Noor Ahmad Shaik

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.

Estrogen Receptor-α gene (T/C) Pvu II Polymorphism in Endometriosis and Uterine Fibroids

Endometriosis and fibroids are estrogen-dependent benign pathologies of the uterus, which account for infertility and pelvic pain along with dysmenorrhea in women. Suppression of the disease and recurrence after discontinuing hormone therapy strongly suggests that these are responsive to hormones, especially estrogen, which acts via its receptor. A T/C SNP in intron 1 and exon 2 boundary of estrogen receptor (ER) α gene recognized by PvuII enzyme has been associated with several female pathologies like breast cancer, osteoporosis, endometriosis and fibroids in various ethnic groups. The aim of the present study was to assess this ER α T/C polymorphism in endometriosis and fibroid patients from Asian Indian population. Genomic DNA was isolated from 367 women, who included 110 cases of endometriosis, 142 cases of uterine fibroids and 115 healthy age matched women volunteers. PCR was carried out to amplify ER α gene followed by restriction digestion with Pvu II. Results indicate a significant association of C allele with both endometriosis [OR = 2.6667, 95% CI = 1.4166 to 5.0199; p < 0.05] and fibroids [2.0833, 95% CI = 1.1327 to 3.8319; p < 0.05]. Further studies are needed in larger population to establish ER α C allele as a risk marker for endometriosis and fibroids in Asian Indian women. Ethnicity, race, diet etc may play a role in susceptibility to endometriosis and fibroids and further studies are warranted in this area.

HER-2/neu Status: A Neglected Marker of Prognostication and Management of Breast Cancer Patients in India

BACKGROUND Categorizing breast tumors based on the ER, PR and HER/Neu 2 receptor status is necessary in order to predict outcome and assist in management of breast cancer. Herfe we assessed this question in South Indian patients. MATERIALS AND METHODS A total of 619 formalin fixed paraffin embedded breast tumor tissues were collected from pathology archives after receipt of ethical clearance. With the help of primary and secondary conjugated antibodies, expression status of ER, PR and HER2/neu was determined. All the experimental data were assessed for correlations with histopathological features of tumors and clinical presentation of the subjects. RESULTS In the present study, the ages ranged from 20-87 years with a mean of 50.0±12.q years, and majority of the tumors (84%) were of infiltrating duct cell carcinoma type. Assessment of ER, PR and Her-2/neu expression showed that 46% were triple negative. Interestingly, an inverse relation between ER, PR and HER-2/neu was apparent in 41.2% (p<0.0001) of the tumors, of which 24.5% (p<0.0001) were ER and PR co-negative but HER-2 positive. CONCLUSIONS ER and PR positive tumors are less common (i.e<30%) compared to HER-2/neu positive tumors (i.e>50%) in Indian breast cancer patients, underlining the need for effective diagnostic screening and specific therapeutic managements in order to improve the survival rate of patients in low resource countries such as India.

Investigation of Calpain 10 (rs2975760) gene polymorphism in Asian Indians with Gestational Diabetes Mellitus

Background Type 2 Diabetes Mellitus (T2DM) and Gestational Diabetes Mellitus (GDM) are part of a heterogeneous and complex metabolic group of disorders that share common pathophysiological circumstances, including β-cell dysfunction and insulin resistance. The protein Calpain 10 (CAPN10) plays a role in glucose metabolism, pancreatic β-cell insulin secretion, and thermogenesis. Objective Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR–RFLP) based genotyping of CAPN10 (rs2975760) polymorphism was carried out in T2DM and GDM with suitable controls for each of the pathologies from the same population. Genomic DNA was isolated from 787 participants, including 250 cases of T2DM, 287 pregnant women, of which 137 were identified as having GDM and the remaining 150 were confirmed as non-GDM, and 250 healthy control volunteers, and association analysis was carried out for genotypes and alleles. Results In the present study, T2DM was compared with healthy controls and was not found to be associated with the CAPN10 C allele (odds ratio, OR: 1.09; 95% CI = 0.8011–1.484; p = 0.5821). GDM also did not show any association when compared with non-GDM (OR: 1.124; 95% CI = 0.7585–1.667; p = 0.5606) respectively. Conclusion Our study suggests that the CAPN10 (rs2975760) polymorphism scrutinized in this study is not associated with T2DM and GDM.

First comprehensive in silico analysis of the functional and structural consequences of SNPs in human GalNAc-T1 gene.

GalNAc-T1, a key candidate of GalNac-transferases genes family that is involved in mucin-type O-linked glycosylation pathway, is expressed in most biological tissues and cell types. Despite the reported association of GalNAc-T1 gene mutations with human disease susceptibility, the comprehensive computational analysis of coding, noncoding and regulatory SNPs, and their functional impacts on protein level, still remains unknown. Therefore, sequence- and structure-based computational tools were employed to screen the entire listed coding SNPs of GalNAc-T1 gene in order to identify and characterize them. Our concordant in silico analysis by SIFT, PolyPhen-2, PANTHER-cSNP, and SNPeffect tools, identified the potential nsSNPs (S143P, G258V, and Y414D variants) from 18 nsSNPs of GalNAc-T1. Additionally, 2 regulatory SNPs (rs72964406 and #x26; rs34304568) were also identified in GalNAc-T1 by using FastSNP tool. Using multiple computational approaches, we have systematically classified the functional mutations in regulatory and coding regions that can modify expression and function of GalNAc-T1 enzyme. These genetic variants can further assist in better understanding the wide range of disease susceptibility associated with the mucin-based cell signalling and pathogenic binding, and may help to develop novel therapeutic elements for associated diseases.

