Bushra Chaudhry

Polymorphisms in MTHFR, MS and CBS genes and homocysteine levels in a Pakistani population.

Background Hyperhomocysteinemia (>15 µmol/L) is highly prevalent in South Asian populations including Pakistan. In order to investigate the genetic determinants of this condition, we studied 6 polymorphisms in genes of 3 enzymes - methylenetetrahydrofolate reductase (MTHFR; C677T; A1298C), methionine synthase (MS; A2756G), cystathionine-β-synthase (CBS; T833C/844ins68, G919A) involved in homocysteine metabolism and investigated their interactions with nutritional and environmental factors in a Pakistani population. Methodology/Principal Findings In a cross-sectional survey, 872 healthy adults (355 males and 517 females; age 18–60 years) were recruited from a low-income urban population in Karachi. Fasting venous blood was obtained and assessed for plasma/serum homocysteine; folate, vitamin B12, pyridoxal phosphate and blood lead. DNA was isolated and genotyping was performed by PCR-RFLP (restriction-fragment-length- polymorphism) based assays. The average changes in homocysteine levels for MTHFR 677CT and TT genotypes were positive [β(SE β), 2.01(0.63) and 16.19(1.8) µmol/L, respectively]. Contrary to MTHFR C677T polymorphism, the average changes in plasma homocysteine levels for MS 2756AG and GG variants were negative [β(SE β), −0.56(0.58) and −0.83(0.99) µmol/L, respectively]. The average change occurring for CBS 844ins68 heterozygous genotype (ancestral/insertion) was −1.88(0.81) µmol/L. The combined effect of MTHFR C677T, MS A2756G and CBS 844ins68 genotypes for plasma homocysteine levels was additive (p value <0.001). Odds of having hyperhomocysteinemia with MTHFR 677TT genotype was 10-fold compared to MTHFR 677CC genotype [OR (95%CI); 10.17(3.6–28.67)]. Protective effect towards hyperhomocysteinemia was observed with heterozygous (ancestral/insertion) genotype of CBS 844ins68 compared to homozygous ancestral type [OR (95% CI); 0.58 (0.34–0.99)]. Individuals with MTHFR 677CT or TT genotypes were at a greater risk of hyperhomocysteinemia in folate and vitamin B12 deficiencies and high blood lead (p value <0.05) level. Conclusions Gene polymorphism (especially MTHFR C677T transition), folate and vitamin B12 deficiencies, male gender and high blood lead level appear to be contributing towards the development of hyperhomocysteinemia in a Pakistani population.

Elevated levels of chemerin, leptin, and interleukin-18 in gestational diabetes mellitus.

Abstract Objective: We aimed to assess the levels of adipokine and their relation to gestational diabetic related clinical phenotypes and fetal growth parameters. Material and methods: International Association of the Diabetes and Pregnancy Study criteria was used to classify gestational diabetic cases (n = 208) and euglycemic controls (n = 300). ELISA assays were performed for insulin, chemerin, leptin, and interleukin-18 (IL-18). Mann–Whitney U test, Chi-square/Fisher exact test, multiple regression analyses, and ROC curves were applied with significant p values of <0.05. Results: Levels of chemerin, IL-18, and leptin were seven-, four-, and five-folds higher in cases versus controls, respectively (p < 0.01). The adipokine showed strong positive correlation with fasting blood glucose, homeostasis model assessment of insulin resistance, and fetal weight (p < 0.01). Odds of GDM association remained significant for chemerin (OR 1.522; 1.097–2.110) and leptin (OR 2.579; 1.503–4.425) while all associations were lost for IL-18 (p > 0.05) after multiple adjustments. Raised chemerin levels were identified in 96% cases (n = 201) employing the proposed cut off value >15.49 ng/ml. Conclusion: High chemerin and leptin levels are seen in GDM which may be associated with subclinical inflammation suggesting a role in development of insulin resistance.

Screening of subclinical hypothyroidism during gestational diabetes in Pakistani population

Abstract Objective: The increased prevalence of adverse effects of altered thyroid functions in pregnancy inspired us to study the frequency of subclinical hypothyroidism (SCH) and the relationship with glycaemic control and foetal weight in pregnant females with and without gestational diabetes mellitus (GDM) in Pakistani population. Patients and methods: Five hundred and eight pregnant females were enrolled and grouped as per the International Diabetes Association criteria into GDM (n = 208) and healthy control (n = 300). Random blood glucose (RBG), thyroid function tests, anthropometric analysis and foetal ultra sound scans were performed on all study subjects. Data were analysed using Mann–Whitney U test and Chi-square test wherever applicable. Spearman correlation and multiple regression analysis were performed. p values of <0.05 was considered significant. Results: A total of 61.5% GDM subjects depicted SCH with normal circulating T4 and T3 versus 6.0% healthy controls (p-value < 0.001). Moreover, TSH remained independently associated with RBG (r = 0.109; p < 0.05), poor glycaemic control (r = 0.227; p < 0.001) and negatively associated with foetal growth (r = −0.206; p < 0.001). Conclusion: The detection of high TSH with normal T3 and T4 in females with GDM strongly emphasises the need of thyroid screening as a routine in all antenatal clinics.

