Mutational Assessment in NKX2-5 and ACTC1 Genes in Patients with Congenital Cardiac Septal Defect (CCSD) from Ethnic Kashmiri Population.

(1) Background globe. The etiology of CHDs is complex and involves both genetic and non-genetic factors. Although, significant progress has been made in deciphering the genetic components involved in CHDs, recent reports have revealed that mutations in Nk2 homeobox5 (NKX2-5) and actin alpha cardiac muscle1 (ACTC1) genes play a key role in CHDs such as atrial and ventricular septum defects. Therefore, the present study evaluates the role of key hotspot mutations in NKX2-5 and ACTC1 genes of congenital cardiac septal defect (CCSD) in ethnic Kashmiri population. (2) Methods: A total of 112 confirmed CHD patients were included in the current study, of which 30 patients were evaluated for mutational analysis for hotspot mutations of NKX2-5 and ACTC1 genes. The total genomic DNA was extracted from the samples (cardiac tissue/blood) and were subjected to amplification for NKX2-5 (exon 1 and 2), and ACTC1 (exon 2) genes by using PCR specific primers to analyze the hotspot mutations in respective exons. The amplified products obtained were sent to Macrogen Korea for sequencing by Sanger's method. (3) Results: Our results confirmed that not a single mutation was found in either hotspot exon 1 and 2 of NKX2-5 and exon 2 of ACTC1 in the patients included in the current study. Interestingly, a novel synonymous nucleotide variation leading to G > C transversion (GCG > GCC) was found in exon 2 of NKX2-5 gene of CCSD patient. (4) Conclusions: The current findings demonstrated the role of NKX2-5 and ACTC1 in cardiac development. The study will provide an insight in understanding the genetic etiology and highlights the role of newly identified mutations in patients with CDS's in ethnic Kashmiri population. In silico findings revealed amino acid changes, splice site variation and the creation of new site. Furthermore, the study warrants complete screening of genes involved in CCSDs.

Analysis of dengue specific memory B cells, neutralizing antibodies and binding antibodies in healthy adults from India.

BACKGROUND: The Indian population is facing highest dengue burden worldwide supporting an urgent need for vaccines. For vaccine introduction, evaluation and interpretation it is important to gain a critical understanding of immune memory induced by natural exposure. However, immune memory to dengue remains poorly characterized in this region. METHODS: We enumerated levels of dengue specific memory B cells (MBC), neutralizing (NT) and binding antibodies in healthy adults (n=70) from New Delhi. RESULTS: NT-antibodies, binding antibodies and MBC were detectable in 86%, 86.56% and 81.63% of the subjects respectively. Among the neutralizing positive subjects, 58%, 27%, 5% and 10% neutralized all four, any three, any two and any one dengue serotypes respectively. The presence of the neutralizing antibodies was associated with the presence of the MBC and binding antibodies. However, a massive interindividual variation was observed in the levels of the neutralizing antibodies (range, <1:50-1:30,264), binding antibodies (range, 1:3,000-1:134,000,) as well as the MBC (range=0.006%-5.05%). CONCLUSION: These results indicate that a vast majority of the adults are immune to multiple dengue serotypes and show massive interindividual variation in neutralizing/binding antibodies and MBCs - emphasizing the importance of monitoring multiple parameters of immune memory in order to properly plan, evaluate and interpret dengue vaccines.

Role of the Functional Polymorphism of Survivin Gene (-31G/C) and Risk of Breast Cancer in a North Indian Population.

INTRODUCTION: Survivin is an apoptosis inhibitor and plays a primary role in cancer development and progression. One of the most common polymorphism of the survivin promoter -31G/C (rs9904341) influences its expression and is associated with the risk of cancer development. This study was conducted to explore survivin promoter gene -31G/C (rs9904341) polymorphism and the risk of breast cancer. PATIENTS AND METHODS: The study group included 190 pathologically confirmed breast cancer patients, in addition to 200 distinct cancer-free controls from Jammu and Kashmir region of India, where breast cancer is the most common cancer in women. Single nucleotide polymorphism genotyping for -31G/C polymorphism in the survivin promoter region was done using a polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: The variant genotype/allele was found in 54.1% of the cases compared with 46.5% of controls. The combined prevalence of genotype GC+CC was significantly higher in patients compared with the control group (P = .02). Analyses of odds ratios (ORs) in the patient and control groups indicated that the presence of homozygous CC genotype was associated with increased risk for development of breast cancer (OR, 2.04; 95% confidence interval [CI], 1.07-2.98). The gene frequencies for G and C alleles were statistically different between patient and control groups (OR, 1.37; 95% CI, 1.03-1.84). CONCLUSION: The results suggest the association of -31G/C survivin polymorphism at a genotypic and allelic level in breast cancer.