A Computational Protein Phenotype Prediction Approach to Analyze the Deleterious Mutations of Human MED12 Gene

Genetic mutations in MED12, a subunit of Mediator complex are seen in a broad spectrum of human diseases. However, the underlying basis of how these pathogenic mutations elicit protein phenotype changes in terms of 3D structure, stability and protein binding sites remains unknown. Therefore, we aimed to investigate the structural and functional impacts of MED12 mutations, using computational methods as an alternate to traditional in vivo and in vitro approaches. The MED12 gene mutations details and their corresponding clinical associations were collected from different databases and by text‐mining. Initially, diverse computational approaches were applied to categorize the different classes of mutations based on their deleterious impact to MED12. Then, protein structures for wild and mutant types built by integrative modeling were analyzed for structural divergence, solvent accessibility, stability, and functional interaction deformities. Finally, this study was able to identify that genetic mutations mapped to exon‐2 region, highly conserved LCEWAV and Catenin domains induce biochemically severe amino acid changes which alters the protein phenotype as well as the stability of MED12‐CYCC interactions. To better understand the deleterious nature of FS‐IDs and Indels, this study asserts the utility of computational screening based on their propensity towards non‐sense mediated decay. Current study findings may help to narrow down the number of MED12 mutations to be screened for mediator complex dysfunction associated genetic diseases. This study supports computational methods as a primary filter to verify the plausible impact of pathogenic mutations based on the perspective of evolution, expression and phenotype of proteins. J. Cell. Biochem. 117: 2023–2035, 2016.

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.

In-silico analysis of inflammatory bowel disease (IBD) GWAS loci to novel connections.

Genome-wide association studies (GWASs) for many complex diseases, including inflammatory bowel disease (IBD), produced hundreds of disease-associated loci—the majority of which are noncoding. The number of GWAS loci is increasing very rapidly, but the process of translating single nucleotide polymorphisms (SNPs) from these loci to genomic medicine is lagging. In this study, we investigated 4,734 variants from 152 IBD associated GWAS loci (IBD associated 152 lead noncoding SNPs identified from pooled GWAS results + 4,582 variants in strong linkage-disequilibrium (LD) (r2 ≥0.8) for EUR population of 1K Genomes Project) using four publicly available bioinformatics tools, e.g. dbPSHP, CADD, GWAVA, and RegulomeDB, to annotate and prioritize putative regulatory variants. Of the 152 lead noncoding SNPs, around 11% are under strong negative selection (GERP++ RS ≥2); and ~30% are under balancing selection (Tajima’s D score >2) in CEU population (1K Genomes Project)—though these regions are positively selected (GERP++ RS <0) in mammalian evolution. The analysis of 4,734 variants using three integrative annotation tools produced 929 putative functional SNPs, of which 18 SNPs (from 15 GWAS loci) are in concordance with all three classifiers. These prioritized noncoding SNPs may contribute to IBD pathogenesis by dysregulating the expression of nearby genes. This study showed the usefulness of integrative annotation for prioritizing fewer functional variants from a large number of GWAS markers.

Down regulated expression of Claudin-1 and Claudin-5 and up regulation of β-catenin: association with human glioma progression.

Glioblastoma multiforme is the most common form of intracranial malignancy in humans, and is characterized by aggressive tumor growth, tissue invasion and neurodegenerative properties. The present study investigated the expression status of tight junction associated Claudin 1 (CLDN1), Claudin 5 (CLDN5) and Adheren junction associated β-catenin genes in the light of their critical role in the progression of both low- and high-grade human gliomas. Using quantitative PCR and Western blot methods the mRNA and protein status of CLDN1, CLDN5 and β-catenin genes were studied in a total of 25 human gliomas of World Health Organization (WHO) grades I-IV, non-cancerous control brain tissues and their corresponding model cell lines (C6, U373, U118, T98 and U87MG). Quantitative analysis of the transcript and protein expression data showed that CLDN1 and CLDN5 were significantly down regulated (p=<0.001) in tumors of all four grades and model cell lines. This decrease in expression pattern was in accordance with the increasing grade of the tumor. A 4-fold stronger reduction of CLDN1 when compared to CLDN5 was evident in high-grade tumors. Interestingly, β-catenin was up regulated in all tumor types we studied (p=<0.005). Our findings, suggest that down regulated CLDN1 and CLDN5 genes have potential relevance in relation to the progression of glioblastoma multiforme. Hence, their therapeutic targeting may provide both insight and leads to control the cellular proliferation and subsequent invasiveness among affected individuals.

Enhanced transcription of estrogen receptor α and mitochondrial cytochrome b genes in uterine leiomyomas

The relative expression levels of estrogen receptor α (ERα) and mitochondrial cytochrome b (MTCYB) transcripts and their association with ERα, −397T > C gene polymorphism was determined in premenopausal uterine leiomyomas and myometrium tissues to gain an insight into the role of ER-mediated action of estrogen on mitochondrial gene transcription. Both ERα and MTCYB transcripts were overexpressed in leiomyomas compared with myometrium tissues with 9.18 ± 0.79 folds and 5.24 ± 0.48 folds, respectively. ERα demonstrated ≥1.7 folds overexpression expressed over MTCYB (p < 0.001). Genotype correlation with transcript expression revealed that leiomyomas with CC genotype had significantly increased levels of ERα with 11.9 ± 1.02 folds as compared with 6.46 ± 0.56 folds seen in CT and TT genotypes together (p < 0.001). Interestingly, MTCYB transcript levels were also >1.9 folds overexpressed in leiomyomas with the CC genotype as compared with leiomyomas with other genotypes (p < 0.01).Significant elevation of ERα and MTCYB transcript levels in premenopausal leiomyomas and its association with ERα, −397 CC genotype suggests the mitochondrial-mediated role of estrogen as the promoter of leiomyoma tumorigenesis.