Lack of association of HFE gene polymorphism with high body iron status in Pakistani patients with type 2 diabetes mellitus.

OBJECTIVE: The Aim of this study was to investigate the relationship of 3 common polymorphisms in the HFE gene (C282Y, H63D and S65C) with high body iron status in a population of Pakistani subjects with type 2 diabetes mellitus (DM) and to explore if there is any novel mutation in HFE gene in a sample of Pakistani subjects with type 2 DM. METHODS: In a case-control design, 200 healthy controls and 200 consecutive adult subjects with type 2 DM (both gender; age range of 30-70 years) were enrolled with informed consent. Their serum samples were analyzed for body iron status (ratio of concentration of soluble transferrin receptor to ferritin concentration). DNA from blood was screened for HFE gene polymorphisms via polymerase chain reaction, followed by restriction fragment length polymorphism or via Sanger sequencing to identify any novel mutation(s) in HFE gene. RESULTS: We found that there was lack of any association between HFE polymorphism and body iron status in Pakistani subjects with type 2 DM and healthy controls. H63D was the most common polymorphism found in this population. Single base substitution of G nucleotide instead of C at the codon position 187 in the HFE gene exon 2 was discovered in one subject with DM. There was also a lack of association between D allele (variant allele of H63D) and type 2 DM. A significant relationship was found between CG genotype and abnormal albuminuria in subjects with type 2 DM (p = 0.036). CONCLUSION: In conclusion, HFE gene polymorphism is not associated either with high body iron status or type 2 DM in a hospital based Pakistani population and variant allele of H63D polymorphism appears to be associated with diabetic nephropathy.

Relationship of sociodemographic factors with serum levels of vitamin D in a healthy population of Pakistan.

High prevalence of vitamin D deficiency has been reported from Pakistan. Association of sociodemographic factors with vitamin D status has received little attention in this region. Therefore, we embarked on investigating the relationship of sociodemographic factors with vitamin D levels in a healthy Pakistani population. Venous blood from 226 healthy participants (age range 19-69 years) was collected and analyzed for serum concentrations of 25(OH) vitamin D [25(OH)D] and other related biomarkers. Demographic characteristics of the study participants were collected. Vitamin D deficiency (25(OH)D levels less than 20 ng/ml) was found to be 75% in this cohort. Gender, sunlight exposure and monthly household income emerged as predictors of hypovitaminosis D. Mean serum 25(OH)D levels in the groups with monthly household income less than Pakistani Rupees (PKR) 20,000, between PKR 20,000-50,000 and above PKR 50,000 were found to be 11.0±7.5, 13.9±9.6 and16.9±11.7 ng/ml, respectively. Using logistic regression the odds of having vitamin D deficiency was 3.22 (95% CI, 1.65-6.28) in the group with household income less than PKR 50,000 per month compared to the group with household income more than PKR 50,000 per month when the model was adjusted for gender and exposure to sunlight. There is an association between household income and hypovitaminosis D in a healthy Pakistani population.

Lack of association of statin use with vitamin D levels in a hospital based population of type 2 diabetes mellitus patients.

OBJECTIVE: To investigate the relationship of statins (drug given to reduce serum levels of LDL-cholesterol) on vitamin D levels of Pakistani type 2 diabetes mellitus (DM) patients in a hospital in Karachi. METHODS: In a cross-sectional survey, 312 consecutive patients with type 2 DM (219 males and 93 females, age 22-70 years) were recruited with informed consent. A questionnaire was administered to find out whether they were statin users or non-users. Serum was analyzed for concentrations of 25(OH) vitamin D [25(OH)D] and other related biomarkers such as serum cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, phosphate and calcium using kit methods. Multiple Linear Regression was used to evaluate association of statin use with serum levels of vitamin D while adjusting for related covariates including duration of statin use, duration of type 2 DM and smoking. RESULTS: Mean concentrations of serum cholesterol, and LDL-cholesterol were lower among statin users compared to statin non-users (P < 0.01), while HDL-cholesterol levels were higher (P<0.01). No relationship was observed between statin use and serum levels of vitamin D (P=0.768), when adjusted for age, gender, BMI, duration of type 2 DM, smoking, serum cholesterol and LDL-cholesterol. The adjusted regression coefficient (ß) and standard error [SE(ß)] for statin use duration were 0.012 (0.042), when serum levels of vitamin D was taken as an outcome. CONCLUSION: Lack of association was found between statin use and vitamin D levels in a hospital-based population of Pakistani patients with type 2 DM.

Association of Vitamin D binding protein polymorphism with risk of type 2 diabetes mellitus in a Pakistani urban population: A case control study.