Hypermethylation Status of E-Cadherin Gene in Gastric Cancer Patients in a High Incidence Area.

Gastric cancer (GC) is the fourth most prevalant cancer and the second leading cause of cancer-related mortality worldwide. As in other cancers gastric carcinogenesis is multifactorial involving environmental, genetic and epigenetic components. Epigenetic silencing due to hypermethylation of tumour suppressor genes is one of the key events in gastric carcinogenesis. This study was aimed to analyse the hypermethylation status of the E-Cadherin (CDH1) gene promoter in GCs in the ethnic Kashmiri population. In this study a total of 80 GC patients were recruited. Hypermethylation in tumour tissue was detected by methylation specific PCR (MS-PCR). Hypermethylation of CDH1 promoter was observed in 52 (65%) of gastric carcinoma cases which was significantly much higher than adjacent normal tissue [p≤0.0001]. Further the frequency of CDH1 promoter methylation was significantly different with intestinal and diffuse types of gastric cancer [55.7% vs 82.1%; <0.05]. Moreover females and cases with lymph node invasion had higher frequencies of CDH1 hypermethylation [P≤0.05]. Thus the current data indicate a vital role of epigenetic alteration of CDH1 in the causation and development of gastric cancer, particularly of diffuse type, in our population.

ApoE gene polymorphism and its relationship with coronary artery disease in ethnic Kashmiri population.

Apolipoprotein E is a fundamental component of various lipoproteins and plays substantial role in cholesterol/lipid transport among cells of various tissues. The ApoE gene is polymorphic with three alleles ε2, ε3, and ε4, coding for isoforms E2, E3, and E4 having different binding inclination for corresponding receptors. This work aimed to investigate the association between ApoE gene polymorphism and coronary artery disease (CAD) in Kashmiri population. APOE genotyping was done by polymerase chain reaction-restriction fragment length polymorphism. Our study indicated ApoE ε3/ε3 to be the most common genotype in both CAD and control group. The frequency of ε2, ε3, and ε4 alleles of ApoE gene in cases was observed to be 0.06, 0.72, and 0.20, while in control subjects it was 0.075, 0.82, and 0.11, respectively. A significant difference was found between cases and controls with respect to TC, LDL, and HDL levels. Our data showed that frequency of ε4/ε4, ε4/ε3 genotype and ε4 allele was significantly higher in cases than in controls (p = 0.02, p = 0.004, p < 0.001 respectively). Moreover, the CAD patients carrying ε4 allele had significantly higher TC and LDL levels (p value <0.01). Thus our data showed a significant association of ApoE ε4 allele with the risk of CAD. The data revealed that ApoE ε4 allele is associated with increased risk of CAD and increased levels LDL and TC in Kashmiri population.

Genetic Variation of ApoE Gene in Ethnic Kashmiri Population and Its Association with Outcome After Traumatic Brain Injury.

The outcome from traumatic brain injury (TBI) is variable and only partly explained by known prognostic factors. Genetic factors may influence the brain's susceptibility to injury or capacity for repair and regeneration. ApoE has been implicated in modifying neurological outcome after TBI, although the mechanisms by which this occurs remain poorly defined. Apolipoprotein E is an apparently multifunctional protein involved in the response of the brain to injury and in subsequent repair processes. Several studies have shown that patients with APOE e4 have a poorer outcome after TBI. This study was aimed to analyse the genotypes of ApoE in Kashmiri population and to examine the association of APOE genotype with outcome after TBI. A total of 450 subjects (300 healthy controls and 150 TBI patients) were recruited for the study. Genotyping was done by PCR-restriction fragment length polymorphism (RFLP).Our study indicated Apoe3/e3 to be the most common genotype in this study group. The allele frequency of the Apo E gene in these study subjects was observed to be 0.07 for the e2 allele, 0.82 for the e3 allele and 0.11 for the e4 allele. However, no association between the presence of APOe4 allele and outcome after head injury was observed in this study [p = 0.92]. Thus, genotype containing the e4 allele (e4/e3 and e4/e4) was not associated with unfavourable outcome after TBI in Kashmiri population.

