|Year : 2021 | Volume
| Issue : 3 | Page : 140-145
Association between serum homocysteine levels and methylene-tetrahydrofolate-reductase gene polymorphism in patients with stroke: A study from a tertiary care teaching hospital from Central India
Dinesh Chouksey, Hashash Singh Ishar, Rahul Jain, Sunil Athale, Ajoy Sodani
Department of Neurology, SAMC and PGI, Indore, Madhya Pradesh, India
|Date of Submission||07-Jun-2020|
|Date of Decision||10-Jul-2020|
|Date of Acceptance||27-Jul-2020|
|Date of Web Publication||06-Oct-2020|
Dr. Dinesh Chouksey
Department of Neurology, SAMC and PGI, Indore, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
Background: Hyperhomocysteinemia (HHcy) is a well-established risk factor for vascular thrombosis leading to stroke. Homocysteine (Hcy) is mainly synthesized in the methionine cycle, and it is an intermediate during the conversion of methionine to cysteine. HHcy can develop by polymorphism of several enzymes like methylene-tetrahydrofolate-reductase (MTHFR) mutation and due to deficiency of co-factor Vitamins such as B12, B6, and folic acid. This study was planned to see the association between HHcy and MTHFR gene polymorphism in patients with stroke in populations from central India. Methods: Seventy-two patients diagnosed with stroke aged 18 years and above who met the inclusion criteria were selected. Hcy level and MTHFR mutation were identified among patients of stroke. Results: Forty-two patients (58.33%) were diagnosed with acute ischemic stroke as compared to cerebral venous thrombosis (30, 41.66%) from a total of 72 patients. The statistical analysis projected that the HHcy diagnosed among more (45, 62.5.0%) cases with stroke who had aged ≤45 years as compared to cases who had aged >45 years (17, 23.6%). None of our patients tested positive for either CT (Heterozygous) or TT(Homozygous) genotypes. Conclusions: HHcy is not associated with MTHFR gene mutation in our study group, and this possibly is related to the deficiency of co-factors in Hcy metabolism. Young age group stroke patients have higher Hcy levels.
Keywords: Homocysteine, hyperhomocysteinemia, methylene-tetrahydrofolate-reductase gene mutation, Hyperhomocysteinemia in young stroke
|How to cite this article:|
Chouksey D, Ishar HS, Jain R, Athale S, Sodani A. Association between serum homocysteine levels and methylene-tetrahydrofolate-reductase gene polymorphism in patients with stroke: A study from a tertiary care teaching hospital from Central India. J Med Sci 2021;41:140-5
|How to cite this URL:|
Chouksey D, Ishar HS, Jain R, Athale S, Sodani A. Association between serum homocysteine levels and methylene-tetrahydrofolate-reductase gene polymorphism in patients with stroke: A study from a tertiary care teaching hospital from Central India. J Med Sci [serial online] 2021 [cited 2021 Jun 22];41:140-5. Available from: https://www.jmedscindmc.com/text.asp?2021/41/3/140/297429
| Introduction|| |
Stroke is the second-leading cause of death and neurologic disability worldwide; almost 80%–85% of strokes are ischemic in origin and rest are hemorrhagic. Despite recent advances in the care of patients with acute ischemic stroke (AIS), prevention remains the optimal strategy to reduce the burden of a stroke at the population level. Stroke is due to several mechanisms include cardioembolism, prothrombotic states, and disease of large and small cerebral vessels. Elevated total homocysteine (Hcy) status is a strong and independent risk factor for occlusive arterial disease in the coronary, cerebral and peripheral vessels, and is also a risk factor for venous thrombosis. Hcy is a natural homologous compound of amino acid cysteine. Hcy is not directly obtained from the diet, but actually, it is mainly synthesized during the methionine cycle., Hcy is an intermediate during the conversion of methionine to cysteine. Plasma Hcy levels of ≥15.0 μmol/L are referred to as hyperhomocysteinemia (HHcy). The Hcy concentration increase seems to be caused mainly by the widely diffused polymorphisms of several enzymes. Prolonged exposure to this condition can lead to the development of atherosclerosis, stroke, inflammatory syndromes like osteoporosis and rheumatism, as well as neuronal pathologies, including Alzheimer's and Parkinson's diseases. Studies have shown that when Hcy levels rise by 5 μmol/L, the risk of cerebrovascular disease and coronary heart disease increases by 59% and 32%, respectively. However, when the Hcy level was reduced by 3 micro mol/l, the risk of stroke and coronary heart disease decreased by 24% and 16%, respectively. Indian studies examining the prevalence of HHcy in the community have reported a much higher incidence of 52%–84%., The deficiency of Vitamin B12 and folic acid results in impaired conversion of Hcy to methionine and cysteine, resulting in HHcy.
