Genetic analysis of colorectal carcinoma using high throughput single nucleotide polymorphism genotyping technique within the population of Jammu and Kashmir.

BACKGROUND: SNP genotyping has become increasingly more common place to understand the genetic basis of complex diseases like cancer. SNP-genotyping through MassARRAY™ is a cost-effective method to quantitatively analyse the variation of gene expression in multiple samples, making it a potential tool to identify the underlying causes of colorectal carcinogenesis. METHODS: In the present study, SNP genotyping was carried out using Agena MassARRAY™, which is a cost-effective, robust, and sensitive method to analyse multiple SNPs simultaneously. We analysed 7 genes in 492 samples (100 cases and 392 controls) associated with CRC within the population of Jammu and Kashmir. These SNPs were selected based on their association with multiple cancers in literature. RESULTS: This is the first study to explore these SNPs with colorectal cancer within the J&K population.7 SNPs with a call rate of 90% were selected for the study. Out of these, five SNPs rs2234593, rs1799966, rs2229080, rs8034191, rs1042522 were found to be significantly associated with the current study under the allelic model with an Odds Ratio OR = 2.981(1.731-5.136 at 95% CI); p value = 4.81E-05 for rs2234593,OR = 1.685(1.073-2.647 at 95% CI);; p value = 0.02292 for rs1799966, OR = 1.5 (1.1-2.3 at 95% CI), p value = 0.02 for rs2229080, OR = 1.699(1.035-2.791 at 95% CI); p value = 0.03521 for rs8034191, OR = 20.07 (11.26-35.75); p value = 1.84E-34 for rs1042522 respectively. CONCLUSION: This is the first study to find the relation of Genetic variants with the colorectal cancer within the studied population using high throughput MassARRAY™ technology. It is further anticipated that the variants should be evaluated in other population groups that may aid in understanding the genetic complexity and bridge the missing heritability.

Evaluation of 17 genetic variants in association with leukemia in the north Indian population using MassARRAY Sequenom.

Leukemia is a heterogeneous disorder, characterized by elevated proliferation of white blood cells. In this study, we explored the association of 17 genetic variants with leukemia patients in the Jammu and Kashmir region of north India. The variants were genotyped by using a high-throughput Agena MassARRAY platform in 758 individuals (166 cases and 592 controls). Of the 17 single-nucleotide polymorphisms (SNPs) studied, five SNPs were showing significant association with the high risk of leukemia in the north Indian population, which includes rs10069690 of telomere reverse transcriptase (TERT) with OR = 0.34 (95% CI, 0.20-0.58; p = .0008), rs2972392 (​​​PSCA) with OR 1.86 (95% CI, 1.04-3.81; p = .035), rs4986764 (BRIP1) with OR 1.34 (95% CI, 1.00-1.80; p = .04), rs6990097 (TNKS) with OR 1.81 (95% CI, 1.2-2.6; p = .001) and rs12190287 (TCF21) with OR 2.87 (95% CI, 1.72-4.7; p = .0001) by allelic association using Plink and analyzed by SPSS. This is the first study to explore these variants with leukemia in the studied population.

MassARRAY-based single nucleotide polymorphism analysis in breast cancer of north Indian population.

BACKGROUND: Breast Cancer (BC) is associated with inherited gene mutations. High throughput genotyping of BC samples has led to the identification and characterization of biomarkers for the diagnosis of BC. The most common genetic variants studied are SNPs (Single Nucleotide Polymorphisms) that determine susceptibility to an array of diseases thus serving as a potential tool for identifying the underlying causes of breast carcinogenesis. METHODS: SNP genotyping employing the Agena MassARRAY offers a robust, sensitive, cost-effective method to assess multiple SNPs and samples simultaneously. In this present study, we analyzed 15 SNPs of 14 genes in 550 samples (150 cases and 400 controls). We identified four SNPs of genes TCF21, SLC19A1, DCC, and ERCC1 showing significant association with BC in the population under study. RESULTS: The SNPs were rs12190287 (TCF21) having OR 1.713 (1.08-2.716 at 95% CI) p-value 0.022 (dominant), rs1051266 (SLC19A1) having OR 3.461 (2.136-5.609 at 95% CI) p-value 0.000000466 (dominant), rs2229080 (DCC) having OR 0.6867 (0.5123-0.9205 at 95% CI) p-value 0.0116 (allelic) and rs2298881 (ERCC1) having OR 0.669 (0.46-0.973 at 95% CI), p-value 0.035 (additive) respectively. The in-silico analysis was further used to fortify the above findings. CONCLUSION: It is further anticipated that the variants should be evaluated in other population groups that may aid in understanding the genetic complexity and bridge the missing heritability.

