Circulating levels of cytokines (IL-6, IL-10 and TGF- ß) and CD4+CD25+FOXP3+Treg cell population in recurrent pregnancy loss.

Recurrent pregnancy loss (RPL), a serious reproductive health issue, characterized by two or more pregnancy losses before 20th week of gestation. Globally, it affects 2-5% couples and the basis of the crisis is still unknown in 50% cases. Successful pregnancy is associated with pro and anti-inflammatory gestational phases that tolerate the semi-allogenic foetus, and disturbance leads to pregnancy complications like RPL. This case-control study aimed to assess the inflammatory status in the mid-gestation of ongoing pregnancy of women with (RPL) and without (NRPL) the history of RPL. Blood samples were processed for PBMC isolation, subjected to Flow-cytometry for CD4+CD25+FOXP3+Treg-cell population count and serum samples for IL-6, TGF-ß, IL-10 cytokine levels (ELISA). Significant reduction in the percentage of Treg cells, and elevated values for IL-6/TGF-ß and IL-6/IL-10 ratios were observed in RPL over NRPL group (p = 0.0001). Opposing results were seen with respect to the magnitude of history of RPL (2 vs. >2 losses). ROC curve analysis showed the superior discriminatory ability of cytokine ratios (IL-6/TGF-ß > IL-6/IL-10) for RPL over Treg cells. Our findings are suggestive of pro-inflammatory dominance in mid-gestation of pregnant women with a history of RPL in general and greater than normal anti-inflammatory milieu in cases with > 2 pregnancy loss. As both sterile and infection related inflammation plays a role in pregnancy loss, studies enrolling women with favourable and unfavourable ongoing pregnancies may shed light on the importance of the present study for developing better management/therapeutic strategies.

UP-regulated levels of sHLA-G in women with a history of RPL in mid-gestation presumably to achieve ongoing pregnancy.

PROBLEM: Recurrent Pregnancy Loss (RPL) is a disorder characterized by two or more pregnancy losses within 20th week of gestation. Globally 1-5% of the couples are affected, 50% of these cases are with unknown etiology. HLA-G, an Immuno-modulatory molecule is a non-classical MHC-1 protein, expressed abundantly on extravillous trophoblastic cells, responsible for spiral artery remodeling, maintaining maternal immune tolerance and fetal growth by adjusting pro and anti-inflammatory milieu during different gestational phases. METHOD OF STUDY: In the present case-control study CD4+HLA-G+ tTreg cells were enumerated by flow cytometry and estimation of the circulating levels of sHLA-G in the blood samples of 300 mid-gestation pregnant women with (iRPL) and without history of RPL (nRPL) by Enzyme-linked Immunosorbent assay was done. The cases included 92 primary and 58 secondary RPL cases RESULTS: A significant reduction in number of tTregs and elevated levels of circulating sHLA-G in iRPL (.03, 200.9) versus nRPL (.09, 90.32) was observed. Further, the primary cases showed higher circulating sHLA-G and no difference in relation to CD4+HLA-G+ tTregs compared to the secondary cases. Receiver operating curve (ROC) characteristics of sHLA-G (AUC = .8) was superior to CD4+HLA-G+ (AUC = .7) for iRPL patients over nRPL group. CONCLUSIONS: Our results are suggestive of the over-expression of sHLA-G which may be caused due to its shedding from surface of trophoblast as a compensatory mechanism to save the on-going pregnancy. To realize the present outcome, studies are required on on-going pregnancy follow-up cases with favorable and unfavorable pregnancy outcome.

Can circulating levels of transforming growth factor-ß1 in early pregnancy serve as a predictive marker of unfavourable outcome?

