Association of BHMT (rs 3733890) gene polymorphism with biochemical markers of B12 deficiency in T2DM patients on metformin therapy

Long term administration of metformin interferes with the absorption of vitamin B12 resulting in vitamin B12 deficiency.Among the various genetic variants that are associated vitamin B12 deficiency, we analyzed BHMT gene polymorphism and their association with metformin induced vitamin B12 deficiency in T2DM patients. A cross sectional study was done with 300 participants. Methyl malonic acid, homocysteine and high sensitive C reactive protein were analysed by Mass Spectrometry, Chemiluminescent analyser and Immunoturbidimetric method respectively. Genetic variants were analysed by ARMS-PCR method, Data was analyzed with various statistical tools like ROC, Odds ratio and Likelihood ratio. Significant reduction in folic acid and vitamin B12 in metformin users was found. High sensitive C reactive protein, homocysteine and methyl malonic acid are significantly increased in patients with metformin induced B12 deficiency. ‘A’ allele in BHMT (A allele OR =2.1, AA genotype =2.8) showed risk of vitamin B12 deficiency in T2DM patients on metformin therapy. BHMT gene polymorphism had LR of 2.65 for folic acid (AG genotype), 2.73 for MMA (AA genotype) and 2.63 for Homocysteine (AA genotype). We found an association between single nucleotide polymorphism of BHMT and diagnosis of vitamin B12 deficiency status in metformin users. Folic acid, MMA and homocysteine had high specificity in concordance with BHMT (rs3733890) polymorphism in predicting vitamin B12 deficiency. Early screening of SNP of BHMT in T2DM patients on metformin therapy will help us to identify group of people who are prone for vitamin B12 deficiency.


Association of BHMT (rs 3733890) gene polymorphism with biochemical markers of B12 deficiency in T2DM patients on metformin therapy
B12 decreases with an increase in dosage and also with the duration of administration of metformin. [2] Metformin interferes with vitamin B12 absorption through the alterations in intestinal motility, increased bacterial overgrowth or alterations of vitamin B12-IF complex. The risk of developing metformin induced vitamin B12 deficiency is greatly influenced by the increase in age, metformin dosage, and its duration of use. [3] Vitamin B12 (Cobalamin) plays a vital cofactor role in the synthesis of DNA as well as in two biochemical reactions. Vitamin B12 deficiency prevalence varies from 5.8% to 30% among patients undergoing long-term treatment with metformin. [4,5]. Metformin induces vitamin B12 deficiency by binding its hydrophobic tail of biguanide to the hydrocarbon core of membranes. The biguanide group gives a positive charge to the membrane and displaces the divalent cations such as calcium. Vitamin B12 uptake into the ileal cells is calcium dependent and can thus be impaired by metformin. [6] Clinical observations indicated a prevalence of 30% for vitamin B12 malabsorption among patients undergoing long-term metformin therapy.
[7]A randomized control trial demonstrating relationship between metformin and serum vitamin B12 found 19% reduction in serum vitamin B12 levels after 4 years of metformin treatment. [8] Metformin induced vitamin B12 deficiency leads to hematological and neurological complications. [9]. Serum methyl malonic acid (MMA) and homocysteine (Hcy) are the markers used for the diagnosis of vitamin B12 deficiency. [10]The genetic variants altering vitamin B12 status can be studied for the better understanding of the interplay of diet, genetics, and human health in metformin therapy. A twin study reports that the magnitude of genetic influence on serum vitamin B12 levels to be 59%. [11]. Several genetic studies on vitamin B12 status suggest that it is a multifactorial trait. Single-nucleotide polymorphisms (SNPs) in multiple genes interact with the environment to result in altered B12 status. [12]. BHMT plays role in homocysteine and 1-carbon metabolism. [13][14][15]It utilizes betaine as the 1-carbon donor. Therefore, mutation of this remethylation enzyme results in elevated homocysteine levels. BHMT is indirectly involved in the folate-dependent pathway. It fluxes homo cysteine when the folate-dependent remethylation is disrupted. [16]. This study also assesses the vitamin B12 status (from homocysteine & Methyl malonic acid level) between different genotypes of T2DM patients on metformin. In addition, the sensitivity & specificity of these molecular and biochemical markers are evaluated for predicting vitamin B12 status in T2DM patients on metformin therapy.

