Haplotype analysis and linkage disequilibrium of MTHFR gene polymorphisms associated with recurrent thrombosis

Main Article Content

Mustafa Ihssan Abbas Al-Mahroos
Mahmmod Riyadh Alhaleem
Bahir Abdul Razzaq Mshimesh

Keywords

Haplotype, linkage disequilibrium, haplogenotype, recurrent thrombosis

Abstract

Haplotype analysis is the study of the origin of linked alleles set occurring on the same chromosome. Linkage disequilibrium (LD) is a population-based parameter that determines the inheritance or correlation between alleles of nearby genetic variants within a given population. The measurement of LD is important for biomedical research and is used in a wide range of applications. The study aimed to detect the true genetic effects of MTHFR polymorphisms that require a specific allele at several single nucleotide polymorphisms (SNPs) by using haplotype-based methods for analyzing all SNPs associated with recurrent thrombosis concurrently. This prospective case-control trial included 58 Iraqi patients (43 males and 15 females) aged between 20 and 64 years who suffered from different cardiovascular and thromboembolic disorders. The control group included 52 subjects composed of 41 males and 11 females without any thrombotic event history. The biochemical analysis of baseline homocysteine level, vitamin B12, and folic acid level and the genetic polymorphism of MTHFR for two specific variants C677T and A1298C were performed. The haplotype block CA was more frequent among the control group than in the patient group (55.77% versus 35.34%) with a significant difference (OR [odds ratio] = 0.32, 95%CI [confidence intervals] = 0.17–0.61, p < 0.001). In contrast, the haplotype block CC was more common in the patient group
(41.38%) than in the control group (20.19%) with a significant difference (OR = 3.11, 95%CI = 1.63–5.93, p < 0.001). The SNP C677T was in a strong LD (the measure D’ was 0.83) with A1298C. Seven haplogenotypes were identified: M1M1, M1M2, M1M3, M1M4, M2M2, M3M3, and M2M4. The most common haplogenotype was M1M2 (CC/AC) representing 24.55% of the total participants and was considered as the wild genotype. The haplogenotype M1M1 (CC/AA) was more frequent in the control group than in the patient group (32.69% vs. 6.9%) with a significant difference (OR = 0.14, 95%CI = 0.89–27.6, p = 0.004). In contrast, the haplogenotype M2M2 (CC/CC) was more frequent among the patient group (10.34%) than in the control group (3.85%) with a significant difference (OR = 3.82, 95%CI = 1.26–14.5, p = 0.018). The homozygous mutant genotype (CC) of the A1298C polymorphism was more common in patients with recurrent thrombosis than in those without recurrent thrombosis (33.33% vs. 12.9%) with a significant difference (OR = 3.8, 95%CI = 1.04–17.034). The most common recurrent thrombosis frequency for MTHFR combined genotypes was CC/CC and CT/AA (66.66%) from all cases, despite two-thirds of these cases being treated with aspirin or clopidogrel for secondary prevention of myocardial infarction and ischemic stroke. Other cases included were CC/AC (18.51%) and TT/AA (7.41%), as well as one case (3.71%) for each of CT/AC and CC/AA. The total percentage of CC/AC, TT/AA, CT/AC, and CC/AA for recurrent thrombosis cases was 33.34%, although 77.8% of these cases used aspirin or clopidogrel for secondary prevention of myocardial infarction and ischemic stroke. The median homocysteine in patients with recurrent thrombosis was higher than in those with no such disorder (75.5 μmol/L vs. 35.75 μmol/L), with a significant difference. The presence of C allele from the first SNP (CT) and A allele from the second SNP (AC) and the presence of CC/AA genotypes for two variants in individuals are considered as protective factors for exposure risk of ischemic heart disease, ischemic stroke, and venous thromboembolism. The individuals carrying C allele from the first SNP and C allele from the second SNP and those carrying CC/CC genotypes for two variants are at 3.11- and 3.82-fold risk of disease exposure respectively, compared to those carrying CC/AA genotypes. The SNP C677T was in a strong LD (the measure D’ was 0.83) with A1298C. Individuals carrying CC homozygous genotype of A1298C are at a 3.8-fold risk of recurrent thrombosis than those with AA wild genotype of the same SNPs due to high levels of homocysteine. This highlighted the role of homocysteine in atherogenic process, indicating that moderate-to-severe level of homocysteine is a potential risk factor for hypertension, cardiovascular disease, and stroke.

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