Serum leptin levels, leptin receptor gene (LEPR) polymorphism, and the risk of systemic lupus erythematosus in Kashmiri population.

The study is conducted to evaluate relationship between LEPRQ223R (Gln > Arg) polymorphism, serum leptin levels, soluble leptin receptor (SOb-R) levels and SLE risk in Kashmiri population.LEPR genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in 100 unrelated SLE patients and equal number of healthy control subjects. Leptin and SOb-R levels were measured by ELISA assays. The present study showed higher frequency of variant genotype (AG + GG) in cases compared to controls [OR = 2.52, CI = 1.18-5.35, p = 0.03]. Moreover the rare (G) allele was significantly more predominant in cases than controls [OR = 1.49, p = 0.04]. Interestingly a positive association between the variant genotype and the development of arthritis [OR = 11.8, CI = 1.6-85.1, p = 0.002] and an inverse association with cardiac disorder [OR = 0.09, CI = 0.02-0.46, p = 0.001] was observed in this study. Furthermore the study showed significant differences of leptin levels in SLE patients and controls (23.9 ± 19.5 vs 14.8 ± 10.4, p < 0.001). SLE patients in the highest quartile leptin levels (≥32.5 ng/mL) were significantly more likely to have higher BMI (p = 0.001) and increased risk of developing arthritis (p = 0.02). Furthermore positive association was observed between the variant genotype(AG + GG) and leptin levels (p = 0.001) in SLE patients. Thus, it is evident from our study that LEPRQ223R polymorphism and elevated leptin levels are associated with increased susceptibility of SLE in Kashmiri population.

MGMT gene silencing by promoter hypermethylation in gastric cancer in a high incidence area.

PURPOSE: Inactivation of tumor suppressor and DNA repair genes by promoter hypermethylation does commonly occur in human cancers. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes methyl groups as well as larger adducts at the O(6) position of guanine. In the absence of MGMT activity, O(6)-methylguanine mispairs with thymine during DNA replication, resulting in G:C to A:T transitions. Promoter hypermethylation of the MGMT gene has been observed in various cancers, including gastric cancer. Here, we aimed at assessing the promoter hypermethylation, mutation and expression status of the MGMT gene in patients from a geographic region with a high incidence of gastric cancer (Kashmir, North India) and to investigate their association with various clinicopathological characteristics. METHODS: In this study 82 gastric cancer samples and adjacent normal tissues were included. Mutations in the MGMT gene were detected by single stranded conformational polymorphism (SSCP) analysis and direct sequencing. Methylation-specific polymerase chain reaction (MS-PCR) and Western blot analyses were performed to detect promoter hypermethylation and concomitant (loss of) expression of the MGMT gene. RESULTS: Promoter hypermethylation of the MGMT gene was found in 52.44% (43 of 82) of the tumor samples and loss of MGMT protein expression was detected in 45.12% (37 of 82) of the tumor samples. Hypermethylation and loss of expression were significantly associated with higher tumor grades (moderately/poorly differentiated) (P < 0.05) and higher tumor stages (III/IV) (P < 0.05). In addition, MGMT hypermethylation and loss of expression were found to be significantly associated with high salt tea consumption (P < 0.05). CONCLUSIONS: Our results indicate that MGMT promoter hypermethylation and concomitant loss of MGMT protein expression may play an important role in the development of gastric cancer in the Kashmiri population. High salt tea consumption may be a risk factor.

Study of TLR4 and IL-8 gene polymorphisms in H.pylori-induced inflammation in gastric cancer in an ethnic Kashmiri population.

