MTHFR (Ala 222 Val) polymorphism and AMI in patients with type II diabetes mellitus.

Abstract

The prevalent Ala222Val single nucleotide polymorphism of the MTHFR gene has been shown to be associated with type II diabetes. The objective of the present study was to find out whether there is genetic predisposition for development of acute myocardial infarction in type II diabetes mellitus among South Indian Tamil population. PCR-based restriction enzyme analysis was performed in DNA isolated from 120 acute myocardial infarction patients with diabetes mellitus and 100 non diabetic healthy individuals with no documented cardiovascular diseases. The results indicate that the MTHFR 677TT genotype is absent in both case and controls. The MTHFR 677CT genotype was observed among 32 (26.7 %) cases and 20 (20%) controls and the MTHFR 677CC genotype among 88 (73.3%) cases and 80 (80%) controls. The allelic frequencies were in accordance to Hardy Weinberg equilibrium. There was no statistical difference in genotype distribution between cases and controls. In conclusion, we suggest that the analysis of MTHFR genotyping for C677T polymorphism alone need not be considered to find out whether there is genetic predisposition for development of acute myocardial infarction in type II diabetes mellitus among South Indian Tamil population.

Full text

Page 1

137
Indian Journal of Clinical Biochemistry, 2009 / 24 (2) 137-141
MTHFR (ALA 222 VAL) POLYMORPHISM AND AMI IN PATIENTS WITH TYPE II DIABETES
MELLITUS
T Angeline, G Thiruvarutselvi, W Isabel, Rita Mary Aruna*, Rama Devi** and Nirmala Jeyaraj
PG & Research Department of Zoology & Biotechnology, Lady Doak College, Madurai- 625 002, Tamil Nadu,
* Penang International Dental College, Salem, and ** Institute of Biochemistry, Madras Medical College, Chennai.
ABSTRACT
The prevalent Ala222Val single nucleotide polymorphism of the MTHFR gene has been shown to be associated
with type II diabetes. The objective of the present study was to find out whether there is genetic predisposition
for development of acute myocardial infarction in type II diabetes mellitus among South Indian Tamil population.
PCR-based restriction enzyme analysis was performed in DNA isolated from 120 acute myocardial infarction
patients with diabetes mellitus and 100 non diabetic healthy individuals with no documented cardiovascular
diseases. The results indicate that the MTHFR 677TT genotype is absent in both case and controls. The
MTHFR 677CT genotype was observed among 32 (26.7 %) cases and 20 (20%) controls and the MTHFR
677CC genotype among 88 (73.3%) cases and 80 (80%) controls. The allelic frequencies were in accordance
to Hardy Weinberg equilibrium. There was no statistical difference in genotype distribution between cases
and controls. In conclusion, we suggest that the analysis of MTHFR genotyping for C677T polymorphism
alone need not be considered to find out whether there is genetic predisposition for development of acute
myocardial infarction in type II diabetes mellitus among South Indian Tamil population.
KEY WORDS
MTHFR gene, Type II diabetes, Polymorphism, Genetic predisposition, Acute Myocardial Infarction.
Address for Correspondence :
Dr. T. Angeline
PG & Research, Department of Zoology & Biotechnology,
Lady Doak College,Madurai- 625 002,
Tamil Nadu, India.
E-mail: angelanand@yahoo.co.in
INTRODUCTION
Diabetes mellitus is a complex, multifactorial and polygenic
disease and it is a major life threatening health problem all
over the world. Patients with diabetes mellitus (DM) have 2-6
fold increase in the prevalence of cardiovascular disease (1).
Acute myocardial infarction is a cardiac disease which occurs
due to sudden occlusion of coronary artery due to prolonged
ischemia. Ozmen et al found a high concentration of
homocysteine in patients with Diabetes mellitus and there are
also studies that report an independent association between
homocysteine and cardiovascular disease in patients with
Diabetes mellitus (2,3,4)
Previous study conducted among Tamil population suggests
hyperhomocysteinemia as a significant risk factor for acute
myocardial infarction (AMI) among Tamil population (5).
Methylene tetrahydrofolate reductase (MTHFR) is a key
enzyme in the remethylation cycle of homocysteine
metabolism. Cobalamin, riboflavin and folate are the vitamins
involved in the remethylation of homocysteine to methionine.
It has also been reported that simultaneous presence of
decreased serum cobalamin status, hyperhomocysteinemia
and MTHFR mutant genotypes (C677T and A1298C) might
lead to an increased risk for the occurrence of acute myocardial
infarction among South Indian Tamil population (6).
The single nucleotide polymorphism in the MTHFR gene
results in substitution of Alanine by Valine (Ala222Val) thereby
reducing the activity
hyperhomocysteinemia (7). The C677T polymorphism is
thought to decrease the binding affinity of FAD to MTHFR
and may increase the rate of dissociation of FAD from the
enzyme, leading to the dissociation of the active dimer into
monomers (8, 9,10).
of MTHFR causing

