Effects Of Non-synonymous Variants Research Articles

Characterization of a missense variant in COG5 in a Tunisian patient with COG5-CDG syndrome and insights into the effect of non-synonymous variants on COG5 protein.

The clinical diagnosis of patients with multisystem involvement including a pronounced neurologic damage is challenging. High-throughput sequencing methods remains crucial to provide an accurate diagnosis. In this study, we reported a Tunisian patient manifesting hypotonia and global developmental delay with visual and skin abnormalities. Exome sequencing was conducted followed by segregation analysis and, subsequently additional investigations. In silico analysis of non-synonymous variants (nsSNPs) described in COG5 in conserved positions was made. Results revealed a homozygous missense variant c.298 C > T (p.Leu100Phe) in the COG5 inherited from both parents. This variant altered both protein solubility and stability, in addition to a putative disruption of the COG5-COG7 interaction. This disruption has been confirmed using patient-derived cells in vitro in a COG5 co-immuno-precipitation, where interaction with binding partner COG7 was abrogated. Hence, we established the COG5-CDG diagnosis. Clinically, the patient shared common features with the already described cases with the report of the ichtyosis as a new manifestation. Conversely, the CADD scoring revealed 19 putatively pathogenic nsSNPs (Minor Allele Frequency MAF < 0.001, CADD > 30), 11 of which had a significant impact on the solubility and/or stability of COG5. These properties seem to be disrupted by six of the seven missense COG5-CDG variants. In conclusion, our study expands the genetic and phenotypic spectrum of COG5-CDG disease and highlight the utility of the next generation sequencing as a powerful tool in accurate diagnosis. Our results shed light on a likely molecular mechanism underlying the pathogenic effect of missense COG5 variants, which is the alteration of COG5 stability and solubility.

Genetic variants, haplotype determination, and function of novel alleles of CYP2B6 in a Han Chinese population

Amino acid variants in protein may result in deleterious effects on enzymatic activity. In this study we investigate the DNA variants on activity of CYP2B6 gene in a Chinese Han population for potential use in precision medicine. All exons in CYP2B6 gene from 1483 Chinese Han adults (Zhejiang province) were sequenced using Sanger sequencing. The effects of nonsynonymous variants on recombinant protein catalytic activity were investigated in vitro with Sf12 system. The haplotype of novel nonsynonymous variants with other single nucleotide variants in the same allele was determined using Nanopore sequencing. Of 38 alleles listed on the Pharmacogene Variation Consortium, we detected 7 previously reported alleles and 18 novel variants, of which 11 nonsynonymous variants showed lower catalytic activity (0.00–0.60) on bupropion compared to CYP2B6*1. Further, these 11 novel star-alleles (CYP2B6*39–49) were assigned by the Pharmacogene Variation Consortium, which may be valuable for pharmacogenetic research and personalized medicine.

Prediction of the Effects of Nonsynonymous Variants on SARS-CoV-2 Proteins.

Background:SARS-CoV-2 virus isa highly transmissible pathogen that causesCOVID-19. Theoutbreak originatedinWuhan, China in December 2019. A number of nonsynonymous mutations located atdifferentSARS-CoV-2 proteinshavebeen reportedby multiple studies.However, therearelimitedcomputationalstudies onthebiological impacts of these mutations on the structure and function of the proteins. Methods:In our study nonsynonymous mutations oftheSARS-CoV-2 genome and their frequencieswere identifiedfrom30,229sequences.Subsequently, theeffects ofthetop10nonsynonymous mutations of different SARS-CoV-2 proteinswere analyzed using bioinformatics toolsincluding co-mutation analysis,prediction oftheprotein structure stabilityand flexibilityanalysis,andprediction oftheprotein functions. Results:Atotal of 231 nonsynonymous mutations were identified from 30,229 SARS-CoV-2 genome sequences. The top10nonsynonymous mutationsaffectingnineamino acid residueswere ORF1a nsp5 P108S, ORF1b nsp12 P323L and A423V, S protein N501Y and D614G, ORF3a Q57H, N protein P151L, R203K and G204R. Many nonsynonymous mutations showedahigh concurrence ratio, suggesting these mutations may evolve together and interact functionally. Our result showedthatORF1a nsp5P108S,ORF3aQ57H and N protein P151Lmutations may be deleterious to thefunctionof SARS-CoV-2 proteins.In addition,ORF1a nsp5P108Sand S proteinD614Gmaydestabilizethe protein structureswhileS protein D614Gmay have a more open conformationcompared to the wild type. Conclusion:The biological consequences of these nonsynonymous mutations of SARS-CoV-2 proteins should be further validated byin vivoandin vitroexperimental studies in the future.

