Nutritional environmental and genetic causes of hyperhomocysteinemia in a Pakistan population
Date of Award
Doctor of Philosophy in Health Science (PhD)
Biological and Biomedical Sciences
Introduction: Hyperhomocysteinemia have been found to be associated with cardiovascular disease. The objectives of this study were to find out the prevalence of hyperhomocysteinemia and deficiencies of folate, vitamin B6 and vitamin B12 in a population in Karachi. We also identified major dietary patterns and investigated their association with plasma homocysteine and assessed the relationship of blood lead (Pb) to the risk of hyperhomocysteinemia in this population. Study objectives also included: the determination of the prevalence of C677T and A1298C mutations in MTHFR gene, A2756G mutation in MS gene, and T833C/844ins68 and G919A mutations in CBS gene; investigation of any relationship between these mutations (and their haplotypes) and hyperhomocysteinemia in the study population; study of gene-gene, gene-nutrient and gene-environment interactions with hyperhomocysteinemia; and determination of the impact of vitamin(s) supplementation on plasma homocysteine levels in vitamin-deficient individuals. Methodology: In a cross sectional survey, 872 healthy adults (355 males, 517 females; age 18-60 years) were recruited from a low income urban population of Karachi. Fasting 11111 venous blood was obtained and assessed for blood Pb and plasma/serum homocysteine, folate, pyridoxal phosphate (PLP, a coenzymic form of vitamin B6) and vitamin B12. DNA was isolated and single nucleotide polymorphisms of the genes (MTHFR, MS and 1111 CBS) were studied by polymerase chain reaction followed by restriction fragment length 111110 polymorphism assay. Dietary intake was assessed by a food frequency questionnaire and factor analysis was used to define major dietary patterns. Results: In the study population, prevalence of hyperhomocysteinemia (> 15pmo1/1) was 32%. Similarly, percent folate (< 3.5 ng/ml), vitamin B12 (< 200 pg/ml) and vitamin B6 (< 20 nmol/1) deficiencies in this population were found to be 27.5%, 9.74%, and 33.7%, respectively. Hyperhomocysteinemia was associated with male sex, folate deficiency and vitamin B12 deficiency [OR (95%CI), 8.3(5.7-12.1); 2.5(1.76-3.58); 2.6(1.5-4.5)], respectively. Three major dietary patterns were identified and labeled as "prudent diet", "high animal-protein diet" and "high plant-protein diet". We observed protective effect of a prudent dietary pattern for the highest quartile of intake in comparison with the lowest quartile on hyperhomocysteinemia when the model was adjusted for age, gender, household income, body mass index (BMI), tobacco chewing and smoking; OR = 0.52 (95% CI = 0.30 - 0.90). The high plant-protein diet pattern was inversely related to hyperhomocysteinemia with a higher intake being protective. Compared with the first quartile, the adjusted OR was 0.42 (95% CI = 0.25 — 0.69) for the fourth quartile. The high animal-protein diet showed a positive association with hyperhomocysteinemia with participants in the highest quartile of intake having the highest increase in risk; OR = 2.10 (95% CI = 1.22 — 3.60). The study population had mean ± SD blood Pb of 11.65 ± 5.5 pg/d1. Prevalence of high blood Pb (levels > 10 pg/d1) was higher in males compared to females (62.5% males vs 56% females; p value =0.05). Mean ± SD value of plasma homocysteine was significantly higher in the highest quartile of blood Pb compared to the lowest quartile (16.13 ± 11.2 pmo1/1 vs 13.28 ± 9.7,umo1/1; p value < 0.001). Daily consumption of fruit juice had a positive influence on both levels of plasma homocysteine and blood Pb. Compared with the lowest quartile of blood Pb, the OR for hyperhomocysteinemia was 1.69 (95% CI, 1.00 to 2.85) for the fourth quartile when the model was adjusted for age, gender, folate and vitamin B12. The frequencies of MTHFR 677 and MTHFR 1298 mutant alleles (677T and 1298C) were found to be 0.15 and 0.55, respectively. The prevalence of heterozygote "AG" individuals of MS (A2756G) was 38.8% with mutant allele "G" frequency