Page 2

Indian Journal of Clinical Biochemistry, 2009 / 24 (2)
138
The C677T polymorphism results due to a common C to T
transition at nucleotide 677 of the MTHFR gene (8).
Occurrence of mutant genotype has become the focus due to
its role in the pathogenesis of various diseases including
premature atherosclerosis (8, 11), coronary artery disease (12)
and complications of diabetes mellitus (13). As Diabetes
mellitus is a major risk factor for acute myocardial infarction, it
has been hypothesized that the MTHFR C677T SNP might
be associated with AMI in individuals with diabetes mellitus.
This study has been designed to find out the association
between MTHFR C677T SNP and AMI in individuals with Type
II diabetes mellitus.
MATERIALS AND METHODS
Fresh human blood was collected in EDTA coated tubes from
120 AMI patients (48-65 years) with diabetes mellitus, with no
other conventional cardiovascular
(hypercholesterolemia, hypertension, past history of heart
disease, family history and unhealthy lifestyles such as
smoking and alcoholism) and similar age and sex matched
100 non-diabetic healthy individuals with no cardiovascular
risk factors. The collected blood was stored in –20°C till
analysis. DNA isolation was carried out according to Sambrook
and Russel from the frozen blood (14).
risk factors
DNA was isolated from all the 220 study subjects. PCR-based
restriction enzyme analysis was performed in DNA isolated
from 120 AMI patients with diabetes mellitus and 100 non -
diabetic healthy individuals with no acute AMI.
PCR analysis(6): PCR amplification of a 198bp sequence of
the MTHFR gene was performed as previously described.
Approximately 120 ng of genomic DNA was incubated in a
total reaction volume of 50μL containing both the forward and
reverse primers for the MTHFR C677T (Ala222Val) SNP, using
2.5 U Taq DNA polymerase (Bangalore Genei, India).
Amplification for the MTHFR C677T SNP was performed with
an initial denaturation step at 93°C for 2 min in a thermal cycler
(Eppendorf India Limited). The PCR amplification conditions
were as follows: 34 cycles consisting of 1 min denaturation at
92°C, 1 min annealing at 64°C and 1 min extension at 72 °C.
The final cycle included a 10-min extension step at 72°C.
Restriction enzyme analysis: The MTHFR C677T SNP
creates a Hinf I (Fermentas Life Sciences, Germany) restriction
enzyme recognition sequence. The SNP was detected by
digestion of PCR amplified products with Hinf I for 3 hrs at
37°C. Restriction fragment size analysis was performed by
visualization of digested PCR products after separation by
gel electrophoresis in a 10% polyacrylamide gel.
Statistical analysis: The allele frequencies were calculated
by allele counting. Pearson Chi-square (χ2) test was performed
to find the statistical significance between the genotypes.
RESULTS AND DISCUSSION
After restriction digestion with Hinf I, the following fragment
sizing patterns were observed by the gel electrophoresis: the
677 C/C genotype results in no cleavage of the amplified 198bp
fragments, the 677C/T genotype results in 3 fragments of
198,175 and 23bp. MTHFR 677T/T genotype which will result
in complete cleavage of the 198bp fragment into 175 and 23bp
was not observed in any of the study subjects.
Genotype and allelic frequencies for the C677T SNP of the
MTHFR gene in AMI patients with diabetes mellitus and non
diabetic healthy individuals with no documented cardiovascular
diseases were calculated. Genotype frequencies obtained
between the AMI patients and controls did not differ significantly
from the frequencies predicted on the basis of the Hardy–
Weinberg law of population genetics (p>0.05).
When the prevalence of the MTHFR C677T mutation in the
methylenetetrahydrofolate reductase (MTHFR) gene among
South Indian Tamil population was determined and the
association of the mutant allele with AMI was evaluated, no
association was found between genotype distribution and AMI
(15). This study was conducted to find out association between
MTHFR C677T SNP and AMI in individuals with type II
diabetes mellitus.
The results indicate that the MTHFR 677TT genotype is absent
in both case and controls. The MTHFR 677CT genotype was
observed among 32 (26.7 %) cases and 20 (20%) controls
and the MTHFR 677CC genotype among 88 (73.3%) cases
Table 1: MTHFR C677T Polymorphism - Genotype and Allele
Frequency in AMI Patients and Controls
Genotype/AlleleTotal
(n=220)
Diabetic Patients
(n=120)
Controls
(n=100)
GENOTYPES (C677T)
CC16888 (73.3%)80 (80%)
CT5232(26.7%)20(20 %)
TT———
ALLELES
C 0.880.860.90
T 0.120.14 0.10