KCNQ1 rs2237895 polymorphism is associated with Gestational Diabetes in Pakistani Women

Background and Objective: Genetic studies on gestational diabetes (GDM) are relatively scarce; moreover, limited data is available for KCNQ1 polymorphism in Pakistani pregnant women. We aimed to determine the frequency of KCNQ1 rs2237895 in GDM and normal pregnant controls and its association with GDM-related phenotypes. Methods: A total of 637 pregnant females (429 controls and 208 cases) in their second trimester were classified according to the International Association of the Diabetes and Pregnancy Study criteria in this study. Their blood samples were genotyped for KCNQ1 SNP rs2237895 using PCR-RFLP method and sequencing. Fasting and two hour-post glucose load blood levels, serum HbA1c, insulin, and anthropometric assessment was performed.: Pearson’s Chi Square test, Mann- Whitney U test, and regression analyses were performed. A p-value of <0.05 was considered significant. Results: The variant genotyped was in Hardy-Weinberg equilibrium (p>0.05). The rs2237895 showed an association with GDM (OR 2.281; 1.388-3.746: p <0.001) and remained significant after multiple adjustments for age and body mass index (OR 2.068; 1.430-2.997: p=0.005). The C allele showed positive association with insulin level, and HOMA-IR in study subjects. Conclusions: This study identifies that KCNQ1 rs2237895 polymorphisms might be associated with risk of GDM in Pakistani population and that it is related to higher glucose levels and insulin resistance. Further large scale studies are required to consolidate on the functional aspect of this polymorphism.

Identification of Calpain 10 Isoforms (b, d, e, f & h) Conserved Regions and Possible Functional Prophecy through Bioinformatics

Calpain 10 is an atypical calpain ubiquitously exist in all human tissues. It exhibits eight protein isoforms designated as “ a-h ” which play a vital role in glucose homeostasis but actual mechanism of action is yet to be ascertained. We have predicted the partial roles of Isoform a, c and g previously. They were envisaged to act partially as mu and m-calpain cysteine proteases. Here we predict the function of minor isoforms b, d, e, f and h. We have applied NCBI Blast and Conserved domain tool for nucleotide and protein alignments. Blast query indicated 87%, 84%, 87%, 94% and 34% identity of isoform b, d, e, f and h with canonical sequence of calpain 10 a isoform. Conserved domain analyses of protein sequences revealed significant structural similarities of their N-terminal domain I and II with catalytic domain of cysteine protease superfamily PC1 (e-value:CAPN10 b, d, e = 2.41e-76, CAPN10 f = 1.07e-43 and CAPN10 h = 1.13e-17). Isoform b, d and e have one consecutive domain similar with C2 like subdomain III (e-value=2.92-32, 1.03e-35, 1.88e-14 respectively) and was classified in CAPN10 group of Palb subfamily. Isoform f and h were lacking this domain and had shorter sequences. Although structural similarities are not guaranteed for similar actions but domain homology predicted the existence of similar functions as of calpain I and II.

Tracking down immune markers from alternative system pathway factors in a diabetic population.

Hyperglycemia associated with type 1 diabetes (T1D) alters the host immune system, resulting in a predisposition to infectious diseases. The high risk of infection in the diabetic population may lead to life‐threatening situations. The early proteins of the alternative complement system pathway, constituting factors P, B, and D, have been shown to play an important role in preventing infection because they form a membrane attack complex (MAC)‐C5‐9, which debilitates the target microbes and/or molecules via cytotoxic and cytolytic reactions. Patients who are devoid of or contain low levels of these proteins may be susceptible to developing chronic infections. We have observed striking differences in partially fractionated serum proteins in diabetic patients (type 2) relative to controls, through single and two‐dimensional gel electrophoresis. Our data, obtained from 50 diabetic patients in the age group of 25–45 years, who had the disease for fewer than 5 years, indicated patterns in low‐ and high‐molecular‐weight proteins, which could be grouped into five different categories with minor differences in their respective levels of protein expression. Immunoblot assay could barely detect the presence of properdin expression in diabetic patients. Quantization by ELISA in 99 patients indicated low levels of properdin expression in 70% of 50 diabetic patients (6.5 ± 3 μg/mL) when compared to nondiabetic controls (19.5 ± 8.5 μg/mL). This study concluded that patients with low expression of properdin should be advised to take extensive preventive measures and seek early management with appropriate treatments against infection.