Association of Methylene Tetrahydrofolate Reductase C677T Genotype With Type 2 Diabetes Mellitus Patients With and Without Renal Complications

Introduction

The incidence of type 2 diabetes mellitus (T2DM) has shown an alarming upward trend globally, especially in the Indian population. An estimate suggests that the number of individuals with DM in India would increase from 31.7 million in the year 2000 to 79.4 million by 2030 (Wild et al., 2004). The disease has a strong genetic origin, but lifestyle factors such as obesity and physical inactivity serve as major risk factors for its development. With the increase in DM, there is also a corresponding increase in the related complications such as cardiovascular disease, retinopathy, neuropathy, and nephropathy (Keane and Eknoyan, 1999).

Renal disease/nephropathy is a devastating complication of DM and a major cause of end-stage renal failure (Pastan and Bailey, 1998; Keane et al., 2002). The classical definition of diabetic nephropathy (DN) is of a progressive rise in urine albumin, coupled with increasing blood pressure and declining glomerular filtration (Obineche and Adem, 2005). Microalbuminuria is considered to reflect an early stage in this irreversible process and it may occur because of endothelial dysfunction due to hyperhomocysteinemia (Guldener and Stehouwer, 2002).

Hyperhomocysteinemia is observed in more than 85% of patients with renal disease and is also considered as an independent predictor of cardiovascular disease (Minna et al., 2004; Stam et al., 2005). The elevated total homocysteine levels in end-stage renal disease (ESRD) may result from the deterioration of renal function or may be due to the genetic variation in the 5,10-methylenetetrahydrofolate reductase gene (MTHFR).

Studies have shown that the 677T MTHFR allele plays a significant role in both the macro- and microvascular complication of diabetes (Sun et al., 2003, 2004, 2005). Clearly, it is essential to arrive at a better understanding of the factors that promote diabetes and/or its complications, so that preventive measures may be put in place as rapidly as possible. As there are no reports to date from India correlating the C677T MTHFR mutation with DM or DN, we carried out this case-control study in our population.

Materials and Methods

Three hundred thirty-six individuals, including 236 cases with T2DM clinically established for more than 8 years, with and without history of renal complications, and 100 healthy nondiabetic volunteers as controls, were chosen for the study. The selection of diabetic patients was based on American Diabetes Association Clinical Practice guidelines (fasting plasma glucose level >126 mg/dL) (American Diabetes Association, 1997). Subjects were selected from the departments of nephrology and general medicine from two multispeciality hospitals, Bhagwan Mahavir Hospital and Research Centre and Kamineni Hospitals, Hyderabad, India, between January 2004 and March 2006.

Detailed personal, clinical, and family history along with a three-generation pedigree was collected in a well-designed proforma, which was approved by the institutional ethical committee. Informed consent was obtained from all the subjects involved in the study.

It is commonly believed that the onset of T2DM is above the age of 40 years in our population and the renal complications appear on an average after 6 years of established diabetes. Hence, the cases of T2DM included in the study were confirmed diabetics on treatment (i.e., oral hypoglycemics or insulin injections) for more than 8 years. T2DM cases with a family history of renal complication were excluded from the study. Persistent albumin excretion rate ≥30 mg/day in at least two of three determinations during the last 6 months was taken in the inclusion criteria of DN group. Microalbuminuria was diagnosed if the albumin excretion was between 30 and 299 mg/day. Overt nephropathy was diagnosed if albumin excretion was 300 mg/day. Nephropathy due to conditions such as chronic interstitial nephritis, chronic glomerulonephritis, and pylonephritis other than diabetes were excluded from the study. Of the 136 DN cases, 106 (77.94%) were on hemodialysis three times a week, whereas the remaining were newly diagnosed cases (22.06%) and were being conservatively managed at the time of sampling. None of the nephropathy cases included had any cardiovascular symptom prior to inclusion in the study, whereas 10% had neuropathy. Diabetic retinopathy is considered one of the criteria for selecting DN patients; however, in our study, only 40% of the DN patients showed retinopathy, which is close to the statistics seen in the Indian population (Unnikrishnan et al., 2007).

The controls recruited were healthy volunteers who had random blood sugar levels of <120 mg/dL and were above the age of 40 years. They had no clinical or family history of T2DM or any renal complication.