Materials and Methods
The present Cross Sectional Study was carried out at a Tertiary health care setup in Puducherry after obtaining Institutional Ethical Committee approval The PCR steps were a) initial DNA denaturation at 95ºCfor 5min, b) denaturation at 95ºC for 30sec, c) annealingat 63ºC for 30sec, d) extension at 72ºC for 30sec followed by amplification of steps b to fourby 35cycles and final extension at 72ºC for 5min. The PCR final volume of 20uL reactionsample contained Red dye PCR master mix of 10uL, primer mix 10uL, purified DNA sampleof 5uL. The PCR products were analysed by agarose gel electrophoresis. [ Fig:1] 2.3 Statistics: Descriptive and inferential statistical analysis was carried out in the present study. Results onContinuous measurements were presented as mean ±SD and results on categorical measurementswere presented in number %. The data were entered in MS excel sheet so that it can be retrieved easily for analysis using SPSS-16 software. Shapiro-Wilk test was performed to find out the distribution pattern for all the data collected. To compare the biochemical parameters between the groups, independent't' Test was performed. P value less than 0.05 is considered as significant. Among the B12 deficient group, 11% of them were taking metformin for more than 10 years, 39% for 5 -10 years and 50% for less than 5 years. The mean duration of metformin therapy was 7 years. Fifty five percent of the vitamin B12 deficient group was receiving a dose of 1000mg/day of metformin, 39% were taking 1500mg/day and 6% were taking more than 2000mg/day of metformin. The mean dosage is 1252mg. Among the group 2 patients (T2DM patients without vitamin B12 deficiency) 107 (65%) were males and 58 (35%) were females. Among the 165 T2DM patients whose serum vitamin B12 levels were within the reference range, 93% were on metformin therapy for less than 5 years, 6% for 5 -10 years and 1% of them were taking metformin for more than 10 years. Seventy six percentages of group 2 patients were receiving a dose of 1000mg/day of metformin, 21% of the patients 1500mg/day and 3% of the T2DM patients without B12 deficiency were receiving more than 2000mg/day of metformin. The mean dosage of metformin is found to be 1133.33 mg/day. Chi square analysis was performed to find out the duration and dose dependent effect of metformin on the levels of serum vitamin B12 and the result showed significant difference of X 2 = 68.72, p<0.001 (Fig:2) and X 2 = 14.59, p <0.001 (Fig:3) respectively.    Chi-Square linear trend analysis showing GG genotype distribution of 128 (77%) in group 2 and 81(60%) in group 1.AG genotype was found in 31 (19%) in group 2 and 43(32%) group 1. AA genotype was observed in 06 (04%)in group 2 and 11 (08%) in group 1. A significant association between AA (ORs 2.8 with 95%CI 1.0-8.1) genotype and A allele (ORs 2.1 with 95% CI 1.3-3.2) were found to exist.

