Novel Association between STAT3 Gene Variant and Vitiligo: A Case-Control Study.

Background: Vitiligo is an autoimmune disorder involving inflammatory damage to melanocytes. STAT3 genetic variant (rs744166 T > C) increases inflammatory signaling via JAK/STAT pathway. Aim: The purpose of this study was to check whether this translates into an association between vitiligo and STAT3 gene variant (rs744166 T > C). Materials and Methods: This is a case-control study. A total of 56 vitiligo patients and 90 healthy, age and gender-matched volunteers were recruited for the study. The STAT3 gene variant (rs744166 T > C) was genotyped using the restriction fragment length polymorphism method. Results: The frequency of the minor allele 'C' was higher in vitiligo patients (72.3%) than in healthy volunteers (57.8%). The difference between the two groups was statistically significant (P = 0.006; OR = 1.9 with 95% CI). The genotypic variant showed the highest association with vitiligo in the dominant model (P = 0.001). Conclusion: This study shows that the STAT3 gene variant (rs744166 T > C) is associated with vitiligo. This observation underlines the importance of the JAK/STAT signaling pathway in vitiligo pathogenesis.

Oxidative nucleic acid damage as a biomarker for preeclampsia.

Recent studies have reported that nucleic acid oxidation is elevated in preeclampsia. Furthermore, it is known that products of nucleic acid oxidation are eventually removed through the urine. The aim of this study was to check whether urine can be used as a non-invasive specimen to analyse nucleic acid oxidation in preeclampsia. We carried out a case-control study by enrolling preeclamptic pregnant (n = 31) and normotensive pregnant (n = 31) women. Urine samples from the study participants were collected and the levels of oxidised guanine species (a common product of nucleic acid oxidation) were determined by ELISA method. The urinary levels of oxidised guanine species were significantly higher in the preeclamptic pregnant group compared to the normotensive pregnant group (p = 0.001). The results were also analysed after stratifying the preeclamptic group in terms of severity and gestational age of onset. A significant difference was observed in both mild (p = 0.005) and severe preeclampsia (p = 0.01). However, a significant difference was observed only in the late onset (p = 0.001) and not in the early onset preeclampsia (p = 0.56). The results of this study show that oxidised guanine level is elevated in the urine of preeclamptic pregnant women. IMPACT STATEMENTWhat is already known on this subject? Nucleic acid oxidation is elevated in preeclampsia. However, the utility of urine (a non-invasive specimen) to analyse nucleic acid oxidation is not known.What do the results of this study add? This study shows that the levels of oxidised guanine (a marker of nucleic acid oxidation) are elevated in the urine of preeclamptic women.What are the implications of these findings for clinical practice and/or further research? Urinary levels of oxidised guanine may be developed as a non-invasive biomarker for preeclampsia.

Effect of Vitamin K Epoxide Reductase Complex 1 Polymorphism on Warfarin Dose Requirement among Patients in Tertiary Care Hospital.

BACKGROUND: Warfarin, anticoagulant is used for thromboembolic disorders. Inter-individual variation in clinical response to warfarin is due to various factors, including polymorphism of Vitamin K epoxide reductase complex 1 (VKORC1)-1639G>A. The aim of our study was to evaluate the effect of VKORC1 polymorphism on the maintenance dose of warfarin. MATERIALS AND METHODS: Cross-sectional study conducted by the departments of Pharmacology, Cell Biology and Molecular Genetics on patients attending cardiology clinic, receiving warfarin for at least 2 months. Genomic deoxyribonucleic acid was extracted and genotyping was done by Polymerase Chain Reaction - Restriction Fragment Length Polymorphism. The correlation between VKORC1 gene polymorphism and warfarin maintenance dose was analyzed. RESULTS: A total of 102 patients with a mean age of 47.72 ± 10.31 years, of which 58 (56.86%) were male. The frequency of VKORC1 G>A for GG, GA, and AA genotypes was 74.51%, 19.61%, and 5.88%, respectively. Variant allele AA was less frequent than the wild type. Mean weekly warfarin dose was 23.12 ± 8.08, 22.93 ± 8.21, and 15.6 ± 5.35 mg in patients with GG, GA, and AA genotypes, respectively. Patients with GG genotype required therapeutic dose compared to variant type (P = 0.001). Multiple stepwise regression model showed 26.3% variability in warfarin dose was due to VKORC1 genotype (R = 0.513, R2 = 0.263, adjusted R2 = 0.256, P = 0.0001). CONCLUSION: VKORC1 polymorphism alone influence 26.3% variability in warfarin dose and AA genotype patients required lower dose.

