The Significance of RHD Genotyping and Characteristic Analysis in Chinese RhD Variant Individuals

BackgroundRhD is the most important and complex blood group system because of its highly polymorphic and immunogenic nature. RhD variants can induce immune response by allogeneic transfusion, organ transplantation, and fetal immunity. The transfusion strategies are different for RhD variants formed by various alleles. Therefore, extensive investigation of the molecular mechanism underlying RhD variants is critical for preventing immune-related blood transfusion reactions and fetal immunity.MethodsRhD variants were collected from donors and patients in Zhejiang Province, China. The phenotypes were classified using the serologic method. The full coding regions of RHD gene were analyzed using the PCR-SBT method. The multiplex ligation-dependent probe amplification (MLPA) assay was used to analyze the genotype and gene copy number. SWISS-MODLE and PyMOL software were used to analyze 3D structures of RhD caused by the variant alleles. The effect of non-synonymous substitutions was predicted using Polymorphism Phenotyping algorithm (PolyPhen-2), Sorting Intolerant From Tolerant (SIFT), and Protein Variation Effect Analyzer (PROVEAN) software.ResultsIn the collected RhD variants, 28 distinct RHD variant alleles were identified, including three novel variant alleles. RH-MLPA assay is advantageous for determining the copy number of RHD gene. 3D homology modeling predicted that protein conformation was disrupted and may explain RhD epitope differential expression. A total of 14 non-synonymous mutations were determined to be detrimental to the protein structure.DiscussionWe revealed the diversity of RHD alleles present in eastern Chinese RhD variants. The bioinformatics of these variant alleles extended our knowledge of RhD variants, which was crucial for evaluating their impact to guide transfusion support and avoid immune-related blood transfusion reactions.

In silico analysis of BRCA1 and BRCA2 missense variants and the relevance in molecular genetic testing

Over the years since the genetic testing of BRCA1 and BRCA2 has been conducted for research and later introduced into clinical practice, a high number of missense variants have been reported in the literature and deposited in public databases. Polymorphism Phenotyping v2 (PolyPhen-2) and Sorting Intolerant from Tolerant (SIFT) are two widely applied bioinformatics tools used to assess the functional impacts of missense variants. A total of 2605 BRCA1 and 4763 BRCA2 variants from the ClinVar database were analysed with PolyPhen2 and SIFT. When SIFT was evaluated alongside PolyPhen-2 HumDiv and HumVar, it had shown top performance in terms of negative predictive value (NPV) (100%) and sensitivity (100%) for ClinVar classified benign and pathogenic BRCA1 variants. Both SIFT and PolyPhen-2 HumDiv achieved 100% NPV and 100% sensitivity in prediction of pathogenicity of the BRCA2 variants. Agreement was achieved in prediction outcomes from the three tested approaches in 55.04% and 68.97% of the variants of unknown significance (VUS) for BRCA1 and BRCA2, respectively. The performances of PolyPhen-2 and SIFT in predicting functional impacts varied across the two genes. Due to lack of high concordance in prediction outcomes among the two tested algorithms, their usefulness in classifying the pathogenicity of VUS identified through molecular testing of BRCA1 and BRCA2 is hence limited in the clinical setting.

Predicted Succinated Dehydrogenase Subunit Variant Pathogenicity: Why Are SDHB Variants “Bad”?