Apart from a deficiency of vitamins, a second important cause for HHcy is a genetic defect in enzymes that metabolize Hcy. Methylene-tetrahydrofolate-reductase (MTHFR) is encoded by the MTHFR gene. 5, 10-Methylene tetrahydrofolate is transformed into 5-methyl-tetrahydrofolic acid by MTHFR, which is a necessary step for the transformation of Hcy into methionine, and it is a key enzyme in the methionine circulating pathway.,
The most common genetic reason for HHcy may be the conversion from C to T and A to C in C677T and A1298C, respectively. However, the frequency of the mutation and the activity of the enzyme in MTHFR (C677T and A1298C) are influenced by race and geographical locations., The frequency of C677T mutation, for instance, was found to be 7.8% among the population in India. In contrast to the west, Indian studies have reported a much higher HHcy at the incidence of 52%–84%. The mean Hcy levels too were quite high, varying from 19.5–23.2 mmol/L.,, Given these high levels, it is felt that HHcy can be considered to be an important risk factor for stroke in Indians. This study was planned to see the MTHFR gene mutation leading to HHcy in this part of the world.
| Methods|| |
The aim is to study the relationship between HHcy and MTHFR gene mutations in patients of stroke from central India. The study is approved by SAIMS. The approval number is SAIMS/IEC/22/20. The approval date is 06/06/2020.
- To see the prevalence of HHcy in this part of central India
- To correlate whether the age, sex, smoking status, diabetes mellitus, hypertension, and dietary status are associated with HHcy in patients with stroke.
A total of 73 patients were enrolled in the study initially; however, one patient excluded as her final diagnosis was benign intracranial hypertension. All the participants were patients diagnosed with stroke, ischemic and cerebral venous thrombosis [SIC] and those willing to do the MTHFR mutation test. The participants were recruited from January 2018 to October 2019. The diagnostic criterion of HHcy was a level of Hcy ≥15 micro mol/l. Verbal consents from all the participants were obtained as a part of management. The blood sample was taken for Hcy and MTHFR genetic study by PCR method.
The patients diagnosed with stroke (ischemic) and cerebral venous thrombosis (CVT) (SIC). Patients aged 18 and more.
Age <18 years, hemorrhagic stroke, pregnant females were excluded.
Responses of frequencies were calculated and analyzed using the raw data of 72 subjects. Statistical software, SPSS version 17.0 Trial (SPSS version 17.0 trial by IBM Chicago USA), was used for the analysis. The prevalence of an outcome variable, along with 95% confidence limits, was calculated. Both descriptive and inferential statistics were used to study the clinical profile and selected clinical outcomes in patients with SIC. A descriptive statistic has used to depict the main features and characteristics of the collected samples presented with SIC. Results on continuous measurements are presented on mean ± standard deviation (Min–Max), and results on categorical measurements are presented in numbers (%). Prevalence of HHcy (≥15 μmol/L) concerning age (≤45 and >45 years) and gender (male and female) of patients presented with SIC was calculated. Karl Pearson's coefficient of correlation had used to identify the extent of strength and direction of the relationship of age and gender of patients presented with SIC with HHcy (μmol/L). The nonparametric test, Pearson's Chi-square test used to investigate the association of age, sex, smoking status, diabetes mellitus, hypertension, dietary status, and MTHFR mutation of patients with the level of Hcy.
The following are the notations used to present the significance of observed probability value for various selected parameters.
Insignificant/Not Significant (P value: P > 0.05).
Moderately Significant/Significant (P value: P < 0.05 to P < 0.02).
Highly/Strongly significant (P value: 0.01 < P < 0.001).
| Results|| |
Forty-two patients (58.33%) were diagnosed with AIS, thirty (41.66%) as CVT from a total of 72 patients. AIS was the diagnosis made among approximately half (49.3%) of the patients with stroke with HHcy (≥15 μmol/L) than those diagnosed with CVT (35.6%). Out of a study population diagnosed with SIC, more than three-fourth (86.1%) of the patients were male. The age of all patients with SIC was obtained in the ranges from 18 to 74 years. The mean age (mean ± standard deviation) of all patients with SIC found to be 38.88 ± 10.44 years. The mean age of male (39.85 years) patients with SIC found to be higher as compared to the mean age of female (33.36 years) patients.