Hypoxia responsiveness linked variant in EGLN1 gene is enriched in oral cancer patients.

OBJECTIVE: The aim of this study was to determine the association of EGLN1 gene variant SNP rs479200 (T > C) with the risk of oral cancer. MATERIALS AND METHODS: A case-control study was conducted by involving 103 oral cancer patients and 206 age and gender-matched healthy controls. SNP rs479200 was genotyped by polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: Minor allele frequency was 47 % in oral cancer patients and 35 % in healthy individual (P = 3.0 × 10-3, Odds ratio = 1.61). The association was highest under the additive genetic model (0.0005). CONCLUSIONS: Our results show that the EGLN1 gene variant SNP rs479200 is associated with the risk of developing oral cancer. This relationship highlights the significance of oxygen sensing in the pathophysiology of oral cancer.

Evaluation of HCP5 and Chemokine C Receptor type 5 Gene Polymorphisms in Indian Psoriatic Patients.

BACKGROUND: Genetic variations associated with nonprogression of HIV infection to AIDS are enriched in psoriasis patients. HCP5 gene 335 T > G and chemokine C receptor type 5 (CCR5) gene Δ32 polymorphisms are associated with HIV nonprogression phenotype. AIM: The aim of this study was to determine the association of HCP5 gene 335 T > G (rs2395029) and CCR5 gene Δ32 (rs333) polymorphisms with psoriasis vulgaris (PV). MATERIALS AND METHODS: Genotype of HCP5 gene 335 T > G and CCR5 gene Δ32 polymorphisms were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific PCR methods, respectively. RESULTS: The frequency of HCP5 gene 335 T > G SNP was ~7 times higher in PV patients than in the control group (P = 1.49 × 10-8; odds ratio [OR] = 10.2; 0.95 confidence interval [CI]: 3.9-26.8). OR for the occurrence of HCP5 335 G allele in either homozygous or heterozygous genotype in PV patients was 13.1 (0.95 CI: 4.7-36.1). The strength of association was higher with moderate-to-severe subgroup (P = 3.29 × 10-9; OR = 18.4; 0.95 CI: 6.2-54.9) than with mild subgroup (P = 2.1 × 10-4; OR = 8.3; 0.95 CI: 2.6-23.3). In addition, the strength of association was higher with Type I (P = 9.53 × 10-8; OR = 15.3; 0.95 CI: 5.1-46.5) than with Type II subgroup (P = 6.8 × 10-6; OR = 11.0; 0.95 CI: 3.6-33.9). Type I gene Δ32 polymorphism was observed neither among psoriatic nor among healthy individuals. CONCLUSIONS: Our results indicate that HCP5 gene 335 T > G polymorphism and not CCR5 gene Δ32 polymorphism is associated with the increased risk of developing PV.

Novel association of SNP rs479200 in EGLN1 gene with predisposition to preeclampsia.

OBJECTIVES: Placental hypoxia is a hallmark of preeclampsia. SNP rs479200 in the EGLN1 gene is associated with reduced responsiveness to hypoxia. Whether this translates into an association between SNP rs479200 and preeclampsia is not known. We evaluated the association of SNP rs479200 (T>C) with the risk of preeclampsia. METHODS: This case-control study involved 600 pregnant women of whom 300 were preeclamptic and 300 were normotensive. SNP rs479200 was genotyped by PCR-RFLP method. RESULT: Minor allele frequency was 44% in preeclamptic women and 53% in normotensive pregnant women (P = 1.8 × 10-3; odds ratio = 1.43). The odds ratio was heterogeneous when compared after categorization of the preeclamptic group into clinical sub-groups. The association was significant with both mild (P = 6.2 × 10-5) and severe (3.8 × 10-3) preeclampsia. However, the odds ratio was 0.52 for mild preeclampsia and 1.43 for severe preeclampsia. CONCLUSION: The minor allele of SNP rs479200 is associated with the predisposition to preeclampsia. This association underlines the importance of oxygen sensing in the pathogenesis of preeclampsia.

Urinary protein carbonyl levels and its correlation with protein misfolding in preeclampsia.