Novel SCN5A variants identified in a group of Iranian Brugada syndrome patients.

Brugada syndrome (BrS) is a rare hereditary arrhythmia syndrome that increases an individual's risk for sudden cardiac death (SCD) due to ventricular fibrillation. This disorder is regarded as a notable cause of death in individuals aged less than 40 years, responsible for up to 40% of sudden deaths in cases without structural heart disease, and is reported to be an endemic in Asian countries. Mutations in SCN5A are found in approximately 30% of patients with Brugada syndrome. This study aimed to investigate mutations in the SCN5A gene in a group of Iranian Brugada syndrome patients. Nine probands (n = 9, male, mean age = 39) diagnosed with Brugada syndrome were enrolled in this study. Exon 2 to 29 were amplified by PCR and subjected to direct sequencing. Eight in silico prediction tools were used to anticipate the effects of non-synonymous variants. Seven known polymorphisms and 2 previously reported disease-causing mutations, including H558R and G1406R, were found in the studied cases. Twenty novel variants were identified: 15 missense, 2 frameshift, 2 synonymous, and one nonsense variants. In silico tools predicted 11 non-synonymous variants to have damaging effects, whereas frameshift and nonsense variants were considered inherently pathogenic. The novel variants identified in this study, alongside previously reported mutations, are highly likely to be the cause of the Brugada syndrome phenotype observed in the patient group. Further analysis is required to understand the physiological effects caused by these variants.

Network propagation of rare variants in Alzheimer's disease reveals tissue-specific hub genes and communities.

State-of-the-art rare variant association testing methods aggregate the contribution of rare variants in biologically relevant genomic regions to boost statistical power. However, testing single genes separately does not consider the complex interaction landscape of genes, nor the downstream effects of non-synonymous variants on protein structure and function. Here we present the NETwork Propagation-based Assessment of Genetic Events (NETPAGE), an integrative approach aimed at investigating the biological pathways through which rare variation results in complex disease phenotypes. We applied NETPAGE to sporadic, late-onset Alzheimer's disease (AD), using whole-genome sequencing from the AD Neuroimaging Initiative (ADNI) cohort, as well as whole-exome sequencing from the AD Sequencing Project (ADSP). NETPAGE is based on network propagation, a framework that models information flow on a graph and simulates the percolation of genetic variation through tissue-specific gene interaction networks. The result of network propagation is a set of smoothed gene scores that can be tested for association with disease status through sparse regression. The application of NETPAGE to AD enabled the identification of a set of connected genes whose smoothed variation profile was robustly associated to case-control status, based on gene interactions in the hippocampus. Additionally, smoothed scores significantly correlated with risk of conversion to AD in Mild Cognitive Impairment (MCI) subjects. Lastly, we investigated tissue-specific transcriptional dysregulation of the core genes in two independent RNA-seq datasets, as well as significant enrichments in terms of gene sets with known connections to AD. We present a framework that enables enhanced genetic association testing for a wide range of traits, diseases, and sample sizes.

Abstract 1625: Clinical biomarkers of survival in renal cell carcinoma patients treated with sorafenib

