In silico assessment of DNA damage response gene variants associated with head and neck cancer.

Head and neck cancer (HNC), the sixth most common cancer globally, stands first in India, especially Northeast India, where tobacco usage is predominant, which introduces various carcinogens leading to malignancies by accumulating DNA damages. Consequently, the present work aimed to predict the impact of significant germline variants in DNA repair and Tumour Suppressor genes on HNC development. WES in Ion ProtonTM platform on 'discovery set' (n = 15), followed by recurrence assessment of the observed variants on 'confirmation set' (n = 40) using Sanger Sequencing was performed on the HNC-prevalent NE Indian populations. Initially, 53 variants were identified, of which seven HNC-linked DNA damage response gene variants were frequent in the studied populations. Different tools ascertained the biological consequences of these variants, of which the non-coding variants viz. EXO1_rs4150018, RAD52_rs6413436, CHD5_rs2746066, HACE1_rs6918700 showed risk, while FLT3_rs2491227 and BMPR1A_rs7074064 conferred protection against HNC by affecting transcriptional regulation and splicing mechanism. Molecular Dynamics Simulation of the full-length p53 model predicted that the observed coding TP53_rs1042522 variant conferred HNC-risk by altering the structural dynamics of the protein, which displayed difficulty in the transition between active and inactive conformations due to high-energy barrier. Subsequent pathway and gene ontology analysis revealed that EXO1, RAD52 and TP53 variants affected the Double-Strand Break Repair pathway, whereas CHD5 and HACE1 variants inactivated DNA repair cascade, facilitating uncontrolled cell proliferation, impaired apoptosis and malignant transformation. Conversely, FLT3 and BMPR1A variants protected against HNC by controlling tumorigenesis, which requires experimental validation. These findings may serve as prognostic markers for developing preventive measures against HNC.

Whole exome sequencing identifies the potential role of genes involved in p53 pathway in Nasopharyngeal Carcinoma from Northeast India.

Nasopharyngeal Carcinoma (NPC) found to be dependent on geographical and racial variation and is more prevalent in Northeast (NE) India. WES-based study was conducted in three states (tribes); Nagaland (Naga), Mizoram (Mizo) and Manipur (Manipuri), which provided an overview of germline variants involved inthemajor signaling pathways. Validation and recurrence assessment of WES data confirmed the risk effect of STEAP3_rs138941861 and JAG1_rs2273059, and the protective role of PARP4_rs17080653 and TGFBR1_rs11568778 variants, where STEAP3_rs138941861conferring Arg290His substitution was the only exonic non-synonymous variant and to be located in proximity to the linking region between the transmembrane and oxidoreductasedomainsof STEAP3 protein, andaffectedits structural and functional dynamics by altering the Electrostatic Potential around this connecting region. Moreover, these significantly associated variants having deleterious effect were observed to have interactions in p53 signaling pathway which emphasizes the importance of this pathway in the causation of NPC.

The impact of prehistoric human dispersals on the presence of tobacco-related oral cancer in Northeast India.

