Keywords

MTHFR, c.677C>T and c.1298A>C variants, Type 2 diabetes, Congolese

Introduction

Type 2 diabetes (T2DM), accounts for 80-90% of all diabetes [1].

It is a complex, multifactorial metabolic disease essentially favored by dietary imbalance and genetic predisposition [2]. The severity of its complications, especially cardiovascular, makes T2DM a major public health problem worldwide [1].

In 2021, the International Diabetes Federation (IDF) estimated the global prevalence of T2DM at 10% and it is expected to reach 15% (or 753 million people) in 2045 [3]. In Africa, it affects 22% of the population (or 24 million people) and is expected to reach 55 million by 2045 [3].

In the Congo, according to the national plan to combat non-communicable diseases (2013-2017), diabetes ranks second (10%) among chronic diseases after cardiovascular diseases [4].

Genetically, T2DM is a polygenic disease and there are several predisposing genes, including MTHFR (methylenetetrahydrofolate reductase). This gene, located at 1p36.3, contains 12 exons [5]. It is involved in homocysteine (Hcy) metabolism and several studies have shown that it is associated with T2DM [2,6].

The most commonly reported variants in the MTHFR gene are variant c.677C>T (p.Ala222Val) (rs1801133) on exon 4, and variant c.1298A>C (p.Glu429Ala) (rs1801131) on exon 7 [7-9]. These variants lead to the formation of a heat-labile isoform with reduced enzymatic activity (70% and 30%) for c.677C>T and c.1298A>C, respectively) [10], resulting in a relative deficiency in the remethylation process of Hcy [11] and finally leading to a high plasma Hcy concentration in addition to a drop in plasma folates for c.667C>T [7,9] whereas c.1298A>C is not necessarily associated with higher Hcy concentrations [8].

These variants are also associated with macrovascular complications (arterial hypertension, stroke and heart failure) or microvascular complications (retinopathy, nephropathy, neuropathy, diabetic foot and susceptibility to infections) [12].

In Congo, these variants have never been studied. Given the high prevalence of T2DM in the country and the frequency of these degenerative complications, we were interested in exploring these variants in this population. The objectives of this study were firstly to investigate these variants (allele and genotype frequencies) in Congolese subjects with T2DM compared to controls and, secondly, to establish a correlation between the genotypes identified and the degenerative complications presented by these T2DM subjects.

Patients and Methods

This was a prospective case-control study conducted over a two-year period (October 2020 to December 2022). Patients were recruited from the Department of Metabolic and Endocrine Diseases at Brazzaville University Hospital (Congo) and from the DI@BCARE diabetes management centre. Genetic analyses were performed at the Biochemistry and Molecular Biology Laboratory of the Tours University Hospital (France).

Patients

The study was conducted on one hundred patients with a clinical and paraclinical profile (blood glucose > 1.20 g/l) in favour of T2DM and fifty non-diabetic healthy controls, selected at random. The T2DM patients were divided into two subgroups: Thirty-four with macrovascular and microvascular complications and sixty-six without vascular complications.

Diabetic patients with pathologies (liver disease, hypothyroidism, cancer) that could influence Hcy concentration were not included in the study.

All consenting individuals assumed to be healthy were included as controls in this study.

Methods

Epidemiological investigation: Anthropometric parameters and sociodemographic data were collected on a pre-established form containing a range of data including age, sex and medical history.

Glycaemia testing: All selected patients were first tested for blood glucose using the hexokinase enzymatic method in serum (Abbot Glucose kit ref 3L82-22) [13]. It was performed at the Biochemistry Laboratory of the Brazzaville University Hospital.

Molecular analysis: After collection of venous blood on an EDTA tube, the samples were stored at -80 °C and then shipped to the Biochemistry and Molecular Biology Laboratory at Tours University Hospital (France).

DNA Extraction was carried out from leucocytes using the QIA symphony extractor from the Innovative Transversal Technology Unit of the CHRU Bretonneau Laboratories in Tours.

DNA concentration was measured using Nanodrop 2000 ^® absorption spectrophotometry at 260 nm and DNA quality was assessed using the 260/280 nm ratio.

