Structural prediction, whole exome sequencing and molecular dynamics simulation confirms p.G118D somatic mutation of PIK3CA as functionally important in breast cancer patients.

To understand the structural and functional importance of PIK3CA somatic mutations, whole exome sequencing, molecular dynamics simulation techniques in combination with in silico prediction algorithms such as SIFT, PolyPhen, Provean and CADD were employed. Twenty out of eighty missense somatic mutations in PIK3CA gene were found to be pathogenic by all the four algorithms. Most recurrent mutations found were known hotspot PIK3CA mutations with known clinical significance like p.E545 K, p.E545A, p.E545 G and p.C420R. A missense mutation p.G118D was found to be recurrently mutated in 5 cases. Interestingly, this mutation was observed in one of the patients who underwent whole exome sequencing and was completely absent from the controls. To see the effect of this mutation on the structure of PIK3CA protein, molecular dynamics simulation was performed. By molecular dynamics approach, we have shown that p.G118D mutation deviated from the native structure which was supported by the decrease in the number of hydrogen bonds, difference in hydrogen bond distance and angle, difference in root mean square deviation between the native and the mutant structures.

Computational Analysis of Breast Cancer GWAS Loci Identifies the Putative Deleterious Effect of STXBP4 and ZNF404 Gene Variants.

The genome-wide association studies (GWAS) have enabled us in identifying different breast cancer (BC) susceptibility loci. However, majority of these are non-coding variants with no annotated biological function. We investigated such 78 noncoding genome wide associated SNPs of BC and further expanded the list to 2,162 variants with strong linkage-disequilibrium (LD, r2 ≥0.8). Using multiple publically available algorithms such as CADD, GWAVA, and FATHAMM, we classified all these variants into deleterious, damaging, or benign categories. Out of total 2,241 variants, 23 (1.02%) variants were extreme deleterious (rank 1), 70 (3.12%) variants were deleterious (rank 2), and 1,937 (86.43%) variants were benign (rank 3). The results show 14% of lead or associated variants are under strong negative selection (GERP++ RS ≥2), and ∼22% are under balancing selection (Tajima's D score >2) in CEU population of 1KGP-the regions being positively selected (GERP++ RS <0) in mammalian evolution. The expression quantitative trait loci of highest deleteriously ranked genes were tested on relevant adipose and breast tissues, the results of which were extended for protein expression on breast tissues. From the concordance analysis of ranking system of GWAVA, CADD, and FATHMM, eQTL and protein expression, we identified the deleterious SNPs localized in STXBP4 and ZNF404 genes which might play a role in BC development by dysregulating its gene expression. This simple approach will be easier to implement and to prioritize large scale GWAS data for variety of diseases and link to the potentially unrecognized functional roles of genes. J. Cell. Biochem. 118: 4296-4307, 2017. © 2017 Wiley Periodicals, Inc.

Exome sequencing identifies novel NTRK1 mutations in patients with HSAN-IV phenotype.

Hereditary sensory autonomic neuropathy type IV (HSAN-IV) is a rare autosomal recessive disorder that usually begins in infancy and is characterized by anhidrosis, insensitivity to noxious stimuli leading to self-mutilating behavior, and intellectual disability. HSAN-IV is caused by mutations in the neurotrophic tyrosine kinase receptor type 1 gene, NTRK1, encoding the high-affinity receptor of nerve growth factor (NGF) which maps to chromosome 1q21-q22. Patients with HSAN-IV lack all NGF-dependent neurons, the primary afferents and sympathetic postganglionic neurons leading to lack of pain sensation and the presence of anhidrosis, respectively. Herein, we report nine patients from nine unrelated families with HSAN-IV due to various mutations in NTRK1, five of which are novel. These are three missense and two nonsense mutations distributed in various domains of NTRK1 involved in binding of NGF. The affected patients had variable intellectual deficits, and some had delayed diagnosis of HSAN-IV. In addition to being the first report of HSAN-IV from the Arabian Peninsula, this report expands the mutational spectrum of patients with NTRK1 mutations and provides further insights for molecular and clinical diagnosis.

Evaluation and identification of damaged single nucleotide polymorphisms in COL1A1 gene involved in osteoporosis.