BACKGROUND: Northeast (NE) India is a subject of debate for predicting its involvement in prehistoric anatomically modern human (AMH) dispersal. The unique lifestyle and genetic characteristics of native ethnic groups in this region are believed to be responsible for their susceptibility to tobacco-related oral cancer (TrOC). The present study assessed mitochondrial macro-haplogroup (mHG) diversity and TrOC susceptibility autosomal loci to evaluate the impact of prehistoric AMH dispersal on the present day's high TrOC prevalence in major NE Indian ethnics. METHODS: We considered 175 unrelated individuals from 35 ethnic groups and previously published 374 sequences for sequencing-based assessment of mtDNA-based marker by subsequent analyses like haplogroup diversity, phylogenetic, genetic structure by AMOVA, and MDS, descriptive statistics of demographic parameters, and migration analysis. Besides, we selected prolonged tobacco-chewing 124 case-control individuals from similar ethnic backgrounds for genotyping 115 autosomal loci in Sequenom iPLEX MassARRAY™ platform and mined 1000genome data (n = 398) for consequent global admixture and ancestry-specific allele frequencies-based analyses. RESULTS: Our mtDNA-based findings suggested that NE populations were distinct from other Indian populations, owing to the first wave of migration from ancient southern China (∼54kya) and two successive spatial expansion events at âˆ¼45kya and âˆ¼43kya. Consequently, it probably acted as another source for prehistoric AMH dispersal in N/NE Asia. Besides, the second wave of back-migration from SE Asia (∼40kya) probably replaced the mitochondrial footprints of survivors from the first migrants and introduced the TrOC susceptibility traits in this region. Afterward, the autosomal marker-based observations on the transition of the disease-associated admixture component 'K6' from SE Asia reconfirmed these results. Moreover, we also observed that the mitochondrial mHG 'R' is significantly associated with the risk of TrOC (OR > 9.5) in NE India. Furthermore, the possible onset of the phenotypic expression of those traits was predicted at âˆ¼4kya, thus, contributing to present-day's TrOC prevalence. CONCLUSIONS: This study reflects its uniqueness by revealing an updated AMH dispersal route for the peopling in and out of NE India, which probably introduced the disease-causing traits in the ancestral NE Indian population. Those traits were then imprinted in their genome to get transferred through their respective generations, forming the present-day's TrOC-prevalent NE Indian population.

Assessment of DNA repair susceptibility genes identified by whole exome sequencing in head and neck cancer.

Head and neck cancer (HNC), the sixth most common cancer globally, stands second in India. In Northeast (NE) India, it is the sixth most common cause of death in males and seventh in females. Prolonged tobacco and alcohol consumption constitute the major etiological factors for HNC development, which induce DNA damage. Therefore, DNA repair pathway is a crucial system in maintaining genomic integrity and preventing carcinogenesis. The present work was aimed to predict the consequence of significant germline variants of the DNA repair genes in disease predisposition. Whole exome sequencing was performed in Ion Proton™ platform on 15 case-control samples from the HNC-prevalent states of Manipur, Mizoram, and Nagaland. Variant annotation was done in Ion Reporter™ as well as wANNOVAR. Subsequent statistical and bioinformatics analysis identified significant exonic and intronic variants associated with HNC. Amongst our observed variants, 78.6% occurred in ExAC, 94% reported in dbSNP and 5.8% & 9.3% variants were present in ClinVar and HGMD, respectively. The total variants were dispersed among 199 genes with DSBR and FA pathway being the most mutated pathways. The allelic association test suggested that the intronic variants in HLTF and RAD52 gene significantly associated (P < 0.05) with the risk (OR > 5), while intronic variants in PARP4, RECQL5, EXO1 and PER1 genes and exonic variant in TDP2 gene showed protection (OR < 1) for HNC. MDR analysis proposed the exonic variants in MSH6, BRCA2, PALB2 and TP53 genes and intronic variant in RECQL5 genetic region working together during certain phase of DNA repair mechanism for HNC causation. In addition, other intronic and 3'UTR variations caused modifications in the transcription factor binding sites and miRNA target sites associated with HNC. Large-scale validation in NE Indian population, in-depth structure prediction and subsequent simulation of our recognized polymorphisms is necessary to identify true causal variants related to HNC.

Detection of p16 Promoter Hypermethylation by Methylation-Specific PCR.

DNA methylation plays a decisive role in the regulation and control of gene expression. DNA methylation is a covalent modification, in which a methyl group is attached to the 5th carbon of the cytosine ring of a CpG dinucleotide that is located upstream from the promoter region of a gene. Promoter hypermethylation (gain of DNA methylation) of the p16 gene may cause silencing of gene expression and plays an important role in cancer. Therefore, detection of the methylation status of p16 gene is an important tool in epigenetic studies of various human cancers. The methylation-specific PCR (MSP) is the most commonly used technique for studying DNA methylation. This technique is based on bisulfite modification of DNA, which converts unmethylated cytosine (C) into uracil (U) and leaving methylated cytosine (Cm) unchanged. Here we describe the bisulfite modification of DNA samples and detection of promoter methylation of p16 gene from bisulfite-treated DNA using MSP. In MSP, modified DNA samples are subjected to PCR amplification using methylated and unmethylated specific primers for the p16 gene separately. The PCR amplified products are then analyzed in a 2.5-3% agarose gel containing ethidium bromide. The PCR amplified band generated by specific sets of primers is used to determine the methylation status of the p16 gene.