of 0.29. The mutant allele "I" frequency of CBS 844ins68 insertion was 0.07 with heterozygous "WI" insertion prevalence to be. 13.2%. CBS T833C polymorphism was present in all alleles carrying the CBS 844ins68 insertion. Genotyping for CBS G919A mutation showed that all the individuals had GG genotype. The mean homocysteine concentration was significantly higher in individuals who were either heterozygous or homozygous variant for MTHFR C677T mutation as compared to the wild type CC genotype (p=0.04 and p<0.001, respectively). It has also been observed that serum folate levels were significantly less in MTHFR TT genotype with respect to other genotypes of MTHFR 677 (p=0.004). Compared to the wild type MTHFR 677 genotype "CC", the odds of having hyperhomocysteinemia with homozygous "TT" variant was 10.17 (p< 0.001) after adjustment for age, gender, folate and vitamin B 12 levels. We also observed protective effect towards hyperhomocysteinemia with heterozygous genotype of CBS 833ins64 when compared to wild type CBS 833ins64 [OR (95% CI); 0.58 (0.34-0.99)]. Regarding influence of gene-gene interaction on plasma homocysteine, mean plasma levels in individuals who were carriers of variants of CBS and MS genes (CBS 844ins68 WI; MS 2756 AG) were lower compared to those who were carriers of either variant (CBS 844ins68 WW, MS 2756 AG or CBS844ins68 WI, MS 2756 AA). We have also observed that individuals having MTHFR TT variant along with MTHFR 1298 AC variant had 8.78 times increased risk of hyperhomocysteinemia. MTHFR TT variant along with MS 2756 AA variant increased the risk for hyperhomocysteinemia by 19.1 fold, while MTHFR TT variant along with CBS 833ins64 WW variant had 14 times higher risk for hyperhomocysteinemia. Regarding geneenvironment interaction, the risk also increased significantly with combinations of MTHFR C677T variants (CT or TT) with folate and vitamin B12 deficiency states (OR 4.84 and 4.33, respectively). Individuals who had either heterozygous "CT" or homozygous "TT" mutant genotype of MTHFR 677 and also had blood Pb levels greater than 10 ug/dl, have increased risk for hyperhomocysteinemia [OR (95% CI) 2.4 (1.43- 4.24); p=0.001] compared with those having "CC" genotype of MTHFR 677 and blood Pb levels less than or equals to 10 ug/dl. A three-week supplementation with folic acid (5 mg/day), methycobalamin (0.5 mg/day) and pyridoxine hydrochloride (50 mg/day) in vitamin-deficient subjects (n=194) decreased plasma homocysteine levels by 37%. Conclusions: High prevalence of folate, vitamin B12, and vitamin B6 deficiencies appears to be the major determinant of hyperhomocysteinemia in a general population in Karachi. A diet rich in fruits and uncooked-vegetables decreased the risk of hyperhomocysteinemia, while diets rich in red meat, chicken and tea with milk were found to be positively associated with hyperhomocysteinemia. This study also showed a relationship between blood Pb and hyperhomocysteinemia suggesting that the harmful effect of Pb on cardiovascular system could be due to its association with hyperhomocysteinemia. MTHFR 677 TT genotypes is strongly associated with hyperhomocysteinemia in study population. Current data suggest that environmental factors (exposure to Pb), nutritional factors (diet and B vitamin status) and genetic factors (especially MTHFR 677 TT genotype) are main determinants of the mild hyperhomocysteinemia observed in study population. The overall contribution of blood Pb, folate and vitamin B12 status, male gender and genetic polymorphisms in 3 genes (MTHFR, MS, CBS) towards plasma homocysteine in this population turns out to be 30%. Since hyperhomocysteinemia is a pathogenic risk factor for atherosclerosis, B vitamin supplementation and increased consumption of high plant protein diet and fruit juices would be of extreme importance in reducing the risk of cardiovascular disease which is highly prevalent in Pakistani population.
Yakub, M. (2010). Nutritional environmental and genetic causes of hyperhomocysteinemia in a Pakistan population (Unpublished doctoral thesis). Aga Khan University, Karachi, Pakistan.