OBJECTIVE: To evaluate the association of protein carbonylation with preeclampsia and its correlation with urinary protein misfolding. METHOD: Protein carbonyl and misfolded protein levels were measured in the midstream urine sample (58 preeclamptic and 44 normotensive pregnancy) by ELISA and Congo Red Dot assay respectively. RESULTS: Significant difference was observed in the levels of protein carbonyls (P = 0.002) and misfolded proteins (P = 0.001). Correlation between protein carbonyl and misfolded proteins levels was significant but weak (r = 0.3; P = 0.018). CONCLUSION: Urinary protein carbonyl level is elevated in preeclampsia but plays a minor role in proteins misfolding.

Novel association of oral squamous cell carcinoma with GSTP1 Arg187Trp gene polymorphism.

AIMS: Glutathione S-transferase subtype pi 1 (GSTP1) is an enzyme that is involved in the detoxification of carcinogenic substances. Arg187Trp is a functional polymorphism in the corresponding GSTP1 gene that reduces the enzymatic activity by 45%. We evaluated, for the first time, the association of Arg187Trp with the risk of oral squamous cell carcinoma and compared it with other established GSTP1 polymorphisms viz, Ile105Val and Ala114Val. MATERIALS AND METHODS: We carried out a 1:2 case-control study by recruiting 100 patients with oral squamous cell carcinoma and 200 age and gender-matched healthy individuals. Ile105Val, Ala114Val, and Arg187Trp polymorphisms were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and their distribution in the study groups was compared by chi-squared test (Fisher's exact). RESULTS: The minor allele of Ala114Val and Arg187Trp were more common in patients than in controls. In contrast, the distribution of Ile105Val minor allele was similar in the two groups. The differential distribution was also significant at the level of genotypes. CONCLUSIONS: These results indicate that GSTP1 Arg187Trp is associated with the risk of developing oral squamous cell carcinoma. Our study underlines the importance of detoxification pathway in the risk of carcinogenesis.

Atrial fibrillation associated genetic variation near PITX2 gene increases the risk of preeclampsia.

OBJECTIVES: SNP rs2200733 located near PITX2 gene is associated with the risk of atrial fibrillation. Preeclamptic women are at increased risk of developing cardiovascular disease like atrial fibrillation. Whether this translates into an association between SNP rs2200733 and preeclampsia is not known. Therefore, we determined the association of SNP rs2200733 (C/T) with the risk of preeclampsia. STUDY DESIGN: A hospital based prospective case-control study involving 585 pregnant women of whom 285 were preeclamptic and 300 were normotensive. SNP rs2200733 was genotyped by PCR-RFLP method. MAIN OUTCOME MEASURES: Statistical significance of the difference in the minor allele frequency between case and control groups was determined by Fisher's exact test. RESULTS: Minor allele frequency was 21.4% among preeclamptic pregnant women and 13.7% among normotensive pregnant women (P = 0.00064; odds ratio = 1.72 (0.95 CI: 1.23-2.41). The measures of association were heterogeneous when compared after categorisation of the preeclamptic group into clinical sub-groups. The association was not significant with the eclampsia sub-group (P = 0.39) but relatively higher with the sub-group not superimposed by eclampsia (P = 0.0000048; odds ratio = 2.10 [0.95CI: 1.50-2.92]). Furthermore, the association was relatively higher with the sub-group involving intrauterine growth retardation and intrauterine death (P = 0.00017; odds ratio = 2.89 (0.95CI: 1.65-4.94)]. CONCLUSIONS: Minor allele of SNP rs2200733 is associated with the risk of preeclampsia. SNP rs220073 may represent a common risk factor that predispose women to develop both preeclampsia during pregnancy and cardiovascular disease later on.

Urinary congophilia in preeclampsia: Experience from a rural tertiary-care hospital in India.

OBJECTIVES: To evaluate the presence of urinary congophilia amongIndian patients with preeclampsia. STUDY DESIGN: A prospective case control study in which congophilia of urine samples from preeclamptic pregnant women (n = 62) and normotensive pregnant women (n = 65) was compared by using Congo Red Dot Blot assay. MAIN OUTCOME MEASURES: Presence of urinary congophilia. RESULTS: Mean percentage of Congo Red Retention was 37.9 ±â€¯4.1 in the normotensive pregnant group and 77.9 ±â€¯11.5 in the preeclamptic pregnant group (P < .001). The mean percentage of Congo Red Retention in both early-onset (70.5 ±â€¯9.0) and late-onset (82.7 ±â€¯10.3) groups were significantly higher than in normotensive controls (P < .001). The mean percentage of Congo Red Retention in mild (61.2 ±â€¯3.2) and severe (82.4 ±â€¯8.4) types of preeclampsia were also as significantly higher than in normotensive controls (P < .001). The mean percentage of Congo Red Retention in preeclampsia superimposed by eclampsia (89.4 ±â€¯2.0) and preeclampsia complicated by intrauterine growth restriction and intrauterine death (74.6 ±â€¯5.8) were significantly higher than in normotensive controls (P < .001). CONCLUSIONS: The results of this study confirms the presence of urinary congophilia in Indian pregnant women with preeclampsia. Furthermore, our study shows that urinary congophilia is not affected by clinical variables like gestational age of onset, severity, superimposition by eclampsia and complication by intrauterine growth restriction and intrauterine death. Urinary congophilia can be used to differentially identify preeclamptic pregnant women from normotensive pregnant women.

