiagnosed inherited retinal dystrophies

PURPOSE: To identify, using genome sequencing (GS), likely pathogenic non-coding variants in inherited retinal dystrophy (IRD) genes Methods: Patients with IRD were recruited to the study and underwent comprehensive ophthalmological evaluation and GS. The results of GS were investigated through virtual gene panel analysis and plausible pathogenic variants and clinical phenotype evaluated by multi-disciplinary team (MDT) discussion. For unsolved patients in whom a specific gene was suspected to harbour a missed pathogenic variant, targeted re-analysis of non-coding regions was performed on GS data. Candidate variants were functionally tested including by mRNA analysis, minigene and luciferase reporter assays. RESULTS: Previously unreported, likely pathogenic, non-coding variants, in 7 genes (PRPF31, NDP, IFT140, CRB1, USH2A, BBS10, and GUCY2D), were identified in 11 patients. These were shown to lead to mis-splicing (PRPF31, IFT140, CRB1, USH2A) or altered transcription levels (BBS10, GUCY2D). CONCLUSION: MDT-led, phenotype driven, non-coding variant re-analysis of GS is effective in identifying missing causative alleles

A Dominantly Inherited 5' UTR Variant Causing Methylation-Associated Silencing of BRCA1 as a Cause of Breast and Ovarian Cancer

Pathogenic variants in BRCA1 or BRCA2 are identified in ∼20% of families with multiple individuals affected by early-onset breast and/or ovarian cancer. Extensive searches for additional highly penetrant genes or alternative mutational mechanisms altering BRCA1 or BRCA2 have not explained the missing heritability. Here, we report a dominantly inherited 5′ UTR variant associated with epigenetic BRCA1 silencing due to promoter hypermethylation in two families affected by breast and ovarian cancer. BRCA1 promoter methylation of ten CpG dinucleotides in families who are affected by breast and/or ovarian cancer but do not have germline BRCA1 or BRCA2 pathogenic variants was assessed by pyrosequencing and clonal bisulfite sequencing. RNA and DNA sequencing of BRCA1 from lymphocytes was undertaken to establish allelic expression and the presence of germline variants. BRCA1 promoter hypermethylation was identified in 2 of 49 families in which multiple women are affected by grade 3 breast cancer or high-grade serous ovarian cancer. Soma-wide BRCA1 promoter hypermethylation was confirmed in blood, buccal mucosa, and hair follicles. Pyrosequencing showed that DNA was ∼50% methylated, consistent with the silencing of one allele, which was confirmed by clonal bisulfite sequencing. RNA sequencing revealed the allelic loss of BRCA1 expression in both families and that this loss of expression segregated with the heterozygous variant c.−107A&gt;T in the BRCA1 5′ UTR. Our results establish a mechanism whereby familial breast and ovarian cancer is caused by an in cis 5′ UTR variant associated with epigenetic silencing of the BRCA1 promoter in two independent families. We propose that methylation analyses be undertaken to establish the frequency of this mechanism in families affected by early-onset breast and/or ovarian cancer without a BRCA1 or BRCA2 pathogenic variant

The inner junction protein CFAP20 functions in motile and non-motile cilia and is critical for vision

Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general