[Table 1] statistical analysis projected that the HHcy diagnosed among more (45, 62.5%) cases with stroke those had aged ≤ 45 years as compared to cases had aged >45 years (17, 23.6%). The proportional differences in sex demonstrated that the HHcy found to be more prevalent among males (54, 75.0%) as compared to female (8, 11.1%) patients with stroke. Further, an investigation revealed that the urban population (34, 47.2%) of patients with stroke found to be more prone to HHcy than rural (28, 38.8%). The patients with stroke who were smokers (43, 59.7%) found at a large risk of HHcy than those who did not present with the smoking habit (19, 26.3%). A dietary habit of patients with stroke demonstrated that the HHcy found to be more prevalent among vegetarians (40, 55.5%) as compared to nonvegetarian (22, 30.5%). Nevertheless, the association of age (P > 0.05), sex (P > 0.05), dwelling (P > 0.05), smoking status (P > 0.05), and dietary habit (P > 0.05) of the patient of SIC with Hcy levels could not satisfy the limit of statistical significance and were statistically insignificant. The odds ratio for the dwelling, i.e., urban and rural population (2.83, confidence interval [CI] 0.67–11.98) found to be higher as compared to smoking status (1.51, CI 0.38–5.97), age (1.13, CI 0.26–4.90), dietary habit (1.21, CI 0.31–4.76), and sex (1.69, CI 0.30–9.40) though P value is not statistically significant. Diabetes mellitus, hypertension, and diagnosis in [Table 2] (AIS and CVT) did not observe with statistically significant P value impactions on HHcy in patients of stroke. HHcy reported among 86.1% of patients with stroke diagnosed with a homozygous allele (CC). However, either CT ot TT mutant copy of gene mutation found to be absent in the present study as these types of gene mutations found to be rare in the Indian scenario.
|Table 1: Association of demographic characteristics of stroke patients with homocysteinemia levels|
Click here to view
[Figure 1] shows the correlation between the ages of the patient (X-axis) with Hcy (micro mol/l at Y-axis). It is noted that the correlation of the age of the stroke patient with Hcy found to be poor in a negative direction of the relationship and statistically not significant (P > 0.05).
|Figure 1: The negative correlation of serum homocysteine (mmol/l) with age (years), as the age is increasing the incidence of hyperhomocysteinemia drops|
Click here to view
| Discussion|| |
This is the first study from rural-based teaching institute from central India to study the association between serum Hcy levels and MTHFR gene polymorphism in patients with stroke and CVT. Several studies have been reported on the modulation of risk of stroke by MTHFR C677T polymorphisms because plasma Hcy (main substrate of MTHFR enzyme) levels are considered a major risk factor for stroke. We found that in all patients tested for MTHFR mutation had CC genotype which is considered normal and in one study done in India was found to be present in 797 (80%) out of 1000 healthy patients in contrast to CT, which was present in 192 (19%) and TT, which was present in 11 (1%). None of our patients tested positive for either CT (Heterozygous) or TT (Homozygous) genotypes and those are associated with an increased risk of vascular events. These results were consistent with many studies done in India in the past, in one study from Hyderabad TT genotype was found in only 1.4% of HHcy cases with stroke; in another study done in Kashmir, MTHFR C677T gene polymorphism was not found to be significantly associated with risk of ischemic stroke., However, few reports are indicating the high prevalence of heterozygous genotype (CT) in certain populations and their association with HHcy., In another study done in Tamilians CT genotype was seen in 18.1% and TT genotype in 1.38%. Thus, there is marked geographic variation in the frequency of C677T polymorphisms in Indians. Among studies done worldwide failed to confirm a robust association between HHcy and MTHFR mutation, like in the study of Szolnoki et al. neither CT nor TT mutations were found to be associated with ischemic stroke. In another study by Dikmen et al., there was no significant difference between MTHFR T677T and A1298a genotypes and allele frequencies in stroke patients and controls, and they concluded that the MTHFR genotype might be a modest risk factor for stroke in the Turkish population. Besides, racial-ethnic differences in folate metabolism and localized dietary differences may account for the disparity. In another study done at Ruby Hall Clinic, Pune serum Hcy, B12, and folate were estimated in consecutive cases of ischemic stroke, arterial, or venous