MassARRAY analysis of twelve cancer related SNPs in esophageal squamous cell carcinoma in J&K, India.

BACKGROUND: MassARRAY (Agena Bioscience™) combines competitive PCR with MALDI-TOF mass spectrometry (MS) analysis that gives highly accurate, sensitive, and high-throughput methods for the quantitative analysis of variation of gene expression in multiple samples. SNPs (Single Nucleotide Polymorphisms) have a very high potential of discovering disease-gene relationships. SNP-genotyping through MassARRAY is not only a cost-effective genotyping method but also provides a platform to validate variants observed through a high-throughput Next-generation sequencing (NGS). METHODS: In the present study, we have incorporated the use of matrix-assisted laser desorption/ionization-time of flight, mass spectrometry (MALDI-TOF) as a tool for differentiating genotypes based on the mass of variant. We have performed multiplex PCR and genotyped 12 SNPs in 758 samples (166 cases and 592 controls). The 12 studied SNPs were chosen with a rationale for their association with multiple cancers in literature. RESULTS: This is the first study to explore these SNPs with esophageal cancer within the J&K population. Out of 12 SNPs, two SNPs rs12190287 of TCF21 and rs10046 of CYP19A1 were significantly associated with esophageal cancer with Odds Ratio (OR) 1.412 (1.09-1.8 at 95% CI, p = 0.008) and 1.54 (1.21-2.072 at 95% CI, p = 0.0007) within the population of Jammu and Kashmir. CONCLUSION: We explored 12 SNPs that were found to be associated with multiple cancers in literature with esophageal cancer within the population of J&K. This is the first study to find the relation of these SNPs with ESCC within the studied population. This study explores the relation of genetic and environmental factors with the ESCC susceptibility.

Genetic analysis of polymorphism rs10937405 of TP63 gene in breast and ovarian cancer patients of North Indian Cohort.

Introduction: Ovarian and breast cancers are highly prevalent in the population of Jammu and Kashmir (J&K). However, case-control association studies on breast and ovarian cancers are lacking in this population. Moreover, no case-control study is available on variant rs10937405 of TP63 in breast and ovarian cancers. Thus, we designed to replicate the cancer susceptible variant rs10937405 of TP63 in ovarian and breast cancers in the population of J&K because the TP63 gene act as a tumor suppressor gene and was previously associated with various cancers. Materials and Methods: This case-control association study conducted at the Shri Mata Vaishno Devi University, includes 150 breast, 150 ovarian cancer cases, and 210 healthy controls (age and sex-matched). Variant rs10937405 of the TP63 gene was determined by the TaqMan assay. Hardy-Weinberg equilibrium for the variant was assessed using the Chi-square test. The allele and genotype-specific risks were estimated by odds ratios (ORs) with 95% confidence intervals (CI). Results: In this study, variant rs10937405 of TP63 gene did not show any risk with ovarian and breast cancer with (P-value = 0.70) having OR 0.94, (0.69-1.28 at 95% CI) and (P-value = 0.16) having OR 0.80, (0.59-1.10). Discussion: Our results indicate that the variant rs10937405 of the TP63 gene did not impart any risk of breast and ovarian cancer in the population of J&K. Our results indicate that a larger sample size is needed for further statistical validation. As the study was for a particular variant, it warrants the analysis of other variants of this gene.

Polymorphism in the TP63 gene imparts a potential risk for leukemia in the North Indian population.

BACKGROUND: The role of single nucleotide polymorphism rs10937405 (C>T) of the TP63 gene in cancer including leukemia has previously been studied in different world populations; however, the role of this variant in leukemia in the North Indian population of Jammu and Kashmir is still unknown. OBJECTIVES: In the present study, we investigated the association of genetic variant rs10937405 with leukemic in the Jammu and Kashmir population. METHODS: A total of 588 subjects, (188 cases and 400 controls) were recruited for the study. The rs10937405 variant was genotyped by using the real-time based TaqMan assay. RESULTS: A statistically significant association was observed between the rs10937405 and leukemia [OR of 1.94 (95% CI 1.51-2.48), p=1.2x10-6]. CONCLUSION: The current study concludes that the rs10937405 variant is a risk factor for the development of leukemia in the population of Jammu and Kashmir, North India. However, it would be interesting to explore the contribution of this variant in other cancers as well. Our findings will help in the development of diagnostic markers for leukemia in the studied population and potentially for other North Indian populations.