INTRODUCTION: Single-nucleotide polymorphisms (SNPs) are biomarkers for exploring the genetic basis of many complex human diseases. The prediction of SNPs is promising in modern genetic analysis but it is still a great challenge to identify the functional SNPs in a disease-related gene. The computational approach has overcome this challenge and an increase in the successful rate of genetic association studies and reduced cost of genotyping have been achieved. The objective of this study is to identify deleterious non-synonymous SNPs (nsSNPs) associated with the COL1A1 gene. MATERIAL AND METHODS: The SNPs were retrieved from the Single Nucleotide Polymorphism Database (dbSNP). Using I-Mutant, protein stability change was calculated. The potentially functional nsSNPs and their effect on proteins were predicted by PolyPhen and SIFT respectively. FASTSNP was used for estimation of risk score. RESULTS: Our analysis revealed 247 SNPs as non-synonymous, out of which 5 nsSNPs were found to be least stable by I-Mutant 2.0 with a DDG value of > -1.0. Four nsSNPs, namely rs17853657, rs17857117, rs57377812 and rs1059454, showed a highly deleterious tolerance index score of 0.00 with a change in their physicochemical properties by the SIFT server. Seven nsSNPs, namely rs1059454, rs8179178, rs17853657, rs17857117, rs72656340, rs72656344 and rs72656351, were found to be probably damaging with a PSIC score difference between 2.0 and 3.5 by the PolyPhen server. Three nsSNPs, namely rs1059454, rs17853657 and rs17857117, were found to be highly polymorphic with a risk score of 3-4 with a possible effect of non-conservative change and splicing regulation by FASTSNP. CONCLUSIONS: Three nsSNPs, namely rs1059454, rs17853657 and rs17857117, are potential functional polymorphisms that are likely to have a functional impact on the COL1A1 gene.

Exploration of deleterious single nucleotide polymorphisms in late-onset Alzheimer disease susceptibility genes.

Non-synonymous single nucleotide polymorphisms (nsSNPs) are considered as biomarkers to disease susceptibility. In the present study, nsSNPs in CLU, PICALM and BIN1 genes were screened for their functional impact on concerned proteins and their plausible role in Alzheimer disease (AD) susceptibility. Initially, SNPs were retrieved from dbSNP database, followed by identification of potentially deleterious nsSNPs and prediction of their effect on proteins by PolyPhen and SIFT. Protein stability and the probability of mutation occurrence were predicted using I-Mutant and PANTHER respectively. SNPs3D and FASTSNP were used for the functional analysis of nsSNPs. The functional impact on the 3D structure of proteins was evaluated by SWISSPDB viewer and NOMAD-Ref server. On analysis, 3 nsSNPs with IDs rs12800974 (T158P) of PICALM and rs11554585 (R397C) and rs11554585 (N106D) of BIN1 were predicted to be functionally significant with higher scores of I-Mutant, SIFT, PolyPhen, PANTHER, FASTSNP and SNPs3D. The mutant models of these nsSNPs also showed very high energies and RMSD values compared to their native structures. Current study proposes that the three nsSNPs identified in this study constitute a unique resource of potential genetic factors for AD susceptibility.

In silico analysis of Single Nucleotide Polymorphisms (SNPs) in human BRAF gene.

BRAF gene mutations are frequently seen in both inherited and somatic diseases. However, the harmful mutations for BRAF gene have not been predicted in silico. Owing to the importance of BRAF gene in cell division, differentiation and secretion processes, the functional analysis was carried out to explore the possible association between genetic mutations and phenotypic variations. Genomic analysis of BRAF was initiated with SIFT followed by PolyPhen and SNPs&GO servers to retrieve the 85 deleterious non-synonymous SNPs (nsSNPs) from dbSNP. A total of 5 mutations i.e. c.406T>G (S136A), c.1446G>T (R462I), c.1556 A>G (K499E), c.1860 T>A (V600E) and c.2352 C>T (P764L) that are found to exert benign effects on the BRAF protein structure and function were chosen for further analysis. Protein structural analysis with these amino acid variants was performed by using I-Mutant, FOLD-X, HOPE, NetSurfP, Swiss PDB viewer, Chimera and NOMAD-Ref servers to check their solvent accessibility, molecular dynamics and energy minimization calculations. Our in silico analysis suggested that S136A and P764L variants of BRAF could directly or indirectly destabilize the amino acid interactions and hydrogen bond networks thus explain the functional deviations of protein to some extent. Screening for BRAF, S136A and P764Lvariants may be useful for disease molecular diagnosis and also to design the molecular inhibitors of BRAF pathways.