infarction. They found HHcy in 80.95% of patients and hypovitaminosis B12 (<200 pg/ml) in 75.8% in the pure vegetarian group. Thus, in the Indian population, deficiency of B12 and B6 group of a vitamin can be more responsible for HHcy as compared to MTHFR mutations. In this study, out of a total of 72, around 40 HHcy patients were vegetarians and as we know that a vegetarian diet is a poor source of Vitamin B12. In this study, HHcy was present more in young, 45 patients were <45 years of age, thus, as the age is increasing, the Hcy levels got decreased, and it can be postulated that other risk factors come into picture resulting in a stroke. In this study, all 15 diabetic patients had HHcy; this was consistent with some studies done worldwide, in the Hoorn study, the effect of HHcy on the 5-year mortality in diabetic patients was compared to nondiabetic patients. For each five mmol/l rise in Hcy levels, the risk of 5-year mortality rise by 17% in nondiabetic and 60% in diabetic participants. Thus, HHcy appears to be a stronger (1.9-fold) risk factor for mortality in type 2 diabetic patients than in nondiabetic patients. In another study done by Wadia et al. HHcy was recorded in 76% of diabetic patients; another study was done in Jaslok hospital Mumbai, recorded significantly higher Hcy levels in diabetic patients as compared to controls., In our study, HHcy was present in 36 out of 42 patients with AIS and 26 out of 30 patients with CVT, which is statistically insignificant between the two groups and is consistent with studies done in the past. In Ruby Hall study, they found HHcy among 109 out of 135 cases (80.74%) diagnosed with AIS and among 21 out of 28 (75.6%) diagnosed with CVT.
The mechanism by which HHcy is causing a stroke is cerebral endothelial cell dysfunction by oxidative stress. Acid sphingomyelinase and ceramide are mediators of cell death caused by the number of cellular events and exogenous stimuli. A study by Lee et al. demonstrated that Hcy and C2-ceramide could cause murine cerebral endothelial cell death. Hcy induces cerebral endothelial cell death, possibly by activating the Acid sphingomyelinase–ceramide pathway.
Limitations of the study are a local, regional group of the study population from a single center with a small number of patients and associated risk factors of stroke other than HHcy.
| Conclusions|| |
HHcy is not associated with MTHFR gene mutation in our study group, and this is possibly related to low co-factor, especially Vitamin B12 and folic acid, needed for Hcy metabolism that might be due to dietary habits and might be due to other factors like GI metabolism and absorption of these vitamins. Patients of younger age, smoking habits, and diabetes are more prone to HHcy with the risk of vascular thrombosis. These specific groups should be targeted by our health-care team to prevent devastating disabled illnesses like stroke, and we also look for a more vigorous approach toward diagnosing HHcy and low Vitamin B12 levels in patients of ≤ 45 years of age with the hope that by reducing HHcy levels we could reduce the rate of young stroke.
Declaration of patient consent
I am as corresponding author certify that we had obtained all appropriate patients consents and their demographic data and investigations were used for publication. Patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Campbell BCV, De Silva DA, Macleod MR, Coutts SB, Schwamm LH, Davis SM, et al
. Ischaemic stroke. Nat Rev Dis Primers 2019;5:70.
Rozen R. Genetic modulation of homocysteinemia. Semin Thromb Hemost 2000;26:255-61.
Zaric BL, Obradovic M, Bajic V, Haidara MA, Jovanovic M, Isenovic ER. Homocysteine and Hyperhomocysteinaemia. Curr Med Chem 2019;26:2948-61.
Finkelstein JD. Methionine metabolism in mammals. J Nutr Biochem 1990;1:228-37.
Hankey GJ, Eikelboom JW. Homocysteine and vascular disease. Lancet 1999;354:407-13.
Tinelli C, Di Pino A, Ficulle E, Marcelli S, Feligioni M. Hyperhomocysteinemia as a Risk Factor and Potential Nutraceutical Target for Certain Pathologies. Front Nutr 2019;6:49.
Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: Evidence on causality from a meta-analysis. BMJ 2002;325:1202.
Refsum H, Yajnik CS, Gadkari M, Schneede J, Vollset SE, Orning L, et al
. Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asian Indians. Am J Clin Nutr 2001;74:233-41.
Sonkar SK, Kumar S, Singh NK, Tandon R. Hyperhomocysteinemia induced locked-in syndrome in a young adult due to folic acid deficiency. Nutr Neurosci 2019 Oct 24:1-3.
Richard E, Desviat LR, Ugarte M, Pérez B. Oxidative stress and apoptosis in homocystinuria patients with genetic remethylation defects. J Cell Biochem. 2013;114:183-91.