The technique used for genotyping the MTHFR gene was the PCR/Sanger method described by Smith [14].

The primers used were as follows:

F: 5'-CCTCTCCTGACTGTCATCCC-3'; R: 5'-GCCTTCACAAAGCGGAAGAA-3’ for the c.677C>T variant.

F:5'-TACCTGAAGAGCAAGTCCCC-3'; R:5'-ACAGGATGGGGAAGTCACAG-3' for the identification of the c.1298A>C variant.

Exon amplification was performed on an AB Applied Biosystems Veriti ^® thermal cycler. PCR products were read after migration on a 1% agarose gel using the BIORAD Gel Doc™ XR+ Molecular Imager ^© .

The amplicons were then purified on a Macherey-Nagel™ plate to remove excess primers and nucleotides not incorporated during the PCR reaction.

Sequencing of the amplification products of the MTHFR gene was performed on the Biometra TRIO™ Analytikjena thermal cycler, using the Big Dye Terminator Cycle Sequencing kit with the same primers as for PCR. A purification phase was then performed on Millipore™ plate and then transferred to a MicroAmp™ plate to finally be placed in an Applied Biosystems™ 3500xL Genetic Analyzer DNA sequences.

The results were analysed using SeqScape Software v4 and Sequencing analysis Software v7 from Applied Biosystems™.

Statistical analysis

Statistical analyses were performed using Excel 2013 and SPSS ^® 20.0 software. Data were expressed as percentages and allelic frequencies for qualitative data and as mean ± standard deviation for quantitative data. Comparisons were made using the Student's t test for quantitative variables and the χ ² test for qualitative variables (such as allelic frequencies). Statistical tests were considered significant for a value of (p) < 0.05.

Results

Age and sex of the study population

 The mean age of the T2 DM subjects (Figure 1) was 52.2 ± 10.8 years, with extremes ranging from 30 to 83 years. Control cases had a mean age of 42.3 ± 7.1 years, with extremes ranging from 28 to 61 years. We found no significant difference between the ages of patients and controls (p = 0.184).

The gender distribution (Figure 2) for T2DM patients was 50/100 females, with a sex ratio of 1. In the controls, there was a clear predominance of females (36/50). There was a statistically significant difference between controls and controls with p = 0.01.

Identification of genotypes in the MTHFR gene

c.677C>T variant: 677C>T variation was found in 17% (17/100) of T2DM in heterozygous form only. It was found in 4% of controls (2/50). It should be noted that the homozygous 677TT genotype was not found in our series. The difference in frequency of genotypes identified was statistically significant between controls and T2DM (p = 0.024) with an increased risk of T2DM of odds ratio (OR) = 4.9 (95% CI: 1.1-22.2) (Table 1).

Variant c.1298A>C: This variation was identified in both heterozygous and homozygous form. The heterozygous1298AC genotype was present in 18% (18/100) of T2DM and in 6% (3/50) of controls (Table 1). The homozygous 1298CC genotype was found only in T2DM (2%). As with the previous variation, the frequency of the latter genotypes was also significantly different (p = 0.024).

The A1298C variation was associated with T2DM at an OR risk of 3.99 (95% CI: 1.1-13.8).

Combinations of C677T and A1298C mutations

Sequencing of the MTHFR gene also identified an association of mutated genotypes in T2DM: (677CT; 1298AC) in 1% (2/100) of cases; (677CC; 1298CC) in 2% of cases.

The other combinations identified in T2DM: (677CC; 1298AC) in 17% of cases and (677CT; 1298AA) in 16% of cases; p = 0.274 showing no difference.

Correlation of identified mutations with degenerative complications

Degenerative complications were observed in 34% (34/100) of T2DM patients. Of these patients, 58.8% (20/34) had the 677C>T variation in heterozygous form, while 41.2% (14/34) had the 1298AC variation. The latter was heterozygous in 93.3% (14/15) of cases and homozygous in 6.9% (1/15) (Table 2).

Discussion

MTHFR variants

Numerous studies conducted in various countries on the c.677C>T and c.1298A>C mutations show a significant association with T2DM [2,5,15,16].

