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.

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.

Foxp3 promoter polymorphism (rs3761548) in breast cancer progression: a study from India.

Breast cancer is the most common female neoplasm that drives the transformation of normal mammary epithelial cells into highly malignant derivatives. Forkhead Box Protein3 (Foxp3), a tumor suppressor/immunomodulatory gene, which controls the function of Treg cells and oncogenes is down regulated in breast cancer. The main aim of the present study is to evaluate the potential influence of Foxp3-3279 C>A polymorphism (rs3761548) and -2383 C>T polymorphism (rs3761549) in 202 breast cancer patients and 130 normal healthy women of Indian origin. The genotypes were determined using ARMS-PCR for rs3761548 and PCR-RFLP method for rs3761549 using specific primers. The results revealed lack of association of these two polymorphisms with breast cancer susceptibility. However, with respect to AA genotype of rs3761548, we found highly significant association with the advanced stage (T3-4) of the tumor (OR = 3.90; 95% confidence interval (CI) = 1.56-9.70; p = 0.03). Stratified data also revealed an association of homozygous mutant genotype with advanced stage of tumor in premenopausal women (OR = 4.56; 95% CI = 1.07-19.38; p = 0.04) with disease duration of <6 months (OR = .10; 95% CI = 1.80-20.50; p = 0.002) suggestive of modulating effect of rs3761548 in tumor progression. We conclude that Foxp3 rs37161548 has a potential to be a polymorphic marker for tumor progression in premenopausal breast cancer patients in Indian women.

NPHS2 mutation analysis and primary nephrotic syndrome in southern Indians.

AIM: Nephrotic syndrome is one of the most commonly diagnosed primary kidney diseases and its progressive forms can lead to chronic kidney disease and or end-stage renal disease. Steroid-resistant nephrotic syndrome is defined by resistance to standard steroid therapy and it remains one of the most intractable causes of kidney failure. Mutations in NPHS2, which encodes for podocin, an integral membrane protein of the glomerular epithelial cells (podocytes), represent a frequent cause of steroid-resistant nephrotic syndrome worldwide. This study was aimed at screening for known NPHS2 mutations in Indians with nephrotic syndrome. METHODS: We screened a cohort of 484 subjects from the southern Indian population for the presence of four missense mutations G92C, P118L, R138Q and D160G within the NPHS2 gene using tetra primer ARMS PCR. RESULTS: Our results revealed that these mutations were seen only among the patients (14.02%) and were absent in the controls, suggesting their disease-causing nature. Further categorization revealed that these mutations were together responsible for 18.5% of steroid-resistant cases in our study group. Conversely, the studied mutations were not found in the controls as well as in the patients with steroid-sensitive nephrotic syndrome. CONCLUSION: This is the first such report from India. More studies are warranted to establish the frequency of NPHS2 mutations in the Asian-Indian population and such analysis may help in developing mutation(s)-specific therapeutic interventions in the future.

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.

Association of FOXP3 (rs3761548) promoter polymorphism with nondermatomal vitiligo: A study from India.

BACKGROUND: The rs3761548 polymorphism (-3279 C>A) of FOXP3 gene is associated with several autoimmune disorders. OBJECTIVE: We sought to determine whether rs3761548 polymorphism is associated with nondermatomal vitiligo in Indian subjects. METHODS: Genomic DNA was isolated from blood samples of 303 patients and 305 control subjects and genotyping was done by allele-specific primers. Data analysis was carried out for the entire cohort and separately for male and female participants as FOXP3 is an X-linked marker. Statistics were performed using software. RESULTS: The genotype frequencies differed significantly from patients to control subjects (P = .002). Further analysis demonstrated female participants with CC genotype were protected (CC vs CA+AA; odds ratio 0.38, 95% confidence interval 0.238-0.615) and those with CA genotype were at higher risk to develop vitiligo (CA vs CC+AA; odds ratio 2.634, 95% confidence interval 1.604-4.325). However, no such statistical difference was observed in male participants. LIMITATIONS: Our study is, to our knowledge, the first report from India with respect to vitiligo and rs3761548; however, we lack adequate literature assistance. CONCLUSIONS: The rs3761548 of FOXP3 gene in our population may be associated with susceptibility to vitiligo because of altered expression. CC genotype appears to be protective and CA genotype seems to impart nearly 3-fold risk to develop vitiligo in women and girls.