Peripheral blood (1 mL) was taken for DNA isolation, and analysis was blinded to the origin of the group to which the sample belonged. Genomic DNA was isolated from peripheral blood and genotyped for C677T MTHFR mutation (Mtiraoui et al., 2007) (Table 1). Briefly, a 3-step polymerase chain reaction (PCR) was standardized using Bioer (Xp cycler) and carried out with initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 68°C for 30 s, and extension at 72°C for 45 s. A final extension at 72°C for 5 min was carried out. The amplified product of 198 bp was digested with HinfI restriction endonuclease (Fermentas) to obtain two fragments of 175 and 23 bp if the T allele was present. Appropriate negative and positive controls for PCR and restriction enzyme analysis were included during genotyping. Randomly, five PCR products from each of the three groups (DM, DN, and controls) were confirmed by sequencing.

Data are presented as mean ± standard deviation. The chi-square test was used for comparison of expected and observed frequencies of categorical variables. A p-value (two-tailed) of <0.05 was considered statistically significant. The odds ratio was also calculated. Statistical analysis was performed using MedCalc for Windows, version 7.4.1.0 (MedCalc Software). The relative risk and the cohort attributable risk percentage were calculated. Hardy-Weinberg equilibrium test was performed.

Results

The mean age at sampling was 57.62 ± 9.09 years for DM and 57.44 ± 8.79 years for DN. The known duration of diabetes in the DM and DN groups was 12.16 ± 4.07 and 14.85 ± 5.19 years, respectively. The mean duration of diabetes prior to developing nephropathy was 13.76 ± 5.14 years and the mode was 10 years. There was more number of men (∼67%) than women in both the DM and DN groups (∼33%). The mean serum creatinine and serum albumin values were 5.31 ± 2.89 and 224.69 ± 139 mg/dL, respectively, for the DN group, compared with 0.98 ± 0.26 and 13.56 ± 5.40 mg/dL, respectively, for the DM group (Table 2).

MTHFR C677T genotyping analysis was performed unaware of the clinical data. The DM group had 68% CC and 32% CT genotypes, whereas the group with DN showed 77.94% CC and 22.05% CT, which was different from the controls, which had 91% CC and 9% CT genotypes. None of the cases or controls had the TT genotype (Table 3). A chi-square test between the DN and control groups for the T allele showed a significant difference (p < 0.05) and the odds ratio was highly significant (2.6309; 95% confidence interval = 1.2197-5.6746). DM cases when compared with controls also showed a significant difference with a p-value of <0.05. However, the frequency of T allele was not significantly different between the DN and DM groups (p > 0.05). The relative risk value for the CT genotype was found to be 1.85 and the cohort attributable risk percentage was 29%.

Gender-specific genotype distribution was performed with the CT and CC genotypes and it was found that women with CT genotype were similar to men in the T2DM group, whereas they were significantly more in the DN group (p = 0.009; Table 4).

Discussion

In the general population, determinants of plasma homocysteine level are age, sex, diet, plasma vitamin B6 and B12 levels, smoking, coffee consumption, and polymorphisms of the MTHFR gene (Nygard et al., 1998). The human MTHFR gene located on chromosome 1p36.3 is 1980 bp long and has a common polymorphism, C677T. This C to T transition in exon 4 results in the substitution of alanine with valine (A222V) in the N-terminal catalytic domain, which renders the enzyme thermolabile and less active, elevating homocysteine levels (Frosst et al., 1995). A number of studies have been carried out in India on MTHFR C677T polymorphism and its role in various diseases, such as deep vein thrombosis, atlantoaxial dislocation, and ischemic stroke, showing a positive association of this allele with these pathologies (Alluri et al., 2005; Panigrahi et al., 2006; Pradhan et al., 2007; Naushad et al., 2008).