Genetic evaluation of the variants using MassARRAY in non-small cell lung cancer among North Indians.

Lung cancer is genetically diverse and a major health burden. Non-small cell lung cancer (NSCLC) accounts for 80% of total lung cancer cases and 20% cases are Small cell lung cancer (SCLC). The present case-control association study focused on the cost effective high throughput genotyping using Agena MassARRAY matrix-assisted laser desorption/ionization-time of flight, mass spectrometry (MALDI-TOF) platform to analyze the genetic association of candidate genetic variants. We performed multiplex PCR and genotyped twelve single nucleotide polymorphisms (SNPs) in 723 samples (162 NSCLC cases and 592 healthy controls). These genetic variants were selected from literature for their association with various cancers worldwide and this is the first study from the region to examine these critically important genetic variants. With prospective case-control association study design, twelve variants from ten genes were evaluated. Amongst these six variants, TCF21 (rs12190287), ERCC1 (rs2298881, 11615), ERCC5 (rs751402), ARNTL (rs4757151), BRIP1 (rs4986764) showed significant association with NSCLC risk (p ≤ 0.003) in Jammu and Kashmir population. In-silico findings of these genetic variants showed remarkable functional roles that needs in-vitro validations. It is further anticipated that such case control studies will help us in understanding the missing heritability of non-small cell lung cancer.

Genetic association of ARID5B with the risk of colorectal cancer within Jammu and Kashmir, India.

Colorectal cancer (CRC), which includes the development of cancer from the colon or rectum, is one of the highly prevalent cancers in the populations of Jammu and Kashmir (J&K) in India. However, case-control genetic association studies on CRC are lacking in this population. Various genome-wide association studies have previously shown that single-nucleotide polymorphisms (SNPs) of the AT-rich interaction domain 5B (ARID5B) gene located on chromosome 10q21.2 contribute substantially to the development of colorectal cancer. The association between ARID5B and CRC risk in north Indian population groups is still unknown. To understand the role of ARID5B SNPs in CRC in the population of J&K, we designed a case-control study to investigate the association of the cancer susceptibility variant rs10740055 of ARID5B with CRC in the population of J&K. The study included 180 cases and 390 healthy controls. Genotyping of the rs10740055 variant was performed by RT-PCR using the TaqMan assay technique. Hardy-Weinberg equilibrium of the variant was assessed using the chi-squared test. The allele- and genotype-specific risks were estimated by odds ratios (ORs) with 95% confidence intervals (CIs). The rs10740055 variant showed a higher risk for colorectal cancer with an OR of 3.35 (1.99-5.65 at 95% CI) and P = 0.000005 corrected for age, gender, ethnicity, BMI, alcohol intake and smoking. Our results indicate that the A allele of rs10740055 imparts risk to the population and also that a larger sample size is needed for further statistical validation. The association of other variants in other ARID family genes should also be tested as their role cannot be ruled out.

Evaluation of newly identified Ikaros family zinc finger 1 loci in colorectal cancer.

AIM: In this study, we evaluated the association of rs6964823 of the Ikaros Family Zinc Finger 1 (IKZF1) gene with the risk of colorectal cancer (CRC) within the population of Jammu and Kashmir (J and K). MATERIALS AND METHODS: The variant rs6964823 of the IKZF1 gene was genotyped using the TaqMan allele discrimination assay for 578 individuals (182 CRC cases and 396 healthy controls). The association of single-nucleotide polymorphisms with the disease was evaluated using logistic regression. RESULTS: It was observed that the variant rs6964823 (IKZF1) showed a significant association with an adjusted allelic odds ratio (OR) of 1.74 (1.34-2.27) at 95% confidence interval (CI), P ≤ 0.05. The dominant model (AA + AG vs. GG) was also applied, where the adjusted OR was 3.096 (2.011-4.76) at 95% CI, P > 0.05. CONCLUSIONS: It was found that the variant rs6964823 of the IKZF1 gene is associated with a higher risk of CRC within the population of J and K.