INTRODUCTION: Transforming Growth Factor (TGF-ß1) is an anti-inflammatory pleiotropic cytokine, crucial for maternal immune tolerance towards semi-allograft. It acts as a mediator in achieving successful implantation and maintenance of pregnancy. METHODS: A total of 300 samples; 150 with Recurrent Pregnancy Loss (RPL) and 150 with no pregnancy loss, in their first trimester were evaluated for circulating levels of TGF-ß1 using Enzyme-Linked Immunosorbent Assay (ELISA). Further, the Receiver Operating Characteristics (ROC) analysis was performed to assess the potential of TGF-ß1 in the risk prediction of RPL and the prognostic importance in the form of favourable and unfavourable outcome in the existing pregnancy. RESULTS: The results showed significant elevated levels in women without the history of RPL compared to those with the history of RPL (4783.60 ± 522.95 vs. 4252.18 ± 672.26 pg/mL, p < 0.0001).Further evaluation of follow up data of women with the history of RPL, based on favourable (78%) and unfavourable (22%) outcome of the existent pregnancy showed significantly higher TGF-ß1 in women with favourable pregnancy outcome in comparison with those who had a foetal loss (4877.12 ± 460.04 vs. 4075.91 ± 616.17 pg/mL, <0.0001). Furthermore, the Receiver Operating Characteristics (ROC) analysis revealed sufficient importance for risk assessment and very good marker to predict unfavourable event (AUC-0.85, SE = 67%, SP = 88%, p < 0.0001). CONCLUSION: Certainly TGF-ß1 appears to have predictive importance; however additional studies with large sample size are warranted for further validation.

Cytokine gene variants of TNF-α and IL-10 in the propensity of type 2 diabetes in south Indian population.

Chronic inflammation plays an important role in type 2 diabetes mellitus (T2DM), a common endocrinological pro-inflammatory disorder associated with insulin resistance. The objective of the present study is to see individual and combined effect of TNF-α (rs361525, rs1800629) and IL-10 (rs1800872, rs1800896) genes on T2DM susceptibility The genotyping was carried out in 200 T2DM patients and 200 healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using suitable primers. The results shown that TNF-α (GA of rs361525 & rs1800629) and IL-10 (AA of rs1800872 & GA of rs1800896) genes are significantly linked with T2DM development. The presence of AA-GA genotype combination for both TNF-α and IL-10 genes were elevating the risk of T2DM. Moreover, individuals bearing haplotypes AAAA, AACA and AAAG experience the increased risk of T2DM. Furthermore, gene-gene interaction analysis shown that TNF-α (GA of rs361525 & rs1800896) gene redundantly confer 3.4-fold elevated risk for T2DM. In gene-environment interaction, GA of TNF-α -1800896, W/H ratio and TG/HDL ratio were redundantly interacted each other and increase the risk of T2DM by 67-times. In conclusion, our results reveal that there is a significant association between foresaid TNF-α, IL-10 gene promoter polymorphisms and T2DM development. To the best of our knowledge this study is the first of its kind in the literature reporting the epistatic association of TNF-α (rs1800629G/A) gene with TG/HDL ratio and W/H ratio over IL-10 gene polymorphisms for T2DM susceptibility among south Indians.

Association of reduced maternal sHLA-G5 isoform levels and elevated TNF-α/IL-4 cytokine ratio with Recurrent Pregnancy Loss: A study on South Indian women.

Inflammation is of critical importance in successful implantation during pregnancy. However, the establishment of maternal immune tolerance towards semi-allograft foetus is more exigent and is achieved predominantly by human leukocyte antigen-G (HLA-G) isoforms with a special emphasis on soluble HLA-G5 (sHLA-G5). Constant inflammation and lack of resolution by anti-inflammatory milieu, due to aberrant expression of critical immunoregulatory molecules such as sHLA-G5 and dysfunctional T helper cells 1 and 2 (Th1-Th2) cytokine shift, can lead to adverse pregnancy outcomes including recurrent pregnancy loss (RPL). Serum samples of 270 pregnant women (135 healthy parous and 135 with a history of RPL) were evaluated for the concentrations of sHLA-G5, interleukin-4 (IL-4) and tumour necrosis factor-alpha (TNF-α) using sandwich enzyme-linked immunosorbent assay (ELISA) and found elevated levels of sHLA-G5 and IL-4 in controls and higher TNF-α levels and TNF-α:IL-4 ratio in patients (P < .05). Stratified data analysis based on the time of sample collection, that is the first and second trimesters exhibited higher sHLA-G5 and IL-4 in both first and second trimesters in controls than patients, while they displayed lower levels concerning TNF-α and TNF-α:IL-4 ratio (P < .05). However, within patients and controls in the first or second trimesters, there was a significant variation concerning sHLA-G5 alone. Further, the outcome of pregnancies studied in the present investigation revealed a significant elevation in sHLA-G5 levels among women with successful pregnancies compared with women who experienced pregnancy loss, therefore, concluding the potential application of sHLA-G5 isoform as a marker in assisting improved pregnancy outcomes.