Page 3

139
and 80(80%) controls (Table 1). There is no deviation in the
“T” allele frequency (0.1) observed in the present study to
that established in the previous study (0.1) which was
conducted among the same south Indian Tamil population (15).
As allele frequencies are population specific, there is variation
in allelic frequencies in different populations (16) (Table 2).
A study conducted in Sweden by Zetterberg et al indicated
that the MTHFR polymorphisms may have a major impact on
foetal survival (18). Another study from India reported the
impact of MTHFR variants on foetal viability and gender. They
identified that high mortality was observed in individual with
one “T” allele (19). Similarly, several other studies conducted
among other populations including Canadians (20), have also
concluded that increase in mutant allele in MTHFR causes
decrease in foetal viability. Thus the absence of MTHFR 677
TT genotype in the study population may be explained by the
fact that the occurrence of the 677TT genotype may be
deleterious and it might have its impact on foetal viability as
described previously (19).
In the light of the above data, we conclude that the presence
of mutant TT genotype may be deleterious and this might be
the reason for the absence of TT genotype (2 mutant alleles)
among the study population in the present study.
Maeda et al found an association between mutant homozygous
genotype for MTHFR C677T and Diabetic retinopathy in
individuals with type II diabetes mellitus (21). Similarly, Ksaizek
et al have also found that the MTHFR C677T mutation in the
MTHFR gene predisposes type 2 diabetes patients to the
development of diabetic retinopathy. They observed a high
(13.5%) prevalence of mutant genotype in DM patients when
compared to the controls (9.5%) (22).
A study relating the MTHFR gene C677T mutation and left
ventricular hypertrophy (LVH) suggested the MTHFR mutant
genotype as a possible risk factor for the development of
LVH in the type II diabetes mellitus (23). Agullo-Ortuno et al
found an association between homocysteine levels and
diabetic complications such as macroangiopathy, retinopathy
and nephropathy in type I diabetes, whereas such an
association was not seen among type II diabetic
subjects (24).
Another study conducted among Israeli Jewish population with
Table 2: Prevalence of the MTHFR gene C677T mutation in different populations (17)
Country Ethnic populationNo. of subjects Genotype frequencyT allele
C/C C/T T/Tfrequency
India SouthIndians(Tamil)
Present study
100 802000.10
IndonesiaJavanese68 571100.81
JapanMongolian244 96116320.37
Australia Caucasians225 88113 240.36
BrazilAmerindian 129 7742100.24
Canada Caucasians414172 183 590.36
CanadaInuit 1741551720.06
ChinaMongolian 121 5153 170.35
EnglandCaucasians 2229697290.34
HollandCaucasians503 22423445 0.32
KoreaMongolian124 33 8290.40
Ireland Caucasians1309 600 568141 0.32
Italy (North)Caucasians130 4271 170.40
Italy (South) Caucasians431130223780.44
MexicoMexican250 44119 870.59
South Africa African 10785 220 0.10
Sub-Sahara African 3012633800.63
USAAfrican-American 49636312760.14
USAHispanics 169 63 71350.42
MTHFR Polymorphism and Diabetes Mellitus

