Association between the methylenetetrahydrofolate reductase ( MTHFR ) gene 677 C > T and 1298 A > C polymorphisms and the risk of diabetic nephropathy ; a meta-analysis

Implication for health policy/practice/research/medical education: Diabetic nephropathy (DN) (diabetic kidney disease) is the leading cause of chronic kidney disease and is characterized by the albuminuria. Homocysteine levels have been found elevated in patients with DN. The methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in the homocysteine metabolism. MTHFR gene polymorphisms were analyzed in many studies, but their results are inconclusive. We performed a meta-analysis of MTHFR 677C>T and 1298A>C studies. Our results support the association of MTHFR 677C>T and 1298A>C variants with the risk of DN. Please cite this paper as: Gupta A, Sharma S, Lakkakula S, Bhaskar LV. Association between the methylenetetrahydrofolate reductase (MTHFR) gene 677C>T and 1298A>C polymorphisms and the risk of diabetic nephropathy; a meta-analysis. J Renal Inj Prev. 2019;8(3):175-184. DOI: 10.15171/jrip.2019.33. Methylenetetrahydrofolate reductase (MTHFR) is involved in the homocysteine metabolism. Two common variants of MTHFR gene (677C>T and 1298A>C), have been reported to reduce the MTHFR enzyme activity and leading to plasma hyperhomocysteinemia. There are a number of recent case-control studies that investigated the association between the MTHFR polymorphism and diabetic nephropathy (DN), albeit with inconsistent results. The aim of this meta-analysis is to evaluate the associations between the genetic polymorphisms of MTHFR with susceptibility to DN. A literature search was conducted on PubMed, Embase and Google scholar from inception till March 18, 2019. For MTHFR 677C>T analysis, a total of 23 studies including DM controls (3095 cases and 3187 DM controls) and 12 studies including non-DM controls (1590 cases and 2052 nonDM controls) were taken. For MTHFR 1298A>C analysis, a total of 7 studies using DM controls (959 cases and 1209 DM controls) and 3 studies using non-DM controls (400 cases and 802 nonDM controls) were taken. Meta-analysis showed that mutant genotypes of the 677C>T (OR: 1.58; 95%CI: 1.16-2.14) and 1298A>C (OR: 1.38; 95%CI: 1.16-1.65) polymorphisms in the MTHFR gene were associated with increased risk of DN (diabetic kidney disease). MTHFR 677C>T and 1298A>C polymorphisms revealed significant heterogeneity between studies. Further, there was no evidence for publication bias for these polymorphisms. In conclusion, this meta-analysis provides strong evidence that MTHFR 677C>T and 1298A>C polymorphisms may be associated with increased risks of DN. However, further studies are still needed to accurately determine whether MTHFR genetic polymorphisms are associated with susceptibility to DN. A R T I C L E I N F O


Introduction
Diabetes mellitus (DM) is a heterogeneous condition characterized by persistent hyperglycemia (1).Type 2 diabetes accounts for nearly 90%-95% of those with diabetes worldwide.Uncontrolled diabetes in synergy with the other metabolic aberrations is a serious health issue due to the morbidity associated with it (2).Several lines of evidences demonstrated that diabetes negatively affects the macro and microvasculature of various organs leading to the development of life-threatening health complications (3).The vascular complications include, microvascular (retinopathy, neuropathy and nephropathy) and macrovascular (coronary artery disease, peripheral vascular disease, and stroke) complications that increase the risk for cardiovascular diseases (4).Diabetic nephropathy (DN) (diabetic kidney disease)is the leading cause of chronic kidney disease and is characterized by albuminuria (greater than 300 mg/dL), hypertension and a persistent decline in glomerular filtration rate leading to renal failure (5).Development and progression of nephropathy in diabetes patients seems to result from the interaction of genetic susceptibility with metabolic and hemodynamic changes (6).
The etiology of DN is multi-factorial and involves both environmental and genetic factors.Microalbuminuria is considered to reflect an early stage in the process of DN, and it may occur because of endothelial dysfunction due to hyperhomocysteinemia. Previous studies demonstrated that the elevated plasma levels of homocysteine (Hcy) have been related to insulin resistance and DN (7).MTHFR is an enzyme involved in the conversion of Hcy to methionine and catalyzes the reduction reaction of 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate (8).MTHFR enzyme plays a vital role in folate metabolism and is involved in DNA synthesis, DNA repair and DNA methylation (9).The gene coding for MTHFR is located on chromosome 1 (1p36.3)and possessing 677C>T and 1298A>C polymorphisms that contribute to the inactivation of this enzyme (10).Several studies have examined the association between MTHFR gene polymorphisms and DN risk, however, the available proof reported to date is inconclusive (11)(12)(13)(14)(15).We performed the meta-analysis to investigate the relationship between MTHFR gene polymorphisms and DN susceptibility.In this meta-analysis, pooled estimate of the association under different genetic models for both 677C>T and 1298A>C polymorphisms were obtained.In addition, the heterogeneity between studies and the existence of publication bias were studied to understand the reliability of this purported association.