OBJECTIVE: To assess if genotypes/diplotypes of vitamin D binding protein have any association with type 2 diabetes mellitus. METHODS: This case-control study was conducted from January 2013 to July 2015 at the endocrinology clinics of the Aga Khan University Hospital, Karachi, and comprised adult patients with type 2 diabetes and their age- and gender-matched healthy controls. Venous blood was obtained and assessed for serum/plasma 25 hydroxyvitamin D, parathyroid hormone, calcium, alkaline phosphatase and creatinine. Deoxyribonucleic acid was isolated and genotyping was done by polymerase chain reaction-restriction fragment length polymorphism procedures. RESULTS: Of the 330 participants, there were 165(50%) cases and as many controls. There were 116(70.3%) males and 49(29.7%) females in each group. The mean age of the patients was 48.82±9.23 years and that of the controls was 46.27±8.77 years (range: 22-70 years) (p=0.010) Mean serum concentration of 25 hydroxy vitamin D was significantly higher among the patients compared to the controls (p<0.001), but not significantly different by genotypes or diplotypes (p>0.05). Multiple conditional logistic regression revealed an association of group-specific 1-2 genotype with patients when adjusted for age, body mass index, and serum levels of 25 hydroxy vitamin D with matched adjusted odds ratio (95% confidence interval) being 3.1(1.22-7.88). CONCLUSIONS: Group-specific 1-2 genotype of vitamin D binding protein gene was associated with the risk of type 2 diabetes.

Association of vitamin D deficiency and VDBP gene polymorphism with the risk of AMI in a Pakistani population.

OBJECTIVE: To investigate the relationship of vitamin D deficiency and risk of AMI in a Pakistani population, and to find out any association between vitamin D binding protein (VDBP) genotypes and risk of AMI in this population. METHODS: In a comparative cross-sectional study, 246 patients (age: 20-70 years; 171 males and 75 females) with first AMI were enrolled with informed consent. Similarly, 345 healthy adults (230 males and 115 females) were enrolled as controls. Their fasting serum samples were analyzed for 25 (OH) vitamin D, lipids and other biomarkers using kit methods, while DNA was analyzed for VDBP genotypes using PCR-RFLP based methods. Chi-squared test and logistic regression were used for association of vitamin D deficiency and VDBP genotypes with AMI. RESULTS: Mean serum concentration of 25(OH) vitamin D was significantly lower in AMI patients compared to healthy subjects (p=0.015) and percent vitamin D deficiency was higher in AMI patients compared to healthy subjects (p=0.003). VDBP IF-IF genotype was positively associated with the risk of AMI in subject above 45 years after adjusting for potential confounders [OR = 9.86; 95% CI=1.16 to 83.43]. CONCLUSION: Vitamin D deficiency and VDBP IF-IF genotype are associated with AMI in Pakistani adults.

Association of vitamin D deficiency with poor glycaemic control in diabetic patients.

OBJECTIVE: An association between serum levels of vitamin D and glycaemic control in type-2 diabetes mellitus (DM) patients has been reported in some of the studies carried out in the West. However, there are no reports on this relationship in Pakistani diabetic patients. The aim of this study was to ascertain whether vitamin D levels have any influence on glycaemic control in Pakistani patients with type-2 DM. METHODS: In a cross-sectional survey, relationship between serum levels of 25-hydroxy vitamin D (25(OH)D) and glycated haemoglobin (HbA1C) was examined in 141 type-2 diabetic patients including 102 males and 39 females; age range 22 to 70 years, visiting the Aga Khan University Hospital during July 2013-April 2014. Venous blood was collected and analyzed for serum/plasma levels of 25(OH)D and related biomarkers using kit methods. HbA1C levels <7.0% and >7.0% were taken as indicators of good and poor glycaemic control, respectively. An association between 25(OH)D and HbA1C was investigated using regression analysis. RESULTS: Percent vitamin D deficiency (serum level of 25(OH)D < 20 ng/ml) was significantly higher in patients with poor glycaemic control compared to patients with good glycaemic control (58.7% vs. 30.6%; p-value=0.006). Binary logistic regression analysis revealed positive association between vitamin D deficiency and poor glycaemic control while adjusting for BMI, serum levels of albumin, alanine aminotransferase and alkaline phosphatase (OR, 4.86 (95% CI, 1.9-11.9; p-value<0.001). CONCLUSIONS: The association between vitamin D deficiency and abnormal HbA1C in Pakistani diabetic patients is suggestive that patients with hypovitaminosis D could benefit from vitamin D supplementation.

Polymorphisms in MTHFR, MS and CBS genes and premature acute myocardial infarction in a Pakistani population.