Screening and structural evaluation of deleterious Non-Synonymous SNPs of ePHA2 gene involved in susceptibility to cataract formation.

Age-related cataract is clinically and genetically heterogeneous disorder affecting the ocular lens, and the leading cause of vision loss and blindness worldwide. Here we screened nonsynonymous single nucleotide polymorphisms (nsSNPs) of a novel gene, EPHA2 responsible for age related cataracts. The SNPs were retrieved from dbSNP. Using I-Mutant, protein stability change was calculated. The potentially functional nsSNPs and their effect on protein was predicted by PolyPhen and SIFT respectively. FASTSNP was used for functional analysis and estimation of risk score. The functional impact on the EPHA2 protein was evaluated by using SWISSPDB viewer and NOMAD-Ref server. Our analysis revealed 16 SNPs as nonsynonymous out of which 6 nsSNPs, namely rs11543934, rs2291806, rs1058371, rs1058370, rs79100278 and rs113882203 were found to be least stable by I-Mutant 2.0 with DDG value of > -1.0. nsSNPs, namely rs35903225, rs2291806, rs1058372, rs1058370, rs79100278 and rs113882203 showed a highly deleterious tolerance index score of 0.00 by SIFT server. Four nsSNPs namely rs11543934, rs2291806, rs1058370 and rs113882203 were found to be probably damaging with PSIC score of ≥ 2. 0 by Polyp hen server. Three nsSNPs namely, rs11543934, rs2291806 and rs1058370 were found to be highly polymorphic with a risk score of 3-4 with a possible effect of Non-conservative change and splicing regulation by FASTSNP. The total energy and RMSD value was higher for the mutant-type structure compared to the native type structure. We concluded that the nsSNP namely rs2291806 as the potential functional polymorphic that is likely to have functional impact on the EPHA2 gene.

Functional genomics based prioritization of potential nsSNPs in EPHX1, GSTT1, GSTM1 and GSTP1 genes for breast cancer susceptibility studies.

In the present study, nsSNPs in EPHX1, GSTT1, GSTM1 and GSTP1 genes were screened for their functional impact on concerned proteins and their plausible role in breast cancer susceptibility. Initially, SNPs were retrieved from dbSNP, followed by identification of potentially deleterious nsSNPs using PolyPhen and SIFT. Functional analysis was done with SNPs3D, SNPs&GO and MutPred methods. Prediction and evaluation of the functional impact on the 3D structure of proteins were performed with Swiss PDB viewer and NOMAD-Ref servers. On analysis, 13 nsSNPs were found to be highly deleterious and damaging to the protein structure, of which 6 nsSNPs, rs45549733, rs45506591 and rs4986949 of GSTP1, rs72549341 and rs148240980 of EPHX1 and rs17856199 of GSTT1 were predicted to be potentially polymorphic. It is therefore hypothesized that the 6 identified nsSNPs may alter the detoxification process and elevate carcinogenic metabolite accumulation thus modifies the risk of breast cancer susceptibility in a group of women.

Screening and Evaluation of Deleterious SNPs in APOE Gene of Alzheimer's Disease.