Association of FOXP3 rs3761548 polymorphism and its reduced expression with unexplained recurrent spontaneous abortions: A South Indian study.

PROBLEM: Fork Head Box Protein 3 (FOXP3) is an X-linked gene, codes for a master transcription regulatory protein that controls the development and function of immunosuppressive T regulatory (Treg) cells. They are crucial mediators of maternal foetal tolerance and successful pregnancy outcome. The aim of the study is to evaluate the association of FOXP3 rs3761548 functional polymorphism and to assess the serum concentrations of full-length FOXP3 protein in Unexplained Recurrent Spontaneous Abortions (URSA) patients of Southern India. METHOD OF STUDY: The study included blood samples from 150 URSA patients and 150 healthy, pregnant parous women. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism was done for rs3761548 FOXP3 genotyping. Serum concentrations of full-length FOXP3 protein were estimated by enzyme-linked immunosorbent assay. RESULTS: The frequencies of mutant A allele, CA and AA genotypes of rs3761548 functional polymorphism were significantly elevated in patients compared to healthy, pregnant parous women and exhibited a two, three and twofold increased risk respectively towards URSA. Serum concentrations of full-length FOXP3 protein were high in controls compared to patients (10.14 ± .30 vs. 8.84 ± 1.73 ng/ml; p < .05). CONCLUSION: Our results advocate an association of FOXP3 rs3761548 polymorphism and reduced expression of full-length FOXP3 protein with URSA.

Gender bias in the genetic vulnerability towards type 2 diabetes and diabetic nephropathy: Role of forkhead box Protein3 transcription factor gene variants.

Forkhead Box Protein3 Transcription Factor (FOXP3) gene is an essential role player in the function and differentiation of regulatory T cells. Polymorphisms/mutations in FOXP3 gene cause Treg cell dysfunction, promote autoimmunity and inflammation. Based on this presumption, we screened 600 subjects from south India (equal number of diabetic (T2DM), diabetic nephropathy (T2DN) and healthy controls) for promoter and intronic (rs3761548C/A and rs2294021C/T) polymorphisms of FOXP3 gene. PCR-RFLP method used for genotyping, revealed an association of promoter SNP for both T2DM (OR = 2.41, 95% C.I = 1.67-3.49; p < 0.0001) and T2DN (OR = 2.16, 95% C.I = 1.45-3.24; p < 0.005). While intronic polymorphism with T2DN (OR = 1.91, 95% C.I = 1.28-2.84; p < 0.05). Further, in females rs3761548C/A showed 2.6 and 5.5-fold; rs2294021C/T showed 2.2- and 2.5-fold predisposition towards T2DM and T2DN respectively. Males exhibited a twofold risk (OR = 2.01, 95% C.I = 1.22-3.30; p < 0.05) towards T2DM with promoter and no association with intronic polymorphism. The combined genotypes in females with AA-CC; AA-TT predisposed and CA-CC; CA-CT protected heading towards T2DM and T2DN respectively, suggesting irrespective of type of allele at intronic locus AA and CA at promoter locus promote or protect the individual for diabetes and diabetic nephropathy, further confirmed by MLR. To our knowledge, the current study is the first of its kind that revealed an association of these polymorphisms of FOXP3 gene and gender influence on T2DM and T2DN among South Indians. Functional and cell-based studies on Treg cells are warranted to confirm our results that help to develop FOXP3/Treg based therapeutic interventions. Lack of data on Treg cells is the limitation of this study.

Association analysis of FMR1 genetic variants and primary ovarian insufficiency in South Indian women with a novel approach of CGG repeats classification.