Do ACE I/D gene polymorphism serve as a predictive marker for age at onset in PCOS?

BACKGROUND: Polycystic ovary syndrome (PCOS) is an endocrine disorder exhibiting variable age at onset of clinical features allied with complex diseases in the later life. ACE is a pleiotropic molecule associated with various pathophysiological functions. The present study was aimed to establish the frequency of ACE I/D gene polymorphism in patients and controls and to assess the influence of this polymorphism on anthropometric and various clinical features of the condition. METHODS: ACE I/D genotyping was carried out in 259 PCOS patients and 315 healthy ultrasound scanned women of South Indian origin. RESULTS: The distribution of DD, ID and II genotypes in patients was 39, 37 and 24 %, whereas in the controls it was 31, 51 and 18 % respectively. Significant difference was observed in the genotypic frequency distributions between the patients and controls, however the allelic frequencies did not vary between the groups (p>0.05). Quartile analysis revealed preponderance of DD genotype in the first two quartiles and a linear increase of II genotype from first to the last quartiles. Further, Multiple Logistic regression analysis revealed significant association of ACE I/D gene polymorphism with acanthosis and age at onset (AAO) of the syndrome (p<0.05). CONCLUSION: The present study is the first report to highlight the predisposing role of DD and protective role of ID genotype towards PCOS. Patients with single or double dose of D allele may develop PCOS symptoms at an early age and also significantly associated with acanthosis, a marker of insulin resistance.

TNF-α haplotype association with polycystic ovary syndrome - a South Indian study.

BACKGROUND: Tumor Necrosis Factor Alpha (TNF-α), is a proinflammatory cytokine in the pathogenesis of Polycystic Ovary Syndrome (PCOS). In order to investigate the role of rs1800629 and rs1799964 polymorphisms in relation to anthropometric measures, family history of complex diseases, diet and clinical features, we performed a case control study in PCOS women from South India. METHODS: A total of 589 samples comprising of 283 patients and 306 controls were enrolled in the present study. Patients were selected based on Rotterdam criteria and ultrasound scanned normal women were selected as controls. Following extraction of DNA, genotyping for rs1800629 and rs1799964 was performed by polymerase chain reaction using tetra primers and PCR-RFLP respectively. RESULTS: The distribution of genotypes for rs1799964 was significantly different between the groups (p = 0.001), however it was not for rs1800629. Haplotype analysis revealed a significant difference between patients and controls. The predisposing and protective role of haplotype with mutant allele at both loci (combination 3) and haplotype with mutant allele at either loci was reflected by the over representation of combination 3 in patients and combination 2 in controls respectively. In addition, rs1799964 showed an association with dietary habit, clinical hyperandrogenism and AAO. The modifying role of TT genotype on age at onset was noted in quartile analysis. CONCLUSION: Replicative studies on the influence of TNF-α polymorphism in different ethnic groups may identify the potentiality of these polymorphisms as markers of inflammation and in turn may help the clinicians for the better management of the condition.

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.

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.

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.

Angiotensin converting enzyme (ACE) gene polymorphism in vitiligo: protective and predisposing effects of genotypes in disease susceptibility and progression.

Vitiligo is a depigmenting skin disorder with profound heterogenity in its aetio-pathophysiology, and is associated with inter-individual variation in progression of disease. Angiotensin converting enzyme (ACE) is a regulator of renin angiotensin system (RAS) that plays an important role in the physiology of the vasculature, blood pressure, inflammation, adipocyte distribution of various diseases. The present study was carried out in 243 vitiligo patients (132 males and 111 females), aged between 3-62 years with a mean age at onset of 21.6  ±  13.6 yrs, and in 205 healthy controls of south Indian origin. The main objectives of the present study were to evaluate the ACE I/D (insertion/deletion) polymorphism in the patient and control groups. Further, I/D genotypes were compared among the patients with and without the family history of vitiligo as well as the progression of the disease, through polymerase chain reaction (PCR) methods.

The results revealed a highly significant association of DD genotype with disease susceptibility (p < 0.01) in patients with a family history of vitiligo (p < 0.05) in terms of early age at onset. Further, the pre-dominance of ID genotype among patients revealed its association with a slow progression of the disease (p < 0.05). The present study is the first report to highlight the protective role of II genotype and the significant association of ID genotype with slow progression of the disease.

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.