GSTP1 c.341C>T gene polymorphism increases the risk of oral squamous cell carcinoma.

Glutathione S Transferases (GST) are anti-oxidant enzymes involved in detoxification of cellular and exogenous carcinogens and oxidative products of reactive oxygen species. Genetic polymorphisms can attenuate the detoxification capacity of GST and consequently increase the susceptibility to carcinogenesis. There are eight classes of GST enzymes of which pi subtype is the predominant form expressed in the oral mucosa. c.341C > T single nucleotide polymorphism (rs1138272) in GSTP1 gene, is a functional variation that reduces the enzymatic activity of GST pi. We carried out a 1:2 case-control study involving 270 individuals to determine the association of c.341C > T variation with the risk of oral squamous cell carcinoma. GSTP1 c.341C > T variation was genotyped by PCR-RFLP method. GST pi expression in the tumour sample was determined by immunohistochemistry. Tobacco consumption was the major risk factor among cancer patients. The odds ratio for the risk of oral squamous cell carcinoma in individuals with the minor allele was 4.5 (0.95 CI = 2.3-8.9; P = 0.000004). The genotype was found to follow dominant mode of inheritance (OR 4.4 [0.95 CI = 2.1-9.2]; P = 0.00006). Our results support the conclusion that c.341C > T variation in GSTP1 increases the risk of OSCC in patients habituated to tobacco consumption.

Nicotinic acetylcholine receptor gene polymorphism is not associated with tobacco-related oral squamous cell carcinoma.

BACKGROUND: Nicotinic acetylcholine receptor is implicated in carcinogenesis indirectly through increasing nicotine dependence and directly through its impact on cell-cycle regulation. Functional polymorphism in nicotinic acetylcholine receptor alpha-5 subunit gene (CHRNA5 c.1192G>A; rs16969968) is associated with nicotine dependence and risk of lung cancer. AIM: The aim of this study was to evaluate the association of CHRNA5 c.1192G>A polymorphism with the risk of oral squamous cell carcinoma (OSCC). SETTINGS AND DESIGN: This was a rural teaching hospital-based case-control study. MATERIALS AND METHODS: A total of 100 histopathologically confirmed cases of OSCC patients and 100 age- and gender-matched healthy individuals were genotyped for CHRNA5 c.1192G>A polymorphism by polymerase chain reaction-restriction fragment length polymorphism method. Allele and genotype frequencies among case and control groups were compared by Chi-squared test (Fisher's exact). RESULTS: The frequency of CHRNA5 c.1192A allele was 22% in OSCC patients and 26% in control individuals. The difference in the distribution of alleles and genotypes between case and control groups was not significant (P > 0.05). CONCLUSIONS: CHRNA5 c.1192G>A polymorphism is not associated with the risk of developing OSCC.

Profile of Tumour Necrosis Factor Alpha -308 G/A Gene Polymorphism in Psoriatic Patients in Karnataka, India.

INTRODUCTION: Tumour necrosis factor-alpha (TNFα) gene -308G/A polymorphism (rs1800629) are associated with psoriasis in several populations worldwide. Presently, there is no literature on the status of this polymorphism in the South Indian population. AIM: To determine the profile of TNFα -308G/A polymorphism among psoriatic patients. MATERIALS AND METHODS: This case-control study involved 74 patients with Psoriasis Vulgaris (PsV) and 74 age and gender matched healthy individuals. Patients were recruited from the Department of Dermatology of R.L. Jalappa Hospital and Research Center, Tamaka, Kolar, Karnataka, India, from March 2014 to March 2016. TNFα -308G/A polymorphism was genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. RESULTS: The frequency of TNFα -308A allele 7.4% among psoriatic and 8.8% among non-psoriatic individuals. The difference was not statistically significant (p=0.82). CONCLUSION: Our results indicate that TNFα gene -308G/A polymorphism is not a significant marker for the risk of developing PsV among South Indian (Karnataka) psoriatic patients.