Influence of the CYP1A1 T3801C Polymorphism on Tobacco and Alcohol-Associated Head and Neck Cancer Susceptibility in Northeast India.

BACKGROUND: Tobacco and alcohol contain or may generate carcinogenic compounds related to cancers. CYP1A1 enzymes act upon these carcinogens before elimination from the body. The aim of this study was to investigate whether CYP1A1 T3801C polymorphism modulates the relationship between tobacco and alcohol- associated head and neck cancer (HNC) susceptibility among the northeast Indian population. MATERIALS AND METHODS: One hundred and seventy histologically confirmed HNC cases and 230 controls were included within the study. The CYP1A1 T3801C polymorphism was determined using PCR-RFLP, and the results were confirmed by DNA sequencing. Logistic regression (LR) and multifactor dimensionality reduction (MDR) approaches were applied for statistical analysis. RESULTS: The CYP1A1 CC genotype was significantly associated with HNC risk (P=0.045). A significantly increased risk of HNC (OR=6.09; P<0.0001) was observed in individuals with combined habits of smoking, alcohol drinking and tobacco-betel quid chewing. Further, gene-environment interactions revealed enhanced risks of HNC among smokers, alcohol drinkers and tobacco-betel quid chewers carrying CYP1A1 TC or CC genotypes. The highest risk of HNC was observed among smokers (OR=7.55; P=0.009) and chewers (OR=10.8; P<0.0001) carrying the CYP1A1 CC genotype. In MDR analysis, the best model for HNC risk was the three-factor model combination of smoking, tobacco-betel quid chewing and the CYP1A1 variant genotype (CVC=99/100; TBA=0.605; P<0.0001); whereas interaction entropy graphs showed synergistic interaction between tobacco habits and CYP1A1. CONCLUSIONS: Our results confirm that the CYP1A1 T3801C polymorphism modifies the risk of HNC and further demonstrated importance of gene-environment interaction.

Tobacco carcinogen-metabolizing genes CYP1A1, GSTM1, and GSTT1 polymorphisms and their interaction with tobacco exposure influence the risk of head and neck cancer in Northeast Indian population.

Genetic polymorphisms in tobacco-metabolizing genes may modulate the risk of head and neck cancer (HNC). In Northeast India, head and neck cancers and tobacco consumption remains most prevalent. The aim of the study was to investigate the combined effect of cytochrome P450 1A1 (CYP1A1) T3801C, glutathione S-transferases (GSTs) genes polymorphisms and smoking and tobacco-betel quid chewing in the risk of HNC. The study included 420 subjects (180 cases and 240 controls) from Northeast Indian population. Polymorphisms of CYP1A1 T3801C and GST (M1 & T1) were studied by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and multiplex PCR, respectively. Logistic regression (LR) and multifactor dimensionality reduction (MDR) approach were applied for statistical analysis. LR analysis revealed that subjects carrying CYP1A1 TC/CC + GSTM1 null genotypes had 3.52-fold (P < 0.001) increase the risk of head and neck squamous cell carcinoma (HNSCC). Smokers carrying CYP1A1 TC/CC + GSTM1 null and CYP1A1 TC/CC + GSTT1 null genotypes showed significant association with HNC risk (odds ratio [OR] = 6.42; P < 0.001 and 3.86; P = 0.005, respectively). Similarly, tobacco-betel quid chewers carrying CYP1A1 TC/CC + GSTM1 null genotypes also had several fold increased risk of HNC (P < 0.001). In MDR analysis, the best model for HNSCC risk was the four-factor model of tobacco-betel quid chewing, smoking, CYP1A1 TC/CC, and GSTM1 null genotypes (testing balance accuracy [TBA] = 0.6292; cross-validation consistency [CVC] = 9/10 and P < 0.0001). These findings suggest that interaction of combined genotypes of carcinogen-metabolizing genes with environmental factors might modulate susceptibility of HNC in Northeast Indian population.