Variants in the 4 genes encoding subunits A-D of succinate dehydrogenase (SDH) are associated with paraganglioma and pheochromocytoma. Intuitively, loss-of-function variants affecting any of the subunits should equally diminish SDH function leading to succinate accumulation and tumorigenesis after loss of heterozygosity. However, variants in SDHB are associated with a higher prevalence of metastatic disease and a more aggressive clinical course. Evaluation of the SDH protein structure shows the fraction of amino acids in contact with other subunits or essential prosthetic groups to be: 13% (SDHA), 40% (SDHB), 28% (SDHC), and 28% (SDHD). We therefore hypothesized that SDHB missense variants are more penetrant because a larger fraction alter sensitive interfaces with other SDH subunits or essential molecular features (e.g. the three SDHB iron-sulfur clusters). We also wondered if truncating variants are more common for SDHB than other subunits. To test these hypotheses, we combined three databases (Genome Aggregation Database, ClinVar-NCBI-NIH, and Leiden Open Variant Database) and our institution’s data to create a pool of all known SDH variants. We categorized variants as truncating or missense and evaluated missense variants in the context of the SDH protein structure, scoring each variant in relation to important structures/interfaces and the severity of the amino acid change. This provided an ad hoc impact score for each variant, where a higher score predicts a more deleterious effect. We compared these scores to those obtained using the “Sorting Intolerant from Tolerant” (SIFT) tool that predicts impacts of amino acid changes based on evolutionary sequence conservation. SIFT scores of 0 to 0.05 predict deleterious effects. Both mean impact and SIFT scores could be weighted for the prevalence of each variant in the population. Our database included 2333 total SDH variants: SDHA (838, 36%), SDHB (703, 30%), SDHC (381, 16%), and SDHD (412, 18%). The fractions of truncating variants were 38%, 50%, 51%, and 53% for A-D subunits, respectively. When weighted for prevalence, these fractions were 0.39%, 6.8%, 8.2%, and 0.2%. The number of truncating variants per coding region length and the distribution of locations were similar between subunits. Ad hoc impact scores for A-D subunits were 3.08, 14.9, 9.93, and 11.0, respectively and, when weighted for prevalence, were 0.28, 3.25, 6.32, and 1.15. Mean SIFT scores for subunits A-D were: 0.185, 0.162, 0.238, and 0.410 respectively, and, when weighted for prevalence, were 0.58, 0.70, 0.22, and 0.018. Our results do not support the hypothesis SDHB variants predict a worse clinical outcome because average SDHB variants are, by chance, more biochemically severe. This suggests that SDHB loss may uniquely impact SDH biochemical function.

A Novel Pathogenic HSPG2 Mutation in Schwartz–Jampel Syndrome

Schwartz–Jampel syndrome is a rare autosomal recessive disease caused by mutation in the heparan sulfate proteoglycan 2 (HSPG2) gene. Its cardinal symptoms are skeletal dysplasia and neuromuscular hyperactivity. Herein, we identified a new pathogenic mutation site (NM_005529.6:c.1125C>G; p.Cys375Trp) of HSPG2 leading to Schwartz–Jampel syndrome by whole-exome sequencing. This mutation carried by the asymptomatic parents was previously registered in a single-nucleotide polymorphism database of the National Institutes of Health as a coding sequence variant rs543805444. The pathogenic nature of this missense mutation was demonstrated by in silico pathogenicity assessment, clinical presentations, and cellular function of primary fibroblast derived from patients. Various in silico software applications predicted the mutation to be pathogenic [Sorting Intolerant From Tolerant (SIFT), 0; Polyphen-2, 1; CADD (Combined Annotation Dependent Depletion), 23.7; MutationTaster, 1; DANN (deleterious annotation of genetic variants using neural networks); 0.9]. Needle electromyography revealed extensive complex repetitive discharges and multiple polyphasic motor unit action potentials in axial and limb muscles at rest. Short exercise test for myotonia showed Fournier pattern I. At cellular levels, mutant primary fibroblasts had reduced levels of secreted perlecan and impaired migration ability but normal capability of proliferation. Patients with this mutation showed more neuromuscular instability and relatively mild skeletal abnormality comparing with previously reported cases.

Variants in CHRNB2 and CHRNA4 Identified in Patients with Insular Epilepsy

Purpose:Our purpose was to determine the role of CHRNA4 and CHRNB2 in insular epilepsy.Method:We identified two patients with drug-resistant predominantly sleep-related hypermotor seizures, one harboring a heterozygous missense variant (c.77C>T; p. Thr26Met) in the CHRNB2 gene and the other a heterozygous missense variant (c.1079G>A; p. Arg360Gln) in the CHRNA4 gene. The patients underwent electrophysiological and neuroimaging studies, and we performed functional characterization of the p. Thr26Met (c.77C>T) in the CHRNB2 gene.Results:We localized the epileptic foci to the left insula in the first case (now seizure-free following epilepsy surgery) and to both insulae in the second case. Based on tools predicting the possible impact of amino acid substitutions on the structure and function of proteins (sorting intolerant from tolerant and PolyPhen-2), variants identified in this report could be deleterious. Functional expression in human cell lines of α4β2 (wild-type), α4β2-Thr26Met (homozygote), and α4β2/β2-Thr26Met (heterozygote) nicotinic acetylcholine receptors revealed that the mutant subunit led to significantly higher whole-cell nicotinic currents. This feature was observed in both homo- and heterozygous conditions and was not accompanied by major alterations of the current reversal potential or the shape of the concentration-response relation.Conclusions:This study suggests that variants in CHRNB2 and CHRNA4, initially linked to autosomal dominant nocturnal frontal lobe epilepsy, are also found in patients with predominantly sleep-related insular epilepsy. Although the reported variants should be considered of unknown clinical significance for the moment, identification of additional similar cases and further functional studies could eventually strengthen this association.