Genetic polymorphism of hOGG1 ser326cys and its association with breast cancer in Jammu and Kashmir.

BACKGROUND: 8-Oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) is a potent DNA damage marker that leads to cellular oxidative stress. It is a DNA-repair enzyme that participates in "8-oxodG" DNA adducts removal. Previous studies show weak associations of rs1052133 (hOGG1) in breast cancer patients of Northern India. We performed this study to explore the variant rs1052133 (hOGG1) with breast in the population of Jammu and Kashmir (J and K). METHOD: A polymerase chain reaction-restriction fragment length polymorphism -based single-nucleotide polymorphism (SNP) genotypic study was carried out in peripheral blood samples of 165 breast cancer patients and 200 healthy controls, using specific primers. Sanger sequencing verified the results. RESULTS: hOGG1-Ser326Cys polymorphism occurred frequently in cases as compared with controls. Data were evaluated by SPSS V.13 software, following Hardy-Weinberg equilibrium (P = 0.002 at OR 2.57; 95% CI [1.68-3.93]), which showed that the SNP rs1052133 had a significant association with increased risk of breast cancer. CONCLUSION: Overall, the results of this analysis show that the hOGG1-Ser326Cys polymorphism may be associated with an increased risk for breast cancer in the J and K population.

MassArray analysis of genomic susceptibility variants in ovarian cancer.

Ovarian cancer (OC), a multifaceted and genetically heterogeneous malignancy is one of the most common cancers among women. The aim of the study is to unravel the genetic factors associated with OC and the extent of genetic heterogeneity in the populations of Jammu and Kashmir (J&K).Using the high throughput Agena MassARRAY platform, present case control study was designed which comprises 200 histopathological confirmed OC patients and 400 age and ethnicity matched healthy controls to ascertain the association of previously reported eleven single nucleotide polymorphisms (SNPs) spread over ten genes (DNMT3A, PIK3CA, FGFR2, GSTP1, ERCC5, AKT1, CASC16, CYP19A1, BCL2 and ERCC1) within the OC population of Jammu and Kashmir, India. The association of each variant was estimated using logistic regression analyses. Out of the 11 SNPs the odds ratio observed for three SNPs; rs2699887 was (1.72 at 95% CI: 1.19-2.48, p = 0.004), rs1695 was (1.87 at 95% CI: 1.28-2.71, p = 0.001), and rs2298881 was (0.66 at 95% CI: 0.46-0.96, p = 0.03) were found significantly associated with the OC after correction with confounding factors i.e. age & BMI. Furthermore, the estimation of interactive analyses was performed and odds ratio observed was 2.44 (1.72-3.47), p value < 0. 001 suggests that there was a strong existence of interplay between the selected genetic variants in OC, which demonstrate that interactive analysis highlights the role of gene-gene interaction that provides an insight among multiple little effects of various polymorphisms in OC.

ANKLE1 as New Hotspot Mutation for Breast Cancer in Indian Population and Has a Role in DNA Damage and Repair in Mammalian Cells.

Breast cancer has replaced cervical cancer as being the most common and having the highest mortality among women in India. ANKLE gene is conserved among organisms during evolutionary succession and is a member of LEM family proteins in lower metazoans and is involved in critical functions in the nuclear architecture, gene expression and cell signaling. ANKLE1 is the human orthologous of LEM-3 and is involved in DNA damage response and DNA repair. Whole Exome Sequencing (WES) of paired breast cancer samples was performed and ANKLE1 was found to be a new possible hotspot for predisposition of breast cancer. The mass array genotyping for breast cancer variant rs2363956 further confirmed the ANKLE1 association with the studied population of breast cancer. To elucidate the role of ANKLE1 in DNA damage, it was knocked down in MCF-7 breast cancer cell line and the expression of γH2AX was assessed. ANKLE1 knockdown cells displayed elevated levels of γ-H2AX foci in response to the cisplatin induced replication stress. The localization pattern of ANKLE1 further emphasized the role of ANKLE1 in DNA repair process. We observed that ANKLE1 is required for maintaining genomic stability and plays a role in DNA damage and repair process. These findings provided a molecular basis for the suspected role of ANKLE1 in human breast cancer and suggested an important role of this gene in controlling breast cancer development among women in India.