Literature search approach
A search for literature published in the English language was conducted until March 18, 2019, using the PubMed database, the Embase database, and Google Scholar with the following search terms: ('Diabetic nephropathy", "MTHFR", "677C>T", "1298A>C", "rs1801133", "rs1801131", "SNP", "polymorphism", and "genetic variant").In addition, we reviewed the reference lists of retrieved papers and recent reviews.A total of 24 articles were found using the above search terms.Articles were excluded if they had the following characteristics; (i) review articles, (ii) nonepidemiologic articles, (iii) no DN subjects (iv) published in other than the English language and (v) did not include the SNPs 677C>T and 1298A>C.The present meta-analysis was conducted as per the guidelines issued in the Preferred Reporting Items for Systematic Reviews and Meta-Analysis 2009 (16).

Data abstraction
All articles were evaluated independently by two reviewers (AG and SS) who extracted data that included the first author, publication year, geographic areas, ethnicity of study population, number of healthy controls, DM patients, DN patients, and genotypes of MTHFR 677C>T and 1298A>C polymorphisms.To test the population stratification in the controls, a chi-square test was applied to determine if MTHFR 677C>T genotype distribution in the controls conformed to Hardy-Weinberg equilibrium.

Statistical analysis
All analysis were performed using comprehensive metaanalysis is a software package version 2. Heterogeneity across individual studies was assessed by Cochran's Q test and I 2 statistic.The strength of the association between MTHFR polymorphisms and DN risk was measured by the pooled OR with its corresponding 95% CI.In the present meta-analysis, strength of the association was assessed in two different (allelic and dominant) genetic models.Further sensitivity analysis was conducted by excluding one study in each analysis to examine robustness of the method used for the meta-analysis.Potential publication bias was assessed by Begg's funnel plot and Egger's test.

Study characteristics
The literature search strategy and process for the selection of papers for this meta-analysis is shown in Figure 1.A total of 24 articles with 25 studies that analyzed DM controls or non-DM controls or both were included in the final analysis (9,(11)(12)(13)(14)(15)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35).Out of these only 8 articles studied both MTHFR 677C>T and 1298A>C polymorphisms.Further, one of the included article that had included two populations was considered as two independent studies (28).For MTHFR 677C>T analysis, a total of 23 studies including DM controls (3095 cases and 3187 DM controls) and 12 studies including non-DM controls (1590 cases and 2052 non-DM controls) were taken.For MTHFR 1298A>C analysis, a total of 7 studies including DM controls (959 cases and 1209 DM controls) and 3 studies including non-DM controls (400 cases and 802 non-DM controls) were taken.The number of patients and controls, genotypes, HWE and other study characteristics of each study included in the meta-analysis are listed in Tables 1 and 2.

Heterogeneity
When we chose patients with DM as controls or non-DM subjects as controls, the association between 677C>T polymorphism and DN showed a significant heterogeneity between studies in both allelic (P < 0.001, I 2 >0.86) and dominant model (P < 0.001, I 2 > 0.86).
In the subgroup analysis according to ethnicity, there still existed heterogeneity in both genetic models for 677C>T polymorphism.Nevertheless, the association between 1298A>C polymorphism and DN, significant heterogeneity between studies was found only in allelic model (P = 0.026, I 2 0.58) when we chose patients with DM as controls (Tables 1 and 2).