Novel mutations in NOTCH2 gene in infants with neonatal cholestasis

One cause of neonatal cholestasis (NC) is paucity of intrahepatic bile ducts which can be associated with Alagille syndrome or non- syndromic. Alagille syndrome is caused by autosomal dominant mutations in the Notch signaling pathway ligand Jagged1 in 94% of patients and mutations in the NOTCH2 receptor in <1% of patients. This is a retrospective case series studying infants with neonatal cholestasis found to have variants of unknown significance (VOUS) in NOTCH2. Sorting intolerant from tolerant (SIFT) and polymorphism phenotyping (PolyPhen) were utilized to predict a damaging effect. Five infants with NC without other features of Alagille syndrome were found to have one copy of a VOUS in NOTCH2, predicted to be damaging by SIFT and PolyPhen. Our cases support the notion that NOTCH2 mutations may result in hypoplastic biliary system. Further characterization of these variants is important to assist with our clinical approach to NC.

274 Exome analysis to investigate autosomal dominant vasovagal syncope

IntroductionVasovagal syncope (VVS) is the most common cause of syncope in children and adults. Previous studies suggest a genetic component accounts for approximately 20% of cases, although the genes responsible are often unidentified. I studied the DNA of two distantly related individuals with VVS enrolled into the 100,000 Genomes Project to identify causal mutations. MethodDNA was extracted from the patients, and analysed using an Ingenuity Variant Analysis program to detect the presence of mutations. The severity of each detected mutation was subsequently examined using two programs: Sorting Intolerant from Tolerant (SIFT) and Polymorphing Phenotyping v2 (PolyPhen-2). ResultsUsing Ingenuity Variant Analysis, a mutation in the ACE (Angiotensin Converting Enzyme), EPAS1 (Endothelial PAS Domain Protein 1) and PLCG2 (Phospholipase C Gamma 2) genes of the VVS patients were identified. Further analysis using SIFT and PolyPhen-2 indicated the ACE mutation was likely to produce a defective protein whilst the EPAS1 and PLCG2 mutations were unlikely to have any effect on protein function. ConclusionMy results support the involvement of the ACE mutation in the cause of VVS within the two studied patients. This may point towards a novel target for therapy within the individuals, should the findings be successfully validated.

IMPACT OF DELETERIOUS NON-SYNONYMOUS SINGLE NUCLEOTIDE POLYMORPHISMS OF CYTOKINE GENES ON NON-CLASSICAL HYDROGEN BONDS PREDISPOSING TO CARDIOVASCULAR DISEASE: AN IN SILICO APPROACH

Objective: Cardiovascular disease (CVD) is a leading cause of death worldwide. Malfunctioning of genes that are responsible for several inflammatory processes is the major cause for its initiation. Cytokine genes are one such group of genes that are involved in the development of CVD. Hence, the prediction of potential point mutations in these genes is important for diagnostic purposes. Such mutations result in altered protein structure and function when compared to neutral ones.Methods: In this study, interleukin factor 6 (IL6), tumor necrosis factor α (TNF-α), interleukin factor 4 (IL4), and interferon gamma have been analyzed using sorting intolerant from tolerant and PolyPhen 2.0 tools.Results: Several single nucleotide polymorphisms (SNPs), in IL6, TNF-α, and IL4, are found to be potentially deleterious. In addition, bond analysis has also been performed on these SNPs. It has been predicted that L119P and R196H of IL6 as well as K87T and T181N of TNF-α are potential ns-SNP’s that may cause structural and functional variations in the corresponding proteins. The hydrogen and Cation-Pi bond analysis performed in this study provides molecular-based evidence that support the predicted deleterious potential of such SNPs for these CVD candidate genes along with other conventional in silico tools.Conclusion: The study testifies the importance of adopting a computational approach to narrow down potential point mutants for disease predictions.

αIIbβ3 variants defined by next-generation sequencing: Predicting variants likely to cause Glanzmann thrombasthenia

Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin αIIbβ3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising ∼32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting ∼11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting ∼9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of αIIbβ3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. αIIb P176H and β3 C547G severely reduced αIIbβ3 expression, whereas αIIb P943A partially reduced αIIbβ3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69–98% sensitivity in detecting GT mutations, between 27% and 71% of the novel αIIb or β3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on αIIbβ3 and highlight the challenges in predicting the clinical significance of novel missense variants.