Genetic variants of DNAH11 and LRFN2 genes and their association with ovarian and breast cancer.

OBJECTIVE: To investigate the association of newly identified genetic variants G>A (rs2285947) of the DNAH11 gene and G>A (rs2494938) of the LRFN2 gene with ovarian and breast cancers in women belonging to Jammu and Kashmir state, where the prevalence of ovarian and breast cancers is remarkably high in the population. METHODS: A candidate gene prospective case-control association study design was adopted, in which 354 cases (219 cases of ovarian cancer and 135 cases of breast cancer) were histopathologically confirmed and 330 healthy controls matched for age and ethnicity were recruited. The details of cases and controls were also recorded in a predesigned pro forma after their written informed consent. Both variants were genotyped by TaqMan allele discrimination assay using real-time polymerase chain reaction. Logistic regression analysis was performed to estimate the corrected odds ratio (OR), confidence interval (CI), and level of significance (P value) for potential confounding factors. RESULTS: The rs2285947 variant of DNAH11 was found to be significantly associated with both ovarian and breast cancers with adjusted ORs of 1.7 (95% CI 1.2-2.4; P=0.004) and 1.70 (95% CI 1.13-2.54; P=0.0009), respectively. However, no significant association of variant rs2494938 of LRFN2 was observed with ovarian cancer (estimated OR 0.9, 95% CI 0.6-1.4; P=0.919) or breast cancer (estimated OR 1.27, 95% CI 0.8-1.9; P=0.216). CONCLUSIONS: The collected data proposed that the variant rs2285947 of DNAH11 gene is a potential risk factor for ovarian and breast cancers in the studied population.

Association of ARID5B and IKZF1 Variants with Leukemia from Northern India.

BACKGROUND: Leukemia is a heterogeneous disorder, characterized by elevated proliferation of white blood cells. Various genetic studies have assessed the contributory roles of several single nucleotide polymorphisms with the development of leukemia. The role of genetic variation in the ARID5B and IKZF1 genes has previously been identified in various population groups; however, the role of these variants in the north Indian populations of Jammu and Kashmir is unknown. AIM: In this study, we explored the association of the newly identified genetic variants, rs10740055 of ARID5B and rs6964823 of IKZF1, with leukemic patients from Jammu and Kashmir of northern India. METHODS: The variants were genotyped using TaqMan allele discrimination assays for 616 individuals (210 leukemic cases and 406 healthy controls). The association of each SNP with the disease was evaluated using logistic regression. RESULTS: It was observed that the variants rs6964823 (IKZF1) and rs10740055 (ARID5B) showed significant associations with odds ratio (OR) and p-values of 1.5 (1.0-2.3 at 95% confidence interval [CI]) and 0.04; and 2.5 (1.5-4.1 at 95% CI) and 0.0002, respectively. We also evaluated the cumulative effect for both the variants by combining the risk genotypes and obtained and OR of 4.9. DISCUSSION: It was found that the variants rs10740055 of ARID5B and rs6964823 of IKZF1 act individually and additively as risk factors in the development of leukemia in the populations of Jammu and Kashmir in Northern India.

Genetic variant rs10937405 of TP63 and susceptibility to lung cancer risk in north Indian population.

Several studies including genomewide association studies (GWASs) in diverse ethnic populations have reported a significant association of genetic variant rs10937405 of TP63 with nonsmall cell lung cancer (NSCLC). However, no data are available from any Indian population on the association of this variant with NSCLC. Using TaqMan genotyping chemistry, we conducted a case-control study involving 190 NSCLC cases and 400 ethnic, age-matched controls to explore the association of rs10937405 genetic variant with NSCLC in patients from north India. Our data support that the rs10937405 variant is also significantly associated with the NSCLC and is a risk factor in the north Indian populations to develop NSCLC. However, unlike most other studies, the wild-type allele T appears to be the risk allele, as its frequency was significantly higher in the cases than controls (0.439 in cases versus 0.383 in controls. OR=1.95 (1.23-3.09 at 95% CI); P value (adjusted)= 0.004). Genetic association was also observed by applying different genetic models. The present study provides important information of the genetic aetiology of NSCLC and strengthens GWAS findings, highlighting the role of TP63 in lung cancer risk.