Sensitivity analysis and publication bias
The analysis results suggested that no individual studies significantly affected the pooled DN risk of both MTHFR 677C>T and 1298A>C variants (Figures 4A-B and 5A-B), indicating a statistically robust result.The shapes of the funnel plots seemed symmetrical for both MTHFR 677C>T (Figure 6A) and MTHFR 1298A>C (Figure 6B) polymorphisms indicating the absence of publication bias.Egger's test also showed that there was no strong statistical evidence of publication bias for both MTHFR 677C>T and 1298A>C polymorphisms in all genetic models (P > 0.050).

Discussion
About 25%-40% of patients with diabetes are susceptible to develop kidney disease.Although inadequate glycemic control is one of the fundamental risk factors for the development nephropathy, it is not an inevitable complication of diabetes.The involvement of multiple risk factors such as duration and severity of diabetes, environmental factors, life-style stressors and genetic factors make it difficult to determine a person's risk of inheriting nephropathy.Familial aggregation of DN is one of the major evidence for a heritable genetic susceptibility to DN.However, despite long and intensive research efforts to determine the causative genetic components remain elusive (36)(37)(38).
Our meta-analysis revealed a significant association   between MTHFR polymorphisms and DN risk.Metaanalysis of MTHFR polymorphisms revealed significant heterogeneity between studies for both 677C>T and 1298A>C.Further, there was no evidence for publication bias for these polymorphisms.In consistent with our results, a series of earlier published meta-analyses provided evidence of association between MTHFR 677C>T polymorphism and DN risk in various ethnicities (39)(40)(41)(42)(43)(44)(45).With regards to MTHFR 1298A>C only one meta-analysis performed in which no association between MTHFR 1298A>C and DN risk was observed (44).Higher levels of homocysteine in patients with DN were demonstrated in multiple studies (46)(47)(48).This is further supported by presence of increased erythrocyte S-Adenosyl-L-homocysteine, decreased erythrocyte S-Adenosyl methionine and lymphocyte MTHFR activity in patients with advanced nephropathy (49).Compared to subjects without complications, individuals with DN and hypermethylated profile in the MTHFR gene promoter showed higher levels of alpha-1 acid glycoprotein and total antioxidant capacity (50).In individuals carrying 677CC/1298AA haplotype, the hypermethylated profile  was linked with higher fasting glycemia values (51).Further, a direct link between perturbations in 1-carbon metabolism, through an interaction of total homocysteine and the activity of MTHFR enzyme on epigenetic regulation of the genome via DNA methylation (52).

Conclusion
In summary, this meta-analysis provided strong evidence for association between MTHFR 677C>T and 1298A>C polymorphisms and DN risk.As the majority of studies included in this meta-analysis were Caucasians and East Asians and that the distribution of MTHFR genetic polymorphism differs among ethnic groups, the results cannot be extrapolated to patients belonging to any other ethnic groups.However, further studies are still needed to accurately determine whether MTHFR genetic polymorphisms are associated with susceptibility to DN.

Figure 1 .
Figure 1.Identification process for eligible studies.

Figure 2 .
Figure 2. Meta-analysis for the association between DN risk and MTHFR 677C>T polymorphism.

Figure 3 .
Figure 3. Meta-analysis for the association between DN risk and MTHFR 1298A>C polymorphism.A: comparison with DM controls; B: comparison with non-DM controls.

Figure 4 .
Figure 4. Sensitivity analysis of meta-analysis results of MTHFR 677C>T polymorphism.A: comparison with DM controls; B: comparison with non-DM controls.

Figure 5 .
Figure 5. Sensitivity analysis of meta-analysis results of MTHFR 1298A>C polymorphism.A: comparison with DM controls; B: comparison with non-DM controls.

Table 1 .
Distribution of MTHFR 677C>T genotypes and HWE P values of in the eligible studies

Table 2 .
Distribution of MTHFR 1298A>C genotypes and HWE P values of in the eligible studies

Table 3 .
The heterogeneity results for the association of MTHFR 677C>T gene polymorphisms and DN in different genetic models a Random effect model; b fixed effect model.