BACKGROUND: TLRs play an essential role in the initial handling of H. pylori and determine the clinical outcomes that range from simple asymptomatic gastritis to peptic ulcer disease and gastric cancer. Asp299Gly and Thr399Ile polymorphisms in TLR4 have been associated with a variety of inflammatory and infectious conditions including gastric cancer. The T-251A polymorphism in the promoter region of IL-8 gene has been found to be associated with changing the in vitro levels of IL-8 production. IL-8 exhibits angiogenic activity and is responsible for tumor-associated angiogenesis in several cancers. MATERIALS AND METHODS: 130 gastric cancer patients and 200 healthy controls were included in this study. DNA extraction was followed by PCR detection of H. pylori infection, PCR-RFLP for the TLR 4 polymorphism and PCR-CTPP for IL-8 gene polymorphism. RESULTS: The adjusted OR for gastric cancer risk was 1.15 (95% CI, 0.8357-1.3463); 1.39 (0.6964-2.781) and 1.43 (0.954-2.1515) for Asp299Gly, Thr399Ile and IL-8 T_251A respectively. Odds Ratio analysis showed CT genotype and AT and AA genotypes as risk factors for the development of gastric cancer. We found the Asp299Gly polymorphism carrier to be significantly associated (p value 0.03)with the development of tumours in the distal part of the stomach and Thr399Ile polymorphism to be significantly associated(p value 0.008) with the development of well-differentiated gastric adenocarcinoma.The IL-8 T-251A polymorphism was not found to be associated with any of the clinicopathological characteristics. DISCUSSION: No correlation was found between the appearance of disease and HP infection or the presence of TLR4 and IL-8 gene polymorphisms and HP infection.

The XRCC1 Arg399Gln gene polymorphism and risk of colorectal cancer: a study in Kashmir.

BACKGROUND: The DNA repair gene XRCC1 Arg399Gln gene polymorphism has been found to be implicated in the development of various cancers, including colorectal cancer (CRC), in different populations. We aimed to determine any association of this polymorphism with the risk of CRC in Kashmir. MATERIALS AND METHODS: A total of 120 confirmed cases of CRC and 146 healthy cancer free controls from the Kashmiri population were included in this study. Genotyping was carried out by the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: Genotype frequencies of XRCC1 Arg399Gln observed in controls were 34.2%, 42.5% and 23.3% for GG (Arg/Arg), GA (Arg/Gln), AA( Gln/Gln), respectively, and 28.3%, 66.7% and 5% in cases, with an odds ratio (OR)=5.7 and 95% confidence interval (CI) =2.3-14.1 (p=0.0001). No significant association of Arg399Gln SNP with any clinicopathological parameters of CRC was found. CONCLUSIONS: We found the protective role of 399Gln allele against risk to the development of CRC. The XRCC1 heterozygote status appears to be a strong risk factor for CRC development in the Kashmiri population.

Novelty of Axin 2 and lack of Axin 1 gene mutation in colorectal cancer: a study in Kashmiri population.

Colorectal cancer is (CRC) one of the leading causes of mortality and morbidity. Various genetic factors have been reported to be involved in the development of colorectal cancers including Axin gene. Axin, a major scaffold protein, plays an important role in various bio signaling pathways. We aim to study mutational pattern of Axin gene in colorectal cancer patients of Kashmiri population. The paired tumor and adjacent normal tissue specimens of 50 consecutive patients with CRC were used in our study. The DNA preparations were evaluated for the occurrence of Axin 1 and Axin 2 gene mutations by direct DNA sequencing. We analyzed exon 1a, 1b, 1c, 2, 4, 6, and 10 of Axin 1 and exon 7 of Axin 2. In this study, we found a novel mutation of G>T (GCT>TCT) transversion in exon 7 of Axin 2 gene at codon G695T (p.alanine > serine) at a frequency of 6% (3/50). In the same exon of Axin 2 gene a single nucleotide polymorphism (SNP) was detected in codon L688L (CCT>CTT) at a frequency of 36% (18/50). In exon 1c of Axin 1 a SNP was detected at codon D726D (GAT>GAC) at a frequency of 62.5% (31/50). Both the SNPs were synonymous hence do not lead to change of amino acid. Although Axin 1 and Axin 2 gene mutations have been found to be involved in the development of colorectal cancers, it seems to be a relatively rare event in Kashmiri population. However, an interesting finding of this study is the novelty of Axin 2 gene mutations which may be a predisposing factor in ethnic Kashmiri population to CRC.

Procalcitonin as the biomarker of inflammation in diagnosis of appendicitis in pediatric patients and prevention of unnecessary appendectomies.