High prevalence of premature coronary heart disease in Pakistanis compared to other populations points towards the genetic predisposition of this population to develop this disease. Since no investigations have been carried out in Pakistan to study the relationship of polymorphisms in genes involved in homocysteine cycle, the objective of the present study was to find out if there is any association of methylenetetrahydrofolate reductase (MTHFR) C677T, A1298C; methionine synthase (MS) A2756G; cystathionine-ß-synthase (CBS) 844ins68, G919A polymorphisms with premature acute myocardial infarction (AMI) in a population of Pakistani patients with this disease. In a cross-sectional study, DNA samples of 143 AMI patients (age <45 years) and 153 healthy controls were genotyped for the above mentioned polymorphisms using PCR-RFLP methods. Plasma/serum samples of both patients and healthy controls were screened for homocysteine, folate and vitamin B12. One way ANOVA and chi-squared test were used for analysis of data. Mean plasma homocysteine levels in premature AMI patients and healthy controls were found to be 23±17.2 and 23±13.4 µmol/l, respectively which are higher than the upper normal limit of this biomarker (15µmol/l). MTHFR 677 CT genotype in healthy controls and MTHFR 677 TT genotype in AMI patients were found to have significantly increased levels of plasma homocysteine (p value <0.05), while all other polymorphisms did not show any significant difference in mean levels of homocysteine between AMI patients and healthy controls. Moreover, no association was observed between MTHFR C677T, A1298C; MS A2756C; CBS844ins68 polymorphisms and premature AMI in this population. This indicates that common polymorphisms in MTHFR, MS and CBS genes have no role in premature AMI in Pakistani population.

Short Communication: Lack of association between MTHFR gene polymorphisms and response to methotrexate treatment in Pakistani patients with rheumatoid arthritis.

Methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms have been reported to be associated with response to methotrexate (MTX) in certain populations of patients with rheumatoid arthritis (RA). This study aims at investigating any relationship of two single nucleotide polymorphisms (SNPs) in MTHFR gene, C677T and A1298C with response to therapy with MTX in Pakistani RA patients. Allelic frequencies of the two polymorphisms (C677T and A1298C) were determined in 67 RA patients (9 males and 58 females; mean age 42.87 ± 13.5 years) who had previously participated in a prospective clinical trial. Fifty-one patients had received MTX and were followed up for response up to 6 months. Genotyping of the two MTHFR polymorphisms was carried out using PCR-RFLP, while fasting concentration of plasma homocysteine was determined using a kit method. Twenty-eight patients were found to be "good responders", while twenty-three were "poor responders". MTHFR 1298C and MTHFR 677T alleles' frequencies in "good responders" were not different from frequencies in "poor responders" (0.574 vs. 0.521; p=0.6 and 0.197 vs. 0.196; p=0.75, respectively). Plasma homocysteine levels in female RA patients were significantly higher compared to general population in Karachi (13.1 ± 6.7 µmol/l vs. 11.4 ± 5.3 µmol/l; p<0.001). MTHFR C677T and A1298C polymorphisms are not associated with response to MTX in a population of Pakistani RA patients.

Association of body iron status with the risk of premature acute myocardial infarction in a Pakistani population.

BACKGROUND: Coronary artery disease is very common in Pakistani population. Some of the studies carried out on Western populations have shown a relationship between body iron status as determined by the ratio of concentrations of serum soluble transferrin receptor (sTfR) to ferritin and the risk of acute myocardial infarction (AMI). In order to investigate whether increased body iron status has any relationship with the risk of premature AMI in Pakistani population, a case-control study was carried out. METHODOLOGY/PRINCIPAL FINDINGS: In this case-control study, 203 consecutive AMI patients [146 males and 57 females; age range 18-45 years] admitted to the National Institute for Cardiovascular Diseases, Karachi, were enrolled with informed consent. In addition, 205 healthy controls whose gender and age (within 3 years) matched the patients, and who had a similar socio-economic background were recruited. Fasting venous blood was obtained and assessed for plasma/serum folate, vitamin B12, homocysteine, total cholesterol, triglycerides, LDL-cholesterol, HDL-cholesterol, sTfR and ferritin and blood lead. It was found that serum concentration of ferritin and blood lead levels were significantly higher in AMI patients compared to their age and gender-matched healthy controls (p value <0.05), while the concentrations of vitamin B12 and HDL-cholesterol were significantly lower in AMI patients compared to controls (p value <0.01). The ratio of sTfR to ferritin was significantly lower in AMI patients compared to controls [mean ± SD/median (IQR) values 84.7 ± 295/28.9 (38.4) vs 255 ± 836/49.4 (83.8), respectively; p value <0.001]. Compared with the highest quartile of sTfR/ferritin (low body iron status), the OR for the risk of AMI was 3.29(95% CI, 1.54-7.03) for the lowest quartile (quartile 1) when the model was adjusted for vitamin B12 and HDL-cholesterol (p value for trend <0.01). CONCLUSIONS/SIGNIFICANCE: This study shows a positive association between total body iron status and risk of premature AMI in a Pakistani population.