C677T variant

The results of our study revealed that the C677T mutation is more frequent in the heterozygous form in Congolese T2DM (17%) than in controls (4%) with a 95% CI [1.2-22.2], p-value 0.018. Our data are close to those reported in Egypt (18.3%) but significantly lower than those found in the Egyptian control group (18.3%) [16]. It should be noted that these results are not isolated; in fact, the same mutation has already been identified in Tunisia and Morocco in 31.4% and 34.9% of T2DM and in 29.4% and 46.5% of controls respectively [17,18]. In Brazil, 37% of T2DM and 43% of controls [19]. As observed in the literature, this heterozygosity is identifiable in both groups studied.

The homozygous 677TT form was not found in our study cohort. This is normal given the allele frequencies and the fact that the Hardy Weinberg equilibrium was respected in the T2DM group: C677T (chi ² : 1.031; p = 0.597) and the Control group: C677T (chi ² : 0.00; p = 1). However, it has been frequently reported in other studies. For example, in Egypt and Tunisia, it was observed in 20% and 27.5% of T2DM and 6.67% and 5.9% of controls respectively [16,17]. In Brazil, it is present in 9% of T2DM and 12% of controls respectively [19].

It should be noted that low values have been observed in the Afro-Brazilian and Afro-American diabetic community (1 to 2%) [20,21]. Furthermore, in African-Americans, the 677TT genotype has never been identified as in the present study [22].

These variations could be explained by the consequence of the presence of these variants: Hyperhomocysteinemia. In fact, these HHcy are related to ethnicity in that they are linked to diet, particularly folate deficiency [23,24].

Some studies have reported contradictory results, finding no link between the C677T mutation of the MTHFR gene and T2D [25,26].

A1298C variant

Its allelic frequency was 20% in our series and is often reported in the literature [16,27,28]. Yan, et al., whose work was carried out on two types of populations, revealed that there is a predisposition to diabetes in Asian populations carrying this variant. However, in Caucasian populations, the authors found no link between the A1298C variant and diabetes [27].

Genotypically, the 1298AC heterozygosity was detected in our study in 18% of T2DM patients and in 6% of controls. Higher frequencies have also been reported in North African communities: In Egypt (50% in T2DM and 28.3% in controls) and Tunisia (42.5% in T2DM and 7% in controls) [16,28].

The homozygous 1298CC form found in our study in T2DM had low rates (2%). However, higher frequencies have been reported in Egypt (16.6% of T2DM and 8.33% of controls), Tunisia (42.5% of T2DM and 7% of controls) and Morocco (34% of T2DM) [16,18,28].

This low frequency can be explained by the allele frequencies and the fact that the Hardy Weinberg equilibrium was respected in the T2DM group (chi ² : 4.35; p = 0.113) and controls (chi ² : 0.985; p = 0.321).

Finally, our results, combined with those in the literature, suggest that the A1298C mutation predisposes to T2DM at high risk and appears to be more frequent in Caucasians.

Genotypic combinations

There is some evidence in the literature that the combination of the C677T and A1298C mutations may act synergistically to reduce the activity of the MTHFR enzyme in T2DM by 70% and 30% respectively [8,29].

When this association is in the combined forms, they further alter the activity of the MTHFR enzyme.

In the present study, the forms detected were (677CC; 1298CC), (677CT; 1298AC) (677CC; 1298AC) and (677CT; 1298AA). However, the homozygous mutated form (677TT; 1298CC) was not found in this study.

These results are similar to those reported in North Africa (Egypt and Morocco), which have described the same combinations in T2DM, at varying frequencies [16,18].

Several hypotheses have been invoked: environment, lifestyle, ethnicity, genetic predisposition [28]. A future study focusing on an epigenetic analysis would be interesting, in order to investigate the existence of MTHFR gene-environment interactions.

Degenerative complications associated with MTHFR mutations in T2DM

Numerous studies correlate diabetic complications with MTHFR mutations [28,30,31]. Concerning the C677T mutation, it should be noted that some case-control studies show no association between C677T and the occurrence of degenerative complications of T2DM [25,31,32]. For the A1298C mutation, some studies have found no link between the mutation and predisposition to degenerative complications [18,31,33].