Around 20-28% of FMR1gene CGG premutation (PM) carriers are at augmented risk towards an infertility related disorder, Fragile X-associated primary ovarian insufficiency (FXPOI). Except the effect of CGG repeats, reports are not available on the mechanism through which the cis-acting variations, namely, SNPs involved in POI susceptibility. Addressing the hypothesis that the FMR1 gene polymorphisms [CGG repeats, rs25731(T > A) and rs4949(A > G)] might increase their individual and combined impact in disease predisposition, we tested the genetic variants in 200 south Indian DNA samples consists of 100 patients and 100 healthy volunteers. We used gene scan method to score the CGG repeat length, and ARMS and RFLP methods to genotype the SNPs. Only 0.5% of each Gray zone and PM alleles were found among patient group, however, no disease association was noticed with repeat length. The rs25731 showed protection [OR:0.32; (0.13-0.76), p = 0.006] and rs4949 reported a 2.5-fold risk towards the disease predisposition [OR:2.46; (1.06-5.74), p = 0.031] but, both found insignificant after Bonferroni correction was done under different Genetic Models. Novel classification of genotype combinations, 'Normal&Variant Homozygote' [OR:2.89,(1.12-7.9), p < 0.05] and 'Allele2-T-G' haplotype block (6%vs.1%, p = 0.08) were noticed to be at marginal risk for POI. We demonstrated a susceptible role of the combined effect of variant allele-G and Allele-2 (repeat allele outside the normal range) for FXPOI. To support our findings of its first kind, further studies with large samples are warranted in understanding the role of FMR1 genetic variants in FXPOI etio-pathophysiology, the outcome might help in providing better reproductive treatment options for females, who are at risk for FXPOI.

Link between ACE I/D Gene Polymorphism and Dyslipidemia in Diabetic Nephropathy: A Case-control Study from Hyderabad, India.

INTRODUCTION: Diabetic nephropathy (DN) is the commonest single cause of end-stage renal failure, and dyslipidemia is a critical risk factor in the occurrence of DN. In the light of recent reports emphasizing the importance of angiotensin I-converting enzyme (ACE) in the modulation of plasma lipids, we sought to evaluate the influence of ACE I/D gene polymorphism with dyslipidemia status among type 2 diabetic (T2D) patients with and without nephropathy in the genetic predisposition and the progression to DN. METHOD: This study comprised of 600 subjects, which include patients with DN, T2D, and healthy controls (HC). Polymerase chain reaction based genotyping of ACE I/D polymorphism was performed and appropriate statistical analysis was done. RESULTS: Out of the 600 subjects, 20 (10%) of the HC, 73 (36.5%) of the T2D group, and 125 (62.5%) of the DN subjects had dyslipidemia. The D allele (0.62) and DD (42.5) genotype frequencies were higher in the DN group in comparison with T2D and HC (P < 0.05). The genotypes also varied among patients with dyslipidemia (χ2 5.04; P < 0.05) but not in the non-dyslipidemia group. Under the co-dominant model, DD genotype conferred a risk of 1.26 (P < 0.001) toward DN, whereas the ID genotype offered protection from DN among the dyslipidemic subjects (OR = 0.05; P < 0.01). In addition, genotype-dependent difference was seen in the plasma lipid levels among study groups. A multiple logistic regression analysis revealed male gender, BMI, HbA1c, TG, HDL, and ACE DD genotype as independent risk factors for the development of DN. CONCLUSION: The study showed a significant predisposing association of ACE DD genotype with DN and protective effect of ID genotype on DN in the dyslipidemia subgroup.

Genetic confirmation of T2DM meta-analysis variants studied in gestational diabetes mellitus in an Indian population.

BACKGROUND: Meta-analysis is useful for combining the results of different studies statistically to confirm genuine associations in genetics. Based on earlier reports, we aimed to investigate the association between type 2 diabetes mellitus (T2DM) genetic variants identified in a previous meta-analysis in gestational diabetes mellitus (GDM) in an Indian woman. MATERIAL AND METHODS: In this study, 137 pregnant women with GDM and 150 pregnant women were selected on the basis of their serum glucose levels. The six single nucleotide polymorphisms (SNPs) of different genes studied had known involvement in pancreatic ß-cell function, particular pathways linked to T2DM, and other biological functions. Genomic DNA was isolated from the 287 women for polymerase chain reaction and restriction fragment length polymorphism analyses. RESULTS: The rs7903146, rs13266634, rs2283228, rs5210 and rs179881 SNPs were found to be positively associated with GDM when calculated for genotype and allele frequencies (p < 0.05), but rs680 (ApaI) variant did not show statistically significant association (p = 0.31). The rs7903146, rs2283228, rs5210 and rs680 variants showed a strong association with oral glucose tolerance test values. CONCLUSION: The SNPs studied in this GDM had the same role as those identified in a previous T2DM meta-analysis, and showed positive association in the Indian women. Meta-analyses should be implemented to assess the IGF2 gene in GDM subjects.