Vitamin B12 deficiency variables versus gene polymorphism
Vitamin B12 deficiency variables versus BHMT gene polymorphism in Group 1 is shown in Table  3.  Table 3. MMA showed significance (p< 0.01). Post hoc comparisons using the Tukey HSD test indicated that the GG genotype was significant with AG with a p value of 0.0172. HsCRP also showed significance (p< 0.01). Post hoc comparisons using the Tukey HSD test indicated that the GG genotype was significant with AG with a p value of 0.0116. Vitamin B12 deficiency variables versus BHMT gene polymorphism in Group 2 is shown in Table 4. One way ANOVA was conducted to compare the biochemical markers within the genotypic variants of BHMT (GG, AG & AA) in T2DM patients with normal levels of vitamin B12, as shown in Table 4. MMA showed significance (p<0.05). Post hoc comparisons using the Tukey HSD test indicated that the GG genotype was significant with AA with a p value of 0.0446.  This study was carried out to explore the genetic interactions in metformin induced vitamin B12 deficiency in T2DM patients. The introduction of certain biomarkers like methyl malonic acid and homocysteine in clinical practice has made the diagnosis of B12 deficiency easier. In addition, we have studied BHMT gene that is responsible for vitamin B12 deficiency in metformin users. Metformin is one of the frequently prescribed drugs for T2DM patients. It has an excellent safety profile, with improvement in cardiovascular morbidity and mortality. It has multiple beneficial effects like altering advanced glycosylation end products when consumed by diabetes patients. [17][18][19][20]Despite all of metformin's benefits, adverse events may occur. Prolonged usage of this drug results in reduction in vitamin B12 and folate levels with raise in homocysteine and methyl malonic acid level, for which some researchers recommend screening and prevention strategies. After 3-4 months of initiation of metformin therapy, a significant reduction in B12 level occurs quickly, while symptomatic deficiency may take as long as 5-10 years to manifest. [8] This is poorly recognized and not currently screened for or treated by the majority of physicians who prescribe metformin. In view of these considerations, the present study was undertaken to analyze the genetic impact (if any) of vitamin B12 deficiency in T2DM patients who are on metformin therapy. A total of 300 T2DM patients under metformin therapy were included in the study population, based on the inclusion and exclusion criteria. Our study defined biochemical vitamin B12 deficiency as serum levels ≤300 pg/mL. [18,19]When the study population was analyzed for the B12 status, 45% of the population showed a clear picture of vitamin B12 deficiency. Wile et al study in Canada and Chen et al study in Hong Kong showed a prevalence of B12 deficiency in metformin users of about 31% and 37% respectively. [20,21]The deficiency status led to grouping of study population who are having T2DM. Group 1 includes 135 patients who are deficient in vitamin B12 and Group 2 including 165 T2DM patients with normal vitamin B12 level. Among group 1 majority of the population (50%)were taking Metformin for less than 5 years, compared to those patients who were treated for 6 -10 years (39%) and more than 10 years. Serum vitamin B12 levels were negatively associated with the dosage and duration of metformin use. Ting et al study which included 155 diabetic patients with vitamin B12 deficiency, showed similar association of serum vitamin B12 levels with the dosage and duration of metformin usage, after adjusting for confounding factors [22] while Ko et al study showed a negative correlation with B12 and duration of treatment and no correlation with B12 and dosage. [23]The differences may be accountable for the increased mean duration of metformin therapy which is higher in our study which is 7 years, compared with Beulens et al study where the mean duration ranged from 4 to 5.3 years. [24]The most consistent risk factors in metformin users were the dosage and duration of treatment of the drug. [22,25]. We also studied other parameters related to vitamin B12 deficiency, such as folate, MMA and homocysteine. Folate is added to our study as it acts as an intermediate in the methionine synthase reaction where vitamin B12 deficiency can trap folate, resulting in folate deficiency. [26] Homocysteine and MMA were analyzed as additional markers to support the evidence of deficiency status. Studies have shown that vitamin B12 also has an antioxidant property. [27] Deficient status of B12 serves as a trigger factor to increase oxidative stress, particularly in patients with diabetes. Therefore inflammatory marker HsCRP was also analyzed as it correlates with hyperhomocysteinemia in B12 deficient population. [28] Our studies identified mean B12 values to be 205.23pg/ mL, significantly lower in metformin treated patients (group 1) while group 2 showed a mean value of 497.50 pg/mL. The mean serum vitamin B12 concentration was 148.6 pg/mL in B12 deficient group and 466.1 pg/mL in B12 normal group as indicated by Rose Zhao-Wei Ting et al study. [22]Although serum vitamin B12 level is the most used test to diagnose deficiency status since 1950s, its sensitivity is not reliable and it can be in the normal range in a significant proportion of population. In such circumstances, serum homocysteine or methylmalonic acid level was considered more sensitive indicators of vitamin B12 status and allows early identification of vitamin B12 deficiency. [29][30][31][32] Homocysteine and MMA were elevated in relation to the metformin induced lowering of B12. Homocysteine and MMA had a mean value of 25.83 µmol/L and 244.9 nmol/L respectively. In our pilot study with 100 metformin users the mean value of Homocysteine and MMA was found to be 12.3 µmol/L and 154.5 µmol/L respectively. [33-40. When ROC is used to test the diagnostic utility of biochemical markers in the diagnosis of metformin induced B12 deficiency, it showed that Homocysteine and MMA had maximum AUC of 0.808 (CI:0.753-0.863) and 0.690 (CI:0.628-0.751) respectively with maximum sensitivity and specificity to diagnose B12 deficiency when compared to low folate status. Folic acid is reduced to 5.1 ng/L in Group 1 when compared to 11.03 ng/L in group 2. A cross-sectional study with 203 outpatient type 2 diabetic patients, showed a mean range of 17.7ng/L of folate concentration. [4] Interaction between vitamin B12 and folate is in the synthesis of methionine from homocysteine by methionine synthase, in which both 5-methyl tetrahydrofolate and methylvitamin-B12 are cofactors. Thus folate cycle, is essential for methylation reactions and for the synthesis of purines and thymidine. Serum folate measurement is nonspecific. Low levels are seen in patients even without the evidence of deficiency. Vitamin B12 and folate deficiency co exists. Low folate status is a risk factor for development of colorectal cancer and other types of cancer. [34]. A study conducted in two hundred and sixteen patients with T2DM on metformin therapy had a HbA1c of mean value of 7.25 ± 1.13. [35] Similarly, our study had a mean value of 7.9 ± 0.8 in group 1. Data of this study shows that the patients require a higher metformin dose to attain a mean of 7.9%.