Introduction. Apolipoprotein E (APOE) is an important risk factor for Alzheimer's disease (AD) and is present in 30-50% of patients who develop late-onset AD. Several single-nucleotide polymorphisms (SNPs) are present in APOE gene which act as the biomarkers for exploring the genetic basis of this disease. The objective of this study is to identify deleterious nsSNPs associated with APOE gene. Methods. The SNPs were retrieved from dbSNP. Using I-Mutant, protein stability change was calculated. The potentially functional nonsynonymous (ns) SNPs and their effect on protein was predicted by PolyPhen and SIFT, respectively. FASTSNP was used for functional analysis and estimation of risk score. The functional impact on the APOE protein was evaluated by using Swiss PDB viewer and NOMAD-Ref server. Results. Six nsSNPs were found to be least stable by I-Mutant 2.0 with DDG value of >-1.0. Four nsSNPs showed a highly deleterious tolerance index score of 0.00. Nine nsSNPs were found to be probably damaging with position-specific independent counts (PSICs) score of ≥2.0. Seven nsSNPs were found to be highly polymorphic with a risk score of 3-4. The total energies and root-mean-square deviation (RMSD) values were higher for three mutant-type structures compared to the native modeled structure. Conclusion. We concluded that three nsSNPs, namely, rs11542041, rs11542040, and rs11542034, to be potentially functional polymorphic.

Functional analysis and structure determination of alkaline protease from Aspergillus flavus.

Proteases are one of the highest value commercial enzymes as they have broad applications in food, pharmaceutical, detergent, and dairy industries and serve as vital tools in determination of structure of proteins and polypeptides. Multiple application of these enzymes stimulated interest to discover them with novel properties and considerable advancement of basic research into these enzymes. A broad understanding of the active site of the enzyme and of the mechanism of its inactivation is essential for delineating its structure-function relationship. Primary structure analysis of alkaline protease showed 42% of its content to be alpha helix making it stable for three dimensional structure modeling. Homology model of alkaline protease has been constructed using the X-ray structure (3F7O) as a template and swiss model as the workspace. The model was validated by ProSA, SAVES, PROCHECK, PROSAII and RMSD. The results showed the final refined model is reliable. It has 53% amino acid sequence identity with the template, 0.24 Å as RMSD and has -7.53 as Z-score, the Ramachandran plot analysis showed that conformations for 83.4 % of amino acid residues are within the most favored regions and only 0.4% in the disallowed regions.

Comparative analysis of hemagglutinin of 2009 H1N1 influenza A pandemic indicates its evolution to 1918 H1N1 pandemic.

To gain insight into the possible origin of the hemagglutinin of 2009 outbreak, we performed its comparative analysis with hemagglutinin of influenza viral strains from 2005 to 2008 and the past pandemics of 1977, 1968, 1957 and 1918. This insilico analysis showed a maximum sequence similarity between 2009 and 1918 pandemics. Primary structure analysis, antigenic and glycosylation site analyses revealed that this protein has evolved from 1918 pandemic. Phylogenetic analysis of HA amino acid sequence of 2009 influenza A(H1N1) viruses indicated that this virus possesses a distinctive evolutionary trait with 1918 influenza A virus. Although the disordered sequences are different among all the isolates, the disordered positions and sequences between 2009 and 1918 isolates show a greater similarity. Thus these analyses contribute to the evidence of the evolution of 2009 pandemic from 1918 influenza pandemic. This is the first computational evolutionary analysis of HA protein of 2009 H1N1 pandemic.

Analysis of casein alpha S1 & S2 proteins from different mammalian species.

Nowadays, the quality of any food used for human consumption is, to a considerable extent, considered by its possible contribution to the maintenance or improvement of the consumer's health. In developed countries there is increasing interest in goat milk and its derivates, the quality of which is considered of special importance in the light of current tendencies favouring healthy eating. In particular, goat's milk is a hypoallergenic alternative to cow's milk in the human diet. In the present work, we studied the casein alpha S1 and S2 proteins of cow, goat and sheep for comparative analysis. We found that the amino acid sequence of these proteins is almost same in goat and sheep but there are several changes at different base pairs when these two sequences are compared with cow. Prediction of secondary structures (GOR) was performed for alpha s1 and s2 proteins to gain functional insights. Our in silico study revealed considerable identity in chemical properties between goat and sheep but a considerable dissimilarity in cow with goat and sheep casein proteins. Moreover, the effect amino acid change on secondary structures in the three species is discussed. There was no significant difference found between goat and sheep alpha S1 and S2 proteins, so naturally both will be having same properties. The study concludes that sheep milk is another convenient alternative for the cow milk allergic children.