Tumor necrosis factor alpha gene promoter -238G/A polymorphism increases the risk of psoriasis vulgaris in Indian patients.

BACKGROUND: Tumor necrosis factor alpha (TNFα) gene -238G/A polymorphism (rs361525) is associated with psoriasis in several populations worldwide. To the best of our knowledge, there is no information about this polymorphism in Indian psoriatic patients. This study was undertaken to fill the gap in knowledge. METHODS: This case-control study involved 72 patients with psoriasis vulgaris (PsV) and 72 age and gender matched healthy individuals. TNFα -238G/A polymorphism was genotyped by PCR-RFLP method. RESULTS: TNFα -238A allele was 5 times commoner in PsV patients than in the control group (P = 4.1 × 10-7 ; odds ratio [OR] = 6.5 [0.95 CI: 2.9-14.6]). Distribution of the genotypes in the two groups showed statistically significant difference in dominant genetic model (P = 2.3 × 10-7 ) and not in recessive genetic model (P = 2.5 × 10-1 ). Odds ratio for the occurrence of -238A genotype in PsV patients was 8.8 (0.95 CI: 3.5-20.2). The association showed no major difference when PsV patients were subgrouped into type I and type II categories and tested separately. Subgroup analysis on the basis of disease severity showed higher association with the moderate-severe subgroup (P = 2.4 × 10-9 , OR 15.4 [0.95 CI: 5.8-41.0]) than with mild subgroup (P = 1.3 × 10-2 , OR 3.8 [0.95 CI: 1.3-10.9]). CONCLUSIONS: Our results indicate that TNFα gene -238G/A polymorphism increases the risk of developing psoriasis vulgaris among Indians. Also, the data show that severity and not the type affects the strength of association in this population.

Prevalence of human papillomavirus in oral squamous cell carcinoma: A rural teaching hospital-based cross-sectional study.

BACKGROUND: Human papillomavirus (HPV) is a well-established oncogenic agent in the pathogenesis of cervical carcinoma. Its role in the oncogenesis of tumors such as oral squamous cell carcinoma (OSCC) is not clear. Globally, approximately 3% of OSCCs are positive for HPV. Studies conducted in India indicate its prevalence from as low as 0% to as high as 74%. However, a recent Indian study on leukoplakia failed to find any evidence of HPV involvement. This motivated us to reexamine the HPV status in OSCC. AIM: To evaluate the prevalence of HPV in OSCC. SETTINGS AND DESIGN: This was a rural teaching hospital-based cross-sectional study. SUBJECTS AND METHODS: Sixty histopathologically confirmed samples of OSCC were used for the study. Genomic DNA was isolated from frozen, surgically-resected specimens. HPV positivity was tested by polymerase chain reaction-based method using GP5+/6+ primers in the L1 consensus region of the viral genome. RESULTS: None of the samples were HPV positive. CONCLUSIONS: Results of this study indicate that the association between HPV and OSCC may be overestimated. Hence, multicentric studies covering diverse geographical and socioeconomic groups are needed to delineate the profile of HPV infectivity and OSCC in the Indian subcontinent.

Sphingosine, a modulator of human translesion DNA polymerase activity.

Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼ 3000 small molecules, including one comprising ∼ 600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼ 100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress.

Effect of pH on the hydrolytic kinetics of gamma-glutamyl transferase from Bacillus subtilis.

The effect of pH on the steady state kinetics of gamma-glutamyl transferase (GGT) from Bacillus subtilis was examined using glutamyl-(3-carboxyl)-4-nitroanilide as the chromogenic reporter substrate. The enzyme was active in the pH range 7.0-11.0 with the optimum activity at pH 11.0. We noticed a pH dependent transformation in the nature of substrate consumption kinetics. The substrate saturation curves were hyperbolic in the pH range 7.0-9.0 but changed into sigmoid form at pH 10.0 and 11.0. Hill's coefficients were >1. We also analysed the effect of pH on the structure of the enzyme. The circular dichroism spectra of the enzyme sample at pH 9.0 and 11.0 were coincidental in both far and near UV regions indicating conservation of the secondary and tertiary structures, respectively. The molecular weight of the enzyme sample was the same in both pH 7.0 and 11.0 indicating conservation of the quaternary structure. These results show that the kinetic transformation does not involve significant conformational changes. Cooperative binding of multiple substrate molecules may not be the basis for the sigmoid kinetics as only one substrate binding site has been noticed in the reported crystal structures of B. subtilis GGT.