CTLA-4 Genetic Variants (rs11571317 and rs3087243): Role in Susceptibility and Progression of Breast Cancer.

BACKGROUND: Dysfunctional regulation at immune checkpoints may lead to escape of the tumor cells and gives a scope to set in the unresolved Breast cancer (BC). The major anti-tumor retort is cell-mediated response which involves T lymphocytes. CTLA-4 (Cytotoxic T lymphocyte associated protein-4) with immune suppressive function and tolerance is associated with various autoimmune diseases and cancers including BC. The present study deals with CTLA-4 gene selected polymorphisms (rs11571317 C/T and rs3087243G/A) to explore their relation with breast cancer susceptibility and progression in BC patients. METHODS: For the present case-control study, we recruited a total of 570 women which include breast cancer patients and healthy control women from south India. Blood samples were collected, genomic DNA was isolated and genotyped by using PCR-RFLP method, and the data were analysed through suitable statistics. RESULTS: We observed a significant association of rs11571317 with BC in our study group, where CC genotype showed a three-fold increased risk towards BC and CT genotype to be protective. In-silico analyses strengthened our observation revealing the abolition of SP1 binding site in the CTLA-4 promoter by the mutant allele T. The CTLA-4 rs3087243 polymorphism showed an association not with the susceptibility but towards the tumor progression, where GG genotype was coupled with reduced tumor growth (OR = 0.01) and GA (OR = 6.2), AA (OR = 3.4) with increased tumor growth. The T-G haplotype was found to confer protection against breast cancer risk while C-A (OR = 3.6) and T-A (OR = 15.8) haplotypes were associated with disease progression. In-silico analysis for rs3087243 revealed change in threshold values between reference and variant sequences. CONCLUSION: The study suggests varied roles of different polymorphisms of CTLA-4 in the aetiopathogenesis of BC. Understanding the mechanism may help in the CTLA-4 based immunotherapy for BC.

Importance of glucokinase -258G/A polymorphism in Asian Indians with post-transplant and type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) and post-transplant diabetes mellitus (PTDM) are non-synonymous forms of diabetes. Glucokinase (GCK) plays a key role in glucose metabolism. The relationship between the GCK promoter and specific types of diabetes, such as PTDM and T2DM, in the Asian Indian population is unknown. We examined the occurrence of a specific GCK promoter variant (-258G/A) in patients with T2DM and PTDM. The case-control study enrolled 640 Asian Indian subjects, including controls (n = 250) and T2DM (n = 250), PTDM (n = 42), and non-post-transplant diabetes mellitus (non-PTDM) (n = 98) patients. Purified Deoxyribonucleic acid (DNA) was genotyped with the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. The digested PCR products were analyzed on 12% polyacrylamide gels. The anthropometric, biochemical, and clinical details of each group were documented. GCK -258G/A alleles and genotypes were not associated with T2DM. However, among PTDM subjects, we detected a higher frequency of heterozygotes (52.4%) and a positive association with alleles/genotypes. The results suggest that the promoter region (-258G/A) of GCK plays an important role in PTDM in Asian Indians.

Type 2 Diabetes Mellitus and the Association of Candidate Genes in Asian Indian Population from Hyderabad, India.