However, the present study significantly suggests that there is a relationship between degenerative complications and the mutations identified. In our study population, degenerative complications occurred in 34% of T2DM, in whom the mutations were detected either in heterozygous or homozygous form.

The C677T mutation was identified exclusively in heterozygous form in 58.8% of T2DM presenting with degenerative complications. In contrast, the 1298AC mutation was observed in 44.1% (15/34) of T2DM, of which 93.3% (14/15) were heterozygous. The homozygous form was observed in only 6.9% (1/15) of T2DM with degenerative complications.

The genotype combinations (677CC; 1298CC) and (677CT; 1298AC) were the most implicated in the occurrence of degenerative complications in Congolese T2DM.

Sex and age

We used an unpaired sample for the 2 groups.

Concerning gender: Data from the literature show that the C677T and A1298C polymorphisms associated with T2DM are responsible for the hyperhomocysteinemia detected more in male subjects [34,35].

The data from the present study show that, genotypically, the heterozygous forms (677CT; 1298AC) were mainly detected in male subjects (63.2%; 71.4%), while the single homozygous mutated form (1298CC) was found in both sexes (Table 2).

These results suggest that heterozygous forms (677CT; 1298AC) predispose mainly male Congolese subjects to T2DM.

Concerning age: The literature reports that the two mutations studied are mainly observed in young subjects [36].

However, our study reveals that both young and elderly subjects are affected. Indeed, the heterozygous forms (677CT; 1298AC) are observed in the 28-60 age group and the homozygous mutated form (1298CC) in a relatively older age group: 50-71 years. These results corroborate those of other published studies [18,37].

Limitations of the Study

The study relied on a single method of analysis (sequencing of the MTHFR gene) and a small sample, and also the control group was unmatched.

Conclusion

The C677T and A1298C mutations in the MTHFR polymorphism gene are more frequent in type 2 diabetics (compared with control cases).

Their prevalence suggests that these mutations are risk factors predisposing Congolese subjects (especially males) to T2DM in the Congolese population.

In addition, genotype combinations (677CT; 1298AC) and (677CC; 1298CC) are more prone to degenerative complications.

Acknowledgements

Our thanks go to: French Embassy and Campus France for facilitating mobility; Laboratoire de Biochimie et Biologie Moléculaire of CHRU Bretonneau de Tours for genetic analyses; Formation Doctorale Santé Biologie Humaine de la Faculté des Sciences de la Santé de Brazzaville.

Contribution to the Authors

MHG, ME, and AC initiated the project. IMVR, AH, DC, VP, AC, carried out the experimentation. BE, MHG and WBE recruited the patients. IMVR, PH and BALM. AC reviewed the manuscript.

Conflict of Interest

None.