Numerous diseases mimic appendicitis, and it is often difficult to rule it out on the basis of clinical presentation. Concentration of procalcitonin selectively increases in inflammatory conditions and determination of its level can help in the diagnosis of acute appendicitis. A prospective, single centre based observational study carried out at our tertiary care institute. Twenty eight patients were admitted with preliminary diagnosis of acute appendicitis. The control group involved around 12 healthy children. Serum Procalcitonin concentration was measured in all patients using the 'Immunoluminometric Method' (LUMI- Test PCT), besides carrying out clinical examination and other investigations. The serums PCT comes out to be a better diagnostic test than serum CRP measurement as serum PCT was able to differentiate patients who came with abdominal pain but were having normal appendix from the patients who were actual cases of acute appendicitis. In patients with histologically confirmed acute appendicitis the level of PCT was above the normal value of 0.5 ng/ml in most cases. The analysis of procalcitonin in different groups of patients showed the serum procalcitonin test having high sensitivity of 95.65% and a specificity of about 100% on the basis of histopathological diagnosis of the removed appendix taking as the standard. The serum procalcitonin test when combined with reliable clinical signs and symptoms is an excellent diagnostic marker of the disease and should be done in the patients of pediatric appendicitis so that proper handling of the patient can be done and we can prevent unnecessary appendectomies.

Role of TP53 Arg72Pro polymorphism in urinary bladder cancer predisposition and predictive impact of proline related genotype in advanced tumors in an ethnic Kashmiri population.

Among various polymorphic variants of TP53 gene, codon 72 polymorphism (Arg72Pro) has been found to be associated with cancer susceptibility, but only few studies have investigated their effect on bladder cancer risk. A case-control study was conducted and we observed the genotype distribution of TP53 Arg72Pro SNP, to elucidate the possible role of this SNP as risk factor in urinary bladder cancer (UBC) development and to examine its correlation with the clinicopathologic variables of UBC cases. Using the polymerase chain reaction-restriction fragment length polymorphism approach, we tested the genotype distribution of 108 bladder cancer patients in comparison with 138 cancer-free controls from the same geographical region. We observed significant differences between the control and bladder cancer patients with odds ratio = 2.9 and 95% confidence interval = 1.5-4.5 (P = 0.00001). Interestingly, the proline form was abundantly observed in advanced tumors (P < 0.05). We also found a significant association of the variant allele (GC+CC) with male subjects and ever smokers (P = 0.001). Thus, it is evident from our study that Arg72Pro SNP is implicated in bladder cancer, and that the rare, proline-related allele is connected with higher susceptibility to bladder cancer.

Lack of p16 gene mutations in gastric cancers in Kashmir.

BACKGROUND AND AIM: The focus of the study was to investigate the frequencies of homozygous deletions and mutations of p16 gene in gastric carcinomas in the Kashmiri population. METHODS: A total of 84 gastric carcinoma patients were screened by the single strand conformation polymorphism (SSCP) technique and later by DNA sequencing to detect mutations of the p16 gene. Also PCR was applied further to further detect any homozygous deletions. RESULTS: SSCP and DNA sequencing performed encompassing all the three exons of p16 gene could not detect any mutations in any ofl 84 cases. Though we could observe mobility shifts in SSCP of two samples, subsequent DNA sequencing did not show any mutation. Further PCR could not detect any homozygous deletion in P16 in any case. CONCLUSION: Though Kashmir is a high incidence area of gastric carcinomas, p16gene mutations /or deletions do not appear to be involved.

Activated H-ras gene mutations in transitional cell carcinoma of urinary bladder in a Kashmiri population.

AIMS AND BACKGROUND: The primary aim of the study was to evaluate the incidence of H-ras specific point mutations among a group of Kashmiri patients diagnosed with bladder cancer. We also explored the correlation of clinic-pathological status of the illness with these mutations. METHODS AND STUDY DESIGN: The DNA samples of both tumor and normal tissue were evaluated for the occurrence of H-ras activating mutations in exon 1 and 2 by PCR-SCCP and DNA sequencing. In addition, blood was also collected from all the cases to rule out any germ-line mutation. RESULTS: Point mutations of activated H-ras identified in bladder cancer patients were 14.5% (7 of 48), including four transversions (two G-->T and two A-->T) and three transitions (A-->G). Of the mutations, 71.4% were detected in codon 61 and 28.6% in codon 12. The pattern of mutation in the study showed a significant association with smoking in bladder tumors (P < 0.05). No correlation was found between tumor grade and/or stage and the presence of H-ras mutation. CONCLUSIONS: Activation of H-ras by mutation plays a less frequent role than other genetic events in the development of the most transitional cell tumors of the bladder in Kashmiri population.