In our study of 336 individuals, none of them showed a TT genotype, which deviated significantly from the Hardy-Weinberg Equilibrium (HWE) (p > 0.05). A recent population study from northern India also reported a similar significant deviation of MTHFR gene polymorphism from HWE in two population groups (Ahirs and Jats) from Haryana (Saraswathy et al., 2008). A latest study from India on acute lymphoblastic leukemia in relation to MTHFR 677 and 1298 gene mutations has further supported our data on TT genotype indicating the low frequency of this genotype in the Indian population. A possible explanation for the relatively low prevalence of the mutant allele seen in Asian Indians may be due to the higher rate of removal (selection) of the mutant allele than its introduction (mutation) in the population gene pool (Adiga et al., 2010). As all our subjects were above 40 years of age, we have included healthy matched controls of the same age group. It can therefore be speculated that, in our population, individuals with the TT genotype succumb to complications other than diabetes and nephropathy, such as neurological or cardiovascular, prior to reaching the age of 40 years. Support for this comes from our analysis of 136 patients with stroke, of which 3 (2.2%) had the TT genotype and all 3 were below the age of 40 years (Alluri et al., 2005). Another study on Indian ischemic stroke patients also reported that the MTHFR TT genotype is present in the patients; however, none of the controls showed the homozygous TT genotype (Panigrahi et al., 2006). Hence, our recruitment criteria, which included only adults above the age of 40 years in both patient and control categories, may be responsible for the absence of the TT genotype.

This is the first report from India correlating MTHFR C677T gene polymorphism with T2DM and one of its microvascular complications, nephropathy. The present study indicates that the T allele frequency is increased in T2DM as well as DN when compared with controls; however, there was no significant difference between diabetic patients with and without nephropathy in the cohort studied (p = 0.100).

Japanese and Turkish populations have also not shown a significant difference of C677T allele frequency between T2DM and DN. However, in both these studies, controls were not included in the analysis (Eroglu et al., 2007; Maeda et al., 2008). In contrast, a report from China showed that the MTHFR 677T allele is associated with nephropathy when compared with type 2 diabetic patients without nephropathy, whereas the frequency of the T allele was similar in both controls and diabetic patients (Sun et al., 2004). A study by Bruntgens et al. on type 1 and type 2 diabetic patients from German and Polish diabetic clinics showed that there is no difference in allele frequencies between nephropathy, type 2 diabetic patients, and controls (Bruntgens et al., 1999). Another Polish study showed a significant association of C677T gene polymorphism with nephropathy when compared with type 2 diabetics and controls (Ksiażek et al., 2004). The reason for this discrepancy in their findings remains unclear. A study from Germany did not show any significant effect of MTHFR C677T genotype on progression of DN (Boger et al., 2007). They also failed to show any association of the T allele with progression of microalbuminuria to ESRD as well as any deleterious effect of this allele on mortality, but they did not evaluate any association with control or DM.

In our cohort of type 2 diabetics, when the age of onset of diabetes was assessed in CT and CC genotype individuals by categorizing them into three different age groups, it was found that there was no difference. Patients with DN having a CT genotype had diabetes for 18.44 ± 5.31 years prior to developing DN when compared with diabetic patients with a CC genotype who developed DN after 15.30 ± 5.46 years (t-test, p > 0.05). This observation indicates that the T allele does not influence the initiation of nephropathy, which is similar to a recent German study (Boger et al., 2007).

Of the 136 nephropathy cases included in our study, 106 were on hemodialysis with ESRD and 30 were microalbuminuric. The percentage of CT genotype was 19% in the ESRD group and 23% in the group with microalbuminuria. This indicates that the 677T allele does not play a role in the progression of nephropathy. The MTHFR 677T mutation was not significantly different between these two groups, indicating that this mutation does not play a role in the severity or survival of nephropathy patients.

Our data therefore suggest that the MTHFR C677T mutation may not be responsible for initiating or progression of nephropathy but may confer an increased risk for developing diabetes. MTHFR 677T, apart from causing hyperhomocystenemia, is also responsible for regulating levels of S-adenosylmethionine, a key intermediate in methionine metabolism and the lone donor of methyl groups for epigenetic modification of DNA and posttranslational methylation of histones (Fuks et al., 2003). The MTHFR 677T allele may result in reduced production of S-adenosylmethionine, which may affect the methylation status of genes playing a role in the etiology of diabetes and alter their expression.

In conclusion, our study shows that MTHFR 677T allele increases the risk of T2DM by fourfold in the Indian population. However, with reference to the meta-analysis carried out by Zintzaras et al. (2005) in diabetic retinopathy, a larger sample size is warranted prior to making recommendations. Individuals with a family history of diabetes or with risk factors such as obesity, hypertension, and impaired glucose tolerance should be screened for MTHFR C677T mutation and may be prescribed folic acid, vitamin B6, and vitamin B12 to assess if this helps in delaying the onset of diabetes.