INTRODUCTION: Genetic and environmental factors play an important role in susceptibility to type 2 diabetes mellitus (T2DM). Several genes have been implicated in the development of T2DM. Genetic variants of candidate genes are, therefore, prime targets for molecular analysis. AIM: In this study, we have selected 3 candidate genes, namely, TCF7L2, SLC30A8, and IGF2, for assessing their association with T2DM in an Indian population. MATERIALS AND METHODS: Five hundred individuals were enrolled in this case-control study- 250 T2DM patients and 250 healthy control individuals. Clinical characteristics were obtained for all subjects, and genotype analysis was performed by PCR-RFLP analysis. RESULTS: Allele and genotyping frequencies, odds ratios, and 95% confidence intervals were calculated for 3 single nucleotide polymorphisms (SNPs), 1 each from TCF7L2 (rs7903146), SLC30A8 (rs13266634), and IGF2 (rs680) in T2DM patients. The rs7903146 and rs680 polymorphisms were found to be significantly associated with T2DM (p < 0.05), whereas the rs13266634 polymorphism was not (p > 0.05). The multifactor dimensionality reduction method identified the particular polymorphisms associated with an increased risk of disease. CONCLUSION: The present study indicated that the gene-gene interaction model successfully predicted T2DM risk based on TCF7L2 and SLC30A8 polymorphisms. These results provide strong evidence of independent association between T2DM and the 3 SNPs analysed herein.

Association of IFN-γ : IL-10 Cytokine Ratio with Nonsegmental Vitiligo Pathogenesis.

Background and Objectives. Cytokines regulate immune response and inflammation and play a crucial role in depigmentation process of vitiligo. The present study aimed to estimate the serum levels of pro- and anti-inflammatory cytokines, IFN-γ and IL-10, and their ratios in nonsegmental vitiligo patients and healthy individuals from India. Methods. Blood samples were collected from 280 subjects and serum IFN-γ and IL-10 levels were measured using standard ELISA. Results. Nonsegmental vitiligo patients showed increased levels of IFN-γ (12.4 ± 3.2 versus 9.9 ± 4.4 pg/mL) and decreased levels of IL-10 (9.3 ± 1.7 versus 11.5 ± 5 pg/mL) compared to controls. Ratio of IFN-γ : IL-10 differed significantly from patients to controls (p < 0.05). IFN-γ concentrations and IFN-γ : IL-10 ratio varied significantly with respect to clinical variants, disease stability, and social habits (smoking and alcohol consumption) and showed a positive correlation with disease duration. Family history of vitiligo was significantly associated with IFN-γ : IL-10 ratio but not with their individual levels. Conclusion. The ratio of IFN-γ : IL-10 serum levels may be considered as one of the promising immunological markers in nonsegmental vitiligo. This is the first study considering multiple aspects in relation to ratio of cytokine levels. Similar studies with large samples are warranted to confirm our observations.

TGFB1 Functional Gene Polymorphisms (C-509T and T869C) in the Maternal Susceptibility to Pre-eclampsia in South Indian Women.

Pre-eclampsia (PE), a pregnancy-specific vascular disorder characterized by hypertension and proteinuria, is hypothesized to be the result of inadequate placental angiogenesis with attendant systemic inflammation. The pleiotropic cytokine, Transforming Growth Factor-ß1 (TGF-ß1), is considered to be a key candidate gene in the molecular pathogenesis of PE by virtue of its ability to not only regulate angiogenesis and apoptosis of target cells, but also by acting as a master controller of Th1/Th2 cytokine balance and production of the anti-inflammatory peripheral regulatory T cells (FOXP3+ Tregs). Based on this presumption, we screened a total of 469 pregnant women from South India that include 239 patients with PE and 230 healthy controls for the two functional polymorphisms of TGFB1 gene (C-509T and T869C). The genotype frequencies of these two polymorphisms differed significantly between the PE and control groups (P = 0.01 and P = 0.002, for the TGFB1 C-509T and T869C polymorphisms, respectively). Under the over-dominant model, the CT genotype of the TGFB1 C509T polymorphism showed a high protective effect (P = 3e-04), while the TT genotype of the same variant appeared to be the predisposing genotype (P = 0.003). The T-T and C-C haplotypes were found to be the risk haplotypes blocks towards PE (OR = 4.72; P = 0.031, OR = 5.39; P = 0.03), respectively. Strong linkage disequilibrium was seen between the two polymorphisms. Our investigations revealed a significant influence of TGFB1 C-509T and T869C polymorphisms on the PE risk in South Indian women. The study represents one of the first of its kind from the Indian subcontinent.

Validation of the association of TCF7L2 and SLC30A8 gene polymorphisms with post-transplant diabetes mellitus in Asian Indian population.