References

1. http://www.who.int/topics/diabetes_mellitus/fr/
2. Meng Y, Liu X, Ma K, Zhang L, Lu M, et al. (2019) Association of MTHFR C677T polymorphism and type 2 diabetes mellitus (T2DM) susceptibility. Mol Genet Genomic Med 7: e1020.
3. Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas, 10th edition.
4. Integrated National Plan for the Fight against Non-Communicable Diseases in Congo, Ministry of Health, 2013-2017.
5. Elnagar IZ, Pasha HF, Eldaly MM, Hadhoud KM (2023) Methylenetetrahydrofolate reductase gene polymorphisms in Egyptian Patients with type 2 diabetis mellitus. Journal of Pharmaceutical Negative Results 14: 3569-3577.
6. Yako YY, Guewo-Fokeng M, Balti EV, Bouatia-Naji N, Matsha TE, et al. (2016) Genetic risk of type 2 diabetes in populations of the African continent: A systematic review and meta-analyses. Diabetes Res Clin Prac 114: 136-150.
7. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, et al. (1995) A candidate genetic risk factor for vascular disesase: A common mutation in methylenetetrahydrofolate reductase. Nat Genet 10: 111-113.
8. Weisberg IS, Jacques PF, Selhub J, Bostom AG, Chen Z, et al. (2001) The 1298A>C polymorphism in methylenetetrahydrofolate reductase (MTHFR): In vitro expression and association with homocysteine. Atherosclerosis 156: 409-415.
9. de Bree A, Verschuren WMM, Monsen ALB, Vander PNMJ, Heil SG, et al. (2003) Effect of the methylenetetrahydrofolate reductase 677C>T mutation on the relations among folate intake and plasma folate and homocysteine concentrations in a general population sample. Am J Clin Nutr 77: 687-693.
10. Zintzaras E, Chatzoulis DZ, Karabatsas CH, Stefanidis I (2005) The relationship between C677T methylenetetrahydrofolate reductase gene polymorphism and retinopathy in type 2 diabetes: A meta-analysis. J Hum Genet 50: 267-275.
11. Thaler R, Agsten M, Spitzer S, Paschalis EP, Karlic H, et al. (2011) Homocysteine suppresses the expression of the collagen cross-linker lysyl oxidase involving IL-6, Fli1, and epigenetic DNA methylation. J Biol Chem 286: 5578-5588.
12. Sparso T, Grarup N, Andreasen C, Albrechtsen A, Holmkvist J, et al. (2009) Combined analysis of 19 common validated type 2 diabetes susceptibility gene variants shows moderate discriminative value and no evidence of gene-gene interaction. Diabetologia 52: 1308-1314.
13. Fletcher O, Kessling AM (1998) MTHFR association with arteriosclerotic vascular disease? Hum Genet 103: 11-21.
14. Saoud MZ, Mechal Y, Biaz A, Rachid A, El Machtani S, et al. (2019) Verification des performances analytiques du dosage du glucose plasmatique sur Architect ci8200. JIRR-19-BIO009.
15. Smith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, et al. (1986) Fluorescence detection in automated DNA sequence analysis. Nature 321: 674-679.
16. Chango A, Boisson F, Barbe F, Quilliot D, Droesch S, et al. (2007) The effect of 677 CT and 1298 AC mutations on plasma homocysteine and 5,10-methylenetetrahydrofolate reductase activity in healthy subjects. Br J Nutr 83: 593-596.
17. AbdRaboh NR, Badr S, Ali S (2013) Prevalence of methylenetetrahydrofolate reductase C677T and A1298C polymorphisms in Egyptian patients with type 2 diabetes mellitus. The Egyptian Journal of Medical Human Genetics 14: 87-93.
18. Oudi M, Aouni Z, Mazigh C, Essaies O, Zidi B, et al. (2008) Polymorphisme C677T du gène de la MTHFR chez une population tunisienne diabétique de type 2 C677T Polymorphism of MTHFR gene in a Tunisian type 2 diabetic population. IBS 23: 220-223.
19. Benrahma H, Abidi O, Melouk L, Ajjemami M, Rouba H, et al. (2012) Association of the C677T Polymorphism in the Human Methylenetetrahydrofolate Reductase (MTHFR) Gene with the Genetic Predisposition for Type 2 Diabetes Mellitus in a Moroccan Population. Genet Test Mol Biomarkers 16: 383-387.