Abstract Purpose: To discover genetic determinants of overall survival (OS) in metastatic renal cell carcinoma (mRCC) patients treated with sorafenib. Patients and Methods: Information for 11,117 germline DNA variants from 56 genes was obtained, using both Illumina GoldenGate genotyping and imputation, from 295 patients originally enrolled in the phase III TARGET trial (155 sorafenib and 140 placebo patients). OS was this genotyping study's primary endpoint, and associations between variants and OS were tested using multivariate Cox regression. Associations were considered significant if P&amp;lt;0.05 and FDR q&amp;lt;0.1. Bioinformatic resources helped prioritize variants for functional validation. Haploreg v4 and RegulomeDB were used to help mine ENCODE data, and SIFT/PolyPhen-2 predicted functional effects of nonsynonymous variants. Dual reporter gene luciferase assays assessed variant effects on promoter activity. Isogenic cell lines were created, and direct cell proliferation assays elucidated variant effects on cell proliferation/cytotoxicity, while endothelial branch formation assays assessed variant effects on endothelial tube formation. Western blotting evaluated nonsynonymous variant effects on receptor phosphorylation, and pulse-chase experiments assessed variant effects on post-translational processing of proteins. Results: Five intragenic variants significantly associated with OS. Among patients in the sorafenib arm, VEGFA rs1885657 (HR=17.3, 95% CI 5.7-52.7; P=1.4x10-4), ITGAV rs3816375 (HR=5.9, 95% CI 2.1-16.4; P=4.9x10-4), and WWOX rs8047917 (HR=4.1, 95% CI 1.9-8.8; P=3.3x10-4) associated with shorter OS. Among patients from both arms, FLT4 rs307826 (HR=13.8, 95% CI 3.0-62.6; P=1.2x10-4) and VEGFA rs3024987 (HR=3.0, 95% CI 1.7-5.4; P=8.3x10-5) associated with shorter OS. No significant associations were discovered in the placebo arm. Bioinformatic evidence was used to prioritize rs1885657, rs58159269 (in complete linkage disequilibrium with rs1885657), and rs307826. rs1885657 and rs58159269 increased luciferase activity in human endothelial (TIME, LPEC) and mRCC (Caki-1) cells, and rs58159269 increased cell proliferation/reduced sorafenib cytotoxicity, and reduced endothelial tube formation in TIME cells. rs307826 increased VEGFR-3 phosphorylation, and led to post-translational VEGFR-3 processing differences in HUVECs. Conclusions: Regulatory VEGFA and FLT4 variants associated with OS in mRCC patients induce cellular alterations that affect angiogenesis and sorafenib activity. These are novel and potentially impactful clinical biomarkers for mRCC and sorafenib, and their relevance to other multikinase inhibitors used in mRCC should be tested. Citation Format: Daniel James Crona, Andrew D. Skol, Veli-Matti Leppänen, Dylan M. Glubb, Amy S. Etheridge, Eleanor Hilliard, Carol Peña, Yuri K. Peterson, Nancy Klauber-DeMore, Kari K. Alitalo, Federico Innocenti. Clinical biomarkers of survival in renal cell carcinoma patients treated with sorafenib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1625.

Effects of genetic variation in the aldosterone synthase (CYP11B2) gene on enzyme function

Evidence suggests that high levels of aldosterone lead to hypertension and increased risk of cardiovascular disease. Around 15% of patients with essential hypertension have a raised aldosterone to renin ratio (ARR) suggesting that aldosterone production is inappropriately high in relation to its principal agonist angiotensin II. This may be due to increased activity of aldosterone synthase caused by genetic variation in the CYP11B2 gene. We screened the coding region of human CYP11B2 for genetic variants and tested their effects on function in vitro. Normotensive subjects (n = 69) were screened for sequence variants in the coding region of CYP11B2 by single-stranded conformation polymorphism (SSCP) analysis and sequencing. The effects of nonsynonymous variants on enzyme activity were assessed in JEG-3 cells transiently transfected with wild-type or variant expression plasmids. The conversion of the substrate 11-deoxycorticosterone (DOC) to corticosterone (B) and aldosterone was measured. Twenty variants were detected in CYP11B2 and eight analysed functionally (Arg87Gly, Asn281Thr, Gly288Ser, Lys296Asn, Asp335Asn, Gln404Arg, Ala414Pro and His439Tyr). Corticosterone synthesis was unaltered and aldosterone synthesis reduced in variant Arg87Gly; Asn281Thr increased corticosterone and decreased aldosterone production; Gly288Ser increased corticosterone production and abolished aldosterone production; Lys296Asn reduced both corticosterone and aldosterone production; Asp335Asn increased corticosterone synthesis but did not affect aldosterone production. Variants Gln404Arg, Ala414Pro and His439Tyr showed increases in both corticosterone and aldosterone synthesis compared to the wild-type. The study confirms the genetic variability of the CYP11B2 gene and provides us with additional valuable structure-function information.