The rs7903146 and rs13266634 polymorphisms in the TCF7L2 and SLC30A8 genes, respectively, have been reported to be associated with type 2 diabetes. However, little is known about the association of these polymorphisms with post-transplant diabetes mellitus (PTDM). To study this linkage, we determined a distribution of allele and genotype frequencies in Asian Indians. 42 PTDM and 98 non-PTDM subjects were recruited. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was performed to detect for rs7903146 and rs13266634 polymorphisms. The clinical details and statistical analysis for PTDM and non-PTDM subjects were recorded. Our results observed higher frequencies of the minor alleles in rs7903146 and rs13266634 polymorphisms in the PTDM group compared to the non-PTDM subjects. The allele frequencies also found to be significantly associated with PTDM (rs7903146: T vs C: OR-2.6; (95%CI: 1.2-5.6); p = 0.01; rs13266634: T vs C: OR-2.0; (95%CI: 1.1-3.4); p = 0.01). These findings suggest that rs7903146 and rs13266634 polymorphisms are associated with PTDM in the Asian Indian population despite a relatively small study group.

Screening of mitochondrial mutations and insertion-deletion polymorphism in gestational diabetes mellitus in the Asian Indian population.

In this study we scrutinized the association between the A8344G/A3243G mutations and a 9-bp deletion polymorphism with gestational diabetes mellitus (GDM) in an Asian Indian population. The A3243G mutation in the mitochondrial tRNA(Leu(UUR)) causes mitochondrial encephalopathy myopathy, lactic acidosis, and stroke-like episodes (MELAS), while the A8344G mutation in tRNA(Lys) causes myoclonus epilepsy with ragged red fibers (MERRF). We screened 140 pregnant women diagnosed with GDM and 140 non-GDM participants for these mutations by PCR-RFLP analysis. Both A3243G and A8344G were associated with GDM (A3243: OR-3.667, 95% CI = 1.001-13.43, p = 0.03; A8344G: OR-11.00, 95% CI = 0.6026-200.8, p = 0.04). Mitochondrial DNA mutations contribute to the development of GDM. Our results conclude that mitochondrial mutations are associated with the GDM women in our population. Thus it is important to screen other mitochondrial mutations in the GDM women.

Evaluation of Gestational Diabetes Mellitus Risk in South Indian Women Based on MTHFR (C677T) and FVL (G1691A) Mutations.

We aimed to scrutinize the extent to which single amino acid substitutions in the MTHFR and factor V Leiden (FVL) genes affect the risk of gestational diabetes mellitus (GDM) in pregnant women of South Indian descendant. This case-control study was implemented once the ethical approval has been obtained. Overall, 237 women were recruited in this study: 137 had been diagnosed with GDM and the remaining 100 women were used as normal controls or non-GDM. The diagnosis of GDM was confirmed with biochemical analysis, i.e., GCT and oral glucose tolerance tests. Five milliliters of peripheral blood was collected and used for biochemical and molecular analyses. DNA was isolated, and genotyping for MTHFR (C677T) and FVL (G1691A) mutations was performed using PCR-RFLP. FVL (G1691A) locus was not polymorphic in the investigated sample. There was no significant difference in the allele and genotype frequencies of C677T polymorphism between GDM and non-GDM women (p = 0.8892).

Forskolin: genotoxicity assessment in Allium cepa.

Forskolin, a diterpene, 7ß-acetoxy-8,13-epoxy-1α,6ß,9α-trihydroxy-labd-14-en-11-one (C22H34O7) isolated from Coleus forskohlii, exerts multiple physiological effects by stimulating the enzyme adenylate cyclase and increasing cyclic adenosine monophosphate (cAMP) concentrations. Forskolin is used in the treatment of hypertension, congestive heart failure, eczema, and other diseases. A cytogenetic assay was performed in Allium cepa to assess possible genotoxic effects of forskolin. Forskolin was tested at concentrations 5-100 µM for exposure periods of 24 or 48 h. Treated samples showed significant reductions in mitotic index (p < 0.05) and increases in the frequency of chromosome aberrations (p < 0.01) at both exposure times. The treated meristems showed chromosome aberrations including sticky metaphases, sticky anaphases, laggard, anaphase bridges, micronuclei, polyploidy, fragments, breaks, and C-mitosis. Forskolin may cause genotoxic effects and further toxicological evaluations should be conducted to ensure its safety.