20. Helfenstein T, Fonseca FAH, Relvas WGM, Santos AO, Dabela ML, et al. (2005) Prevalence of myocardial infarction is related to hyperhomocysteinemia but not influenced by C677T methylenetetrahydrofolate reductase and A2756G methionine synthase polymorphisms in diabetic and non-diabetic subjects. Clin Chim Acta 355: 165-172.
21. Botto LD, Yang Q (2000) 5,10-methylenetetrahydrofolate reductase gene variants and congenital anomalies: A HuGE review. Am J Epidemiol 151: 862-877.
22. Wilcken B, Bamforth F, Li Z, Zhu H, Ritvanen A, et al. (2003) Geographical and ethnic variation of the 677 C > T allele of 5,10 methylenetetrahydrofolate reductase (MTHFR): findings from over 7000 newborns from 16 areas worldwide. J Med Genet 40: 619-625.
23. Scholtz CL, Hein JO, Thiart R, Loubser L, Hillermann R, et al. (2002) Analysis of two mutations in the mthfr gene associated with mild hyperhomocysteinaemia - heterogeneous distribution in the south African population. S Afr Med J 92: 464-467.
24. Rosenberg N, Murata M, Ikeda Y, Opare-Sem O, Zivelin A, et al. (2002) The frequent 5,10-methylenetetrahydrofolate reductase C677T polymorphism is associated with a common haplotype in whites, Japanese and Africans. Am J Hum Genet 70: 758-762.
25. Saweda AH, Sunarti, Sutomo R, Hayashi C, Lee MJ, et al. (2002) The C677T mutation in the methylenetetrahydrofolate reductase gene among the Indonesian Javanese population. Kobe J Med Sci 48: 137-144.
26. Zhong JH, Rodriguez AC, Yang NN, Li LQ (2013) Methylenetetrahydrofolate reductase gene polymorphism and risk of type 2 diabetes mellitus. PLoS One 8: e74521.
27. Ay A, Alkanli N, Kurt I, Ustundag S, Siphai T, et al. (2002) The roles of MTHFR (C677T, A1298C) and MGP (G-7A, T-138C) gene variations in development of diabetic nephropathy in patients with type 2 diabetes mellitus. J Diabetes Metab Disord 21: 1317-1326.
28. Yan Y, Liang H, Yang S, Xie L, Qin X, et al. (2014) Methylenetetrahydrofolate reductase A1298C polymorphism and diabetes risk: Evidence from a meta-analysis. Ren Fail 36: 1013-1017.
29. Fekih-Mrissa N, Mrad M, Ibrahim H, Akremi I, Sayeh A, et al. (2017) Methylenetetrahydrofolate Reductase (MTHFR) (C677T and A1298C) Polymorphisms and Vascular Complications in Patients with Type 2 Diabetes. Can J Diabetes 41: 366-371.
30. Alghasham A, Settin AA, Ahmad Ali A, Dowaidar M, I Hisham, et al. (2012) Association of MTHFR C677T and A1298C gene polymorphisms with hypertension. Int J Health Sci 6: 3-11.
31. Chehadeh SWEH, Jelinek HF, Mahmeed WAA, Tay GK, Odama UO, et al. (2016) Relationship between MTHFR C677T and A1298C gene polymorphisms and complications of type 2 diabetes mellitus in an Emirati population. Meta Gene 9: 70-75.
32. Chang YH, Fu WM, Wu YH, Yeh CJ, Huang CN, et al. (2011) Prevalence of methylenetetrahydrofolate reductase C677T and A1298C polymorphisms in Taiwanese patients with Type 2 diabetic mellitus. Clin Biochem 44: 1370-1374.
33. Demellawy HH, Farhan HM, Dina EF, Tawfik MS. (2020) Methylene tetrahydrofolate reductase Gene Polymorphism C677T and Risk of Type two Diabetes Mellitus in Beni-Suef Governorate. Arab J Nucl Sci Appl 53: 159-166.
34. Jerbi Z, Abdennebi M, Douik H, Romdhane BH, Harzallah L, et al. (2005) Étude du polymorphisme C677T du gène de la méthylène tétrahydrofolate réductase dans la population tunisienne. Ann Biol Clin 63: 487-491.
35. Guillen M, Corella D, Portoles O, Gonzales JI, Mulet F, et al. (2001) Prevalence of the methylenetetrahydrofolate reductase 677 C > T mutation in the Mediterranean Spanish population. Association with cardiovascular risk factors. Eur J Epidemiol 17: 255-261.
36. Matsushita S, Muramatsu T, Arai H, Matsui T, Higuchi S, et al. (1997) The frequency methylenetetrahydrofolate reductase gene mutation varies with age in the normal population. Am J Hum Genet 61: 1459-1460.
37. Mtiraoui N, Ezzidi I, Chaieb M, Marmouche H, Aouni Z, et al. (2007) MTHFR C677T and A1298C gene polymorphisms and hyperhomocysteinemia as risk factors of diabetic nephropathy in type 2 diabetes patients. Diabetes Res Clin Pract 75: 99-106.

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