Page 4

Indian Journal of Clinical Biochemistry, 2009 / 24 (2)
140
type 2 diabetes mellitus suggested that folate supplementation
in diabetic patients with the C677T mutation and low-normal
serum folate may prevent the onset or retard the progression
of diabetic nephropathy in type II diabetic patients (25).The
MTHFR C677T mutation of MTHFR gene was found to be
common in Chinese population and MTHFR C677T gene
polymorphism associated with a predisposition to increased
plasma homocysteine levels may represent a genetic risk
factor for diabetic nephropathy in Chinese type 2 diabetic
patients (26).
In conclusion, as there was no statistical difference in genotype
distribution between cases and controls, we suggest that the
analysis of MTHFR genotyping for C677T polymorphism alone
need not be considered to find out whether there is genetic
predisposition for development of acute myocardial infarction
in type II diabetes mellitus among South Indian Tamil
population.
ACKNOWLEDGEMENT
Financial assistance was provided to author by CSIR –New
Delhi (India) in the form of Senior Research Fellowship.
REFERENCES
1.Audelin MC, Genest J. Homocysteine and cardiovascular
disease in diabetes mellitus. Atherosclerosis 2001; 159:
497-511.
2.Ozmen B, Ozmen D, Turgan N, Habif S, Mutaf I, Bayindir O.
Association between homocysteinemia and renal function
in patients with type 2 diabetes mellitus. Ann Clin Lab Sci
2002; 32(3):279-86.
3.Chico A, Perez A, Cordoba A, Arcelus R, Carreras G, de
Leiva A, Gonzalez-Sastre F, Blanco-Vaca F. Plasma
homocysteine is related to albumin excretion rate in patients
with diabetes mellitus: a new link between diabetic
nephropathy and cardiovascular disease. Diabetologia
1998;41: 684-93.
4. Hoogeveen EK, Kostense PJ, Beks PJ, Mackaay AJ, Jakobs
C, Bouter LM, Heine
Hyperhomocysteinemia is associated with an increased risk
of cardiovascular disease, especially in non-insulin
dependent diabetes mellitus: a population-based study.
Arterioscler Thromb Vasc Biol 1998 ; 18: 133-8.
RJ, Stehouwer CD.
5.Angeline T, Rita Mary Aruna, Ramadevi K, Mohan G, Nirmala
J. Homocysteine status and acute myocardial infarction
among Tamilians. Ind J Clin Biochem 2005; 20 (1):18-20.
6. Angeline T, Nirmala J, Tsongalis GJ. MTHFR Gene
polymorphisms, B-vitamins and hyperhomocystinemia in
young and middle-aged acute myocardial infarction patients.
Exp Mol Pathol 2007; 227-33.
7. Spotila LD, Jacques PF, Berger PB, Ballman KV, Ellison RC,
Rozen R. Age dependence of the influence of Methylene
tetrahydrofolate reductase genotype on plasma
homocysteine level. Am J Epidemiol 2003; 158: 871-7.
8.Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA,
Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van
den Heuvel LP. A candidate genetic risk factor for vascular
disease: a common mutation in methylenetetrahydrofolate
reductase. Nat Genet 1995; 10 :111 –3.
9.Friedman G, Goldschmidt N, Friedlander Y, Ben-Yehuda A,
Selhub J, Babaey S, Mendel M, Kidron M,
Bar-On H. A common mutation A1298C in human
methylenetetrahydrofolate reductase gene, Association with
plasma total homocysteine and folate concentrations. Am
Soc Nutr Sci 1999; 129:1656–61.
10. Crott JW, Mashiyama ST, Ames BN, Fenech MF.
Methylenetetrahydrofolate reductase C677T polymorphism
does not alter folic acid deficiency-induced Uracil
incorporation into primary human lymphocyte DNA in vitro
Cancer. Epidemiol Biomarkers Prev 2001; 22: 1019 - 25.
11. Kluijtmans LA, van den Heuvel LP, Boers GH, Frosst P,
Stevens EM, van Oost BA, et al. Molecular genetic analysis
in mild hyperhomocysteinemia: a common mutation in the
methylenetetrahydrofolate reductase gene is a genetic risk
factor for cardiovascular disease. Am J Hum Genet 1996;
58: 35–41.
12. Morita H, Taguchi J, Kurihara H, Kitaoka M, Kaneda H,
Kurihara Y, et al. Genetic polymorphism of 5,10-
methylenetetrahydrofolate reductase (MTHFR) as a risk
factor for coronary artery disease. Circulation 1997; 95:
2032–6.
13. Neugebauer S, Baba T, Watanabe T. Defective homocysteine
metabolism as a risk factor for diabetic retinopathy. Lancet
1997; 349: 473–4.
14. Sambrook J, Russell DW. Molecular cloning, A laboratory
manual, 3rd ed. Cold Spring Harbor Laboratory 2001;
5.1-5.86.
15. Angeline T, Nirmala J, Granito S, Tsongalis GJ. Prevalence
of MTHFR gene polymorphisms (C677T and A1298C)
among Tamilians. Exp Mol Pathol 2004;77 (2):85-8.
16. Dekov V, Whincup P, Papacosta O, Ebrahim S, Lennon L,
Ueland PM, Refsum H, Humphries SE, Gudnason V. The
effect of the C677T and A1298C polymorphism in the
Methylene tetrahydrofolate reductase gene on homocysteine
levels in elderly men and women from the British Regional
Heart study. Atherosclerosis 2001; 154: 659-66.
17. Robien K, Ulrich CM. 5,10-Methylenetetrahydrofolate
reductase polymorphisms and leukemia risk: A Human
Genome Epidemology Review minireview. Am J Epidemiol
2003; 157: 571–82.