Födinger M, Hörl WH, Sunder-Plassmann G. Molecular biology of 5,10-methylenetetrahydrofolate reductase. J Nephrol 2000;13:20-33.
Markan S, Sachdeva M, Sehrawat BS, Kumari S, Jain S, Khullar M. MTHFR 677 CT/MTHFR 1298 CC genotypes are associated with increased risk of hypertension in Indians. Mol Cell Biochem 2007;302:125-31.
Esfahani ST, Cogger EA, Caudill MA. Heterogeneity in the prevalence of methylenetetrahydrofolate reductase gene polymorphisms in women of different ethnic groups. J Am Diet Assoc 2003;103:200-7.
Yang B, Liu Y, Li Y, Fan S, Zhi X, Lu X, et al
. Geographical distribution of MTHFR C677T, A1298C and MTRR A66G gene polymorphisms in China: Findings from 15357 adults of Han nationality. PLoS One 2013;8:e57917.
Saraswathy KN, Asghar M, Samtani R, Murry B, Mondal PR, Ghosh PK, et al
. Spectrum of MTHFR gene SNPs C677T and A1298C: A study among 23 population groups of India. Mol Biol Rep 2012;39:5025-31.
Katsiki N, Perez-Martinez P, Mikhailidis DP. Homocysteine and non-cardiac vascular disease. Curr Pharm Des 2017;23:3224-32.
Angeline T, Jeyaraj N, Granito S, Tsongalis GJ. Prevalence of MTHFR gene polymorphisms (C677T and A1298C) among Tamilians. Exp Mol Pathol 2004;77:85-8.
Perry IJ. Homocysteine and risk of stroke. J Cardiovasc Risk 1999;6:235-40.
Yadav U, Kumar P, Gupta S, Rai V. Distribution of MTHFR
C677T Gene polymorphism in healthy north indian population and an updated meta-analysis. Indian J Clin Biochem 2017;32:399-410.
Alluri RV, Mohan V, Komandur S, Chawda K, Chaudhuri JR, Hasan Q. MTHFR C677T gene mutation as a risk factor for arterial stroke: A hospital based study. Eur J Neurol 2005;12:40-4.
Nissar S, Rasool R, Bashir A, Sameer AS. MTHFR C677T polymorphism and risk of ischemic stroke in Kashmiri population. Hereditary Genet 2015;4:155.
Sarinee P, Thanyachai S, Katcharin A, Manisa B, Vichai A. Prevelance of the C677T methylenetetrahydrofolate reductase mutation in Thai patients with deep vein thrombosis. Acta Haematol 2000;103:4:191-6.
Ghosh K, Shetty S, Madikaikar M, Pawar A, Nair S, Khare A, et al
. Venous thromboembolism in young patients from western India: A study. Indian Heart J 2001;2:158-65.
Szolnoki Z, Somogyvári F, Szabó M, Kondacs A, Fodor L, Melegh B. Interactions between the MTHFR C677T and MTHFR A1298C mutations in ischaemic stroke. Ideggyogy Sz 2006;59:107-12.
Dikmen M, Ozbabalik D, Gunes HV, Degirmenci I, Bal C, Ozdemir G, et al
. Acute stroke in relation to homocysteine and methylenetetrahydrofolate reductase gene polymorphisms. Acta Neurol Scand 2006;113:307-14.
Wadia RS, Edul NC, Bhagat S, Bandhishi S, Kulkarni R, Sontakke S, et al
. Hyperhomocysteinaemia and Vitamin B12 defi ciency in ischaemic strokes in India. Ann Indian Acad Neurol 2004;7:387-92. [Full text]
Hoogeveen EK, Kostense PJ, Jakobs C, Dekker JM, Nijpels G, Heine RJ, et al
. Hyperhomocysteinemia increases risk of death, especially in type 2 diabetes: 5-year follow-up of the Hoorn Study. Circulation 2000;101:1506-11.
Ridker PM, Shih J, Cook TJ, Clearfield M, Downs JR, Pradhan AD, et al
. Plasma homocysteine concentration, statin therapy, and the risk of first acute coronary events. Circulation 2002;105:1776-9.
Lee JT, Peng GS, Chen SY, Hsu CH, Lin CC, Cheng CA, et al
. Homocysteine induces cerebral endothelial cell death by activating the acid sphingomyelinase ceramide pathway. Prog Neuropsychopharmacol Biol Psychiatry 2013;45:21-7.
[Table 1], [Table 2]