Genetic variants
The diagnostic approach to B12 deficiency includes the demonstration of the deficiency by biochemical investigation and the identification of the cause of the deficiency. The cause analysis would be better if carried from the genetic study. Vitamin B12 status can be altered by several genetic variants by affecting the proteins involved in vitamin B12 absorption, cellular uptake of the vitamin and intracellular metabolism. [36] A twin study showed that the B12 levels had 59% heritability, indicating the magnitude of genetic influence on vitamin B12 status. [37] The altered B12 status is a cause of multifactorial trait, where several single-nucleotide polymorphisms (SNPs) in multiple genes interact with the environment. [38]There are several genetic variants involved in vitamin B12 deficiency. Among them, BHMT gene is the gene of interest in our study.

BHMT:
Zinc-dependent enzyme, expressed in liver, kidney and lens, takes part in glycine, serine, threonine and methionine metabolism catalyzing the transfer of a methyl group from trimethylglycine and a hydrogen ion from homocysteine to produce dimethylglycine and methionine respectiv ely. Thus it is important in homocysteine and 1carbon metabolism. [39,40] Choline is a source of Betaine and BHMT enzyme substrate. [41] Choline is essential in the formation of S-adenosyl methionine needed for DNA methylation reactions. Defective DNA methylation and impaired DNA repair are the reasons for development of deficiency related complications. [42]Betaine intake can also contribute to reduction in homocysteine level. [43] Mutation occurs at nucleotide 742 in exon 6 of the gene, resulting in replacement of glutamine with arginine in structure of the enzyme. [44,45]. Re-methylation process of homocysteine to methionine is affected due to mutation in Betaine homocysteine methyltransferase enzyme (BHMT) coding gene, leading to decrease in S-adenosyl methionine (SAM) synthesis, that eventually result in hypomethylation of DNA, chromosome instability, and chromosome miss-segregation serving as a risk factor in B12 deficiency. [46]In our study we found that BHMT polymorphism is associated with T2DM patients on metformin therapy. Our results are in accordance with Finkelstein et al. [47] and we have reported 'A' allele as a risk factor in T2DM patients on metformin which was found to be a similar risk factor in Indian population. Current study also tried to associate the gene polymorphism of BHMT with the biochemical markers of vitamin B12 deficiency which showed significant increase in the markers like MMA and Homocysteine. Hyperhomocysteinemia is caused by a combination of genetic and nutritional disturbances. Alteration in the function of BHMT results in raised Hcy levels. Schwahn et al study showed high Hcy levels depletes hepatic betaine stores. [48] The oral supplementation of betaine is said lower Hcy concentrations in severe hyperhomocysteinemic patients. [49] The cysteine residues in BHMT function as zinc-binding domains, required for the binding of Hcy to BHMT. Mutation of cysteine residues with alanine or serine results in complete loss of activity. Thus mutated BHMT may lead to accumulation of Hcy levels. [50,51] In our study the frequency of AA genotype was observed in 08% in group 1 and 04% in group 2 and when compared to 8% in case group in Heil et al study. 'A' allele had a maximum odds ratio of 2.1 (95% CI 1.3-2) with statistically significant p value (<0.01) in chi square test, suggesting its association with B12 deficiency. Presence of the homozygous mutated allele AA may result in significant elevation in MMA and non-significant elevation in Hcy levels as in Heil et al study, where AA allele is associated with significant elevation in Hcy levels. [49]Elevated homocysteine levels are risk factors for cardiovascular disease, osteoporosis and dementia. [52]BHMT is not involved directly in the folate-dependent pathway. Hyperhomocysteinemia due to BHMT pathway is enhanced when folatedependent remethylation is disrupted. [53] The diagnostic efficiency of BHMT polymorphism in predicting vitamin B12 status was evaluated by statistical analysis and the results confirmed that A allele had more specificity than sensitivity for diagnosing the deficiency. [54] A high likelihood ratio (LR) for AA and AG genotype suggests that A allele had more probability in predicting the risk of B12 deficiency status and its associated biochemical markers. . LR of A allele for Folic acid is 2.65; 2.73 for MMA and 1.85 for Hcy.

Conclusion
In our study, we have been able to find the association of rs3733890 in BHMT, genes with vitamin B12 deficiency in T2DM patients who were on metformin therapy. We also observed that the T2DM patients adhering to metformin with homozygous genotypes AA in BHMT genes have significantly lower levels of vitamin B12 together with high risk in developing vitamin B12 deficiency. Decreased Folic acid levels together with increased levels of MMA and Homocysteine were found to have high specificity with the above genetic variations of BHMT (rs3733890) in predicting vitamin B12 deficiency in T2DM patients on metformin therapy. To conclude, the genetic variants of BHMT (rs3733890) are associated with vitamin B12 deficiency in T2DM patients on metformin therapy. Henceforth, BHMT (rs3733890) can be a good predictor for vitamin B12 deficiency in T2DM patients on metformin therapy. Further studies can be carried out to find out the association of other genes associated with vitamin B12 deficiency in T2DM patients on metformin therapy.