Page 5

141
18. Zetterberg H, Regland B, Palmér M, Ricksten A, Palmqvist
L, Rymo L, Arvanitis DA, Spandidos DA, Blennow K.
Increased frequency of combined methylenetetrahydrofolate
reductase C677T and A1298C mutated alleles in
spontaneously aborted embryos. Eur J Hum Genet 2002;
10: 113-8.
19. Devi RR, Govindaiah V, Ramakrishna G, Naushad SM.
Prevalence of methylene tetrahydrofolate reductase
polymorphism in South Indian population. Curr Sci 2004; 89
(3): 440–43.
20. Isotalo PA, Wells GA. Donnelly JG. Neonatal and fetal
methylenetetrahydrofolate
polymorphisms, An examination of C677T and A1298C
mutations. Am J Hum Genet 2000; 67:986–90.
reductase genetic
21. Maeda M, Yamamoto I, Fukuda M, Nishida M, Fujitsu J,
Nonen S, et al. MTHFR Gene Polymorphism as a Risk Factor
for Diabetic Retinopathy in Type 2 Diabetic Patients without
Serum Creatinine Elevation. Diabetes Care 2003; 26:
547-8.
22. Ksiazek P, Bednarek-Skublewska A, Buraczynska M. The
C677T methylenetetrahydrofolate reductase gene mutation
and nephropathy in type 2 diabetes mellitus. Med Sci Monit
2004; 10(2): BR47-51.
23. Yilmaz H, Agachan B, Ergen A, Karaalib ZE, Isbir T.
Methylene tetrahydrofolate reductase C677T mutation and
left ventricular hypertrophy in Turkish patients with type II
diabetes mellitus. J Biochem Mol Biol 2004; 37(2):234-8.
24. Agullo-Ortuno MT, Albaladejo MD, Parra S, Rodriguez-
Manotas M, Fenollar M, Ruiz-Espejo F, Tebar J, Martinez P.
Plasmatic homocysteine concentration and its relationship
with complications associated to diabetes mellitus. Clin Chim
Acta 2002; 326:105-12.
25. Shpichinetsky V, Raz I, Friedlander Y, Goldschmidt N, Wexler
ID, Ben-Yehuda A, Friedman G. The association between
two common mutations C677T and A1298C in human
methylenetetrahydrofolate reductase gene and the risk for
diabetic nephropathy in type II diabetic patients. J Nutr 2000;
130(10):2493-7.
26. Sun J, Xu Y, Zhu Y, Lu H. Genetic polymorphism of
methylenetetrahydrofolate reductase as a risk factor for
diabetic nephropathy in Chinese type 2 diabetic patients.
Diabetes Res Clin Pract 2004; 64(3):185-90.
MTHFR Polymorphism and Diabetes Mellitus