Open Access

5,10‑Methylenetetrahydrofolate reductase (MTHFR) C677T/A1298C polymorphisms in patients with nonsyndromic cleft lip and palate

  • Authors:
    • Yuske Komiyama
    • Chikako Koshiji
    • Waka Yoshida
    • Nagato Natsume
    • Hitoshi Kawamata
  • View Affiliations

  • Published online on: October 13, 2020     https://doi.org/10.3892/br.2020.1364
  • Article Number: 57
  • Copyright: © Komiyama et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Cleft lip with or without cleft palate (CL/P) is considered a multifactorial genetic disorder. Folic acid metabolism has been suggested to underlie the development of CL/P. The gene for the enzyme 5,10‑methylentetrahydrofolate reductase (MTHFR) contributes to folic acid metabolism, and polymorphisms of this gene at C677T (rs1801133) and A1298C (rs1801131) are reported to alter its enzyme activity and are suggested to be involved in CL/P development. We investigated C677T and A1298C polymorphisms of the MTHFR gene in Japanese patients with nonsyndromic CL/P and cleft palate only (CPO). We examined 240 patients with CL/P, 103 fathers and 153 mothers of the patients, and 68 healthy controls. Restriction fragment length polymorphisms (RFLPs) of C677T and A1298C of MTHFR were analyzed. We determined the frequencies of the polymorphisms in the patients and controls and performed a transmission equilibrium test and haplotype analysis of both MTHFR C677T and A1298C. There were no significant differences in the frequencies of MTHFR C677T and A1298C polymorphisms between the patients and controls. We did not observe transmission equilibrium or linkage equilibrium among the cases. In this experimental condition, we did not detect an association of MTHFR C677T and/or A1298C polymorphisms with the development of CL/P in this Japanese cohort.

Introduction

Cleft lip with or without cleft palate (CL/P) is the most frequent congenital abnormality in the maxillofacial region. The prevalence of cleft lip with or without cleft palate in Japan is 1.73 per 1,000 individuals (1). As a disease model for CL/P, a multi-factorial threshold model involving several genetic and environmental factors has been proposed to explain the development of CL/P, but it is difficult to identify key factors (2). Modifications in various genes have been proposed to be related to the development of CL/P, including IRF6, VAX1, 8q24 locus, ABCA4, MAFB, and 17q22 locus (which were identified mainly by genome-wide association studies), and BMP4, FGFR2, FOXE1, MSX1, MYH9, CHIRSPLD2 FGF8, GSTT1, MTHFR, PDGFC, PVRL1, SUMO1, TGFα, and TGFβ3 (which were revealed mainly by candidate gene association or mutation detection studies) (3,4). Smoking, alcohol consumption, drug use, and viral infection during pregnancy have been proposed to be associated with the development of CL/P as environmental factors (2).

It has been known for decades that vitamin deficiencies during pregnancy can cause congenital abnormalities. Folic acid deficiency is well known to affect neural tube closure and the development of orofacial clefts, and supplementation with folic acid was demonstrated to reduce the frequencies of congenital abnormalities (2,5-7). In A/J mice, folic acid administered before and during pregnancy reduced the onset of naturally developed CL/P (8,9). Tolarova and Harris and Shaw et al reported a decrease in the prevalence of CL/P when mothers took vitamin supplements including folic acid before and during pregnancy until the organogenesis stage (10,11).

Regarding the metabolism of folic acid, the enzyme activity of 5,10-methylenetetrahydrofolate reductase (MTHFR) was proposed to be a rate-determining step influencing serum folic acid levels (12). Polymorphisms of the MTHFR gene at base pair 677 and 1298 (C677T; rs1801133, A1298C; rs1801131) were demonstrated to decrease the enzyme activity (13). These polymorphisms were also suggested to be correlated with CL/P (11,14). We conducted the present study to investigate the relationship between MTHFR gene polymorphisms and the development of CL/P in a Japanese population.

Patients and methods

Patients

The patient series included Japanese individuals with non-syndromic CL/P treated at the Department of Oral and Maxillofacial Surgery, Dokkyo Medical University Hospital or the Cleft Lip and Palate Center, Aichi-Gakuin University Dental Hospital. The patients, parents and guardians of the patients and control subjects provided informed consent for the investigation of gene polymorphisms for MTHFR by polymerase chain reaction (PCR) amplification of their DNA fragment. The study was approved by the Clinical Research Ethics Committee of Dokkyo University Hospital (approval no. R-33-22J), and the Research Ethics Committee of Aichi-Gakuin University School of Dentistry (approval no. 55). The samples were collected from April 2004 to March 2007 at Aichi-Gakuin University Dental Hospital and from June 2007 to May 2008 at Dokkyo Medical University Hospital. We were able to analyze 240 patients as well as 103 fathers and 153 mothers of these patients. As controls, 68 volunteers with no congenital abnormalities (including cleft lip and palate) and no family history of congenital abnormality were also enrolled (Table I).

Table I

Demographic data of the study cohort (n=240), parents and controls.

Table I

Demographic data of the study cohort (n=240), parents and controls.

Sex  Total no. of cases
Male  129
Female  111
Demographic data labeled by type of abnormality
Type of abnormalitySex No. of cases
CLA  73
     Right cleftMale 16
  Incomplete12
  Complete3
  Unknown1
 Female 5
  Incomplete3
  Complete2
  Unknown0
     Left cleftMale 18
  Incomplete13
  Complete4
  Unknown1
 Female 21
  Incomplete10
  Complete10
  Unknown1
     Bilateral cleftMale 8
  Incomplete3
  Complete4
  Unknown1
 Female 4
  Incomplete3
  Complete1
  Unknown0
Unidentified  1
     CLP  124
     Right cleftMale 23
  Incomplete6
  Complete15
  Unknown2
 Female 22
  Incomplete3
  Complete17
  Unknown2
     Left cleftMale 32
  Incomplete9
  Complete21
  Unknown2
 Female 22
  Incomplete3
  Complete18
  Unknown1
     Bilateral cleftMale 20
  Incomplete2
  Complete17
  Unknown1
 Female 5
  Incomplete1
  Complete2
  Unknown2
CP  43
Demographic data labeled by family members, and controls
Family membersType of abnormality No of cases
Fathers:  103
 of CLA 32
 of CP 17
 of CLP 54
Mothers:  153
 of CLA 51
 of CP 34
 of CLP 68
Controls:  68
 Male 29
 Female 39

[i] CLA, cleft lip; CLP, cleft lip palate; CP, cleft palate.

PCR amplification and detection of the restriction fragment length polymorphisms (RFLPs)

DNA was extracted from all of the subject peripheral blood samples with the use of the DNA Blood Mini kit (Qiagen GmbH) following the manufacturer's instructions. The extracted DNA was dissolved in Tris-EDTA buffer and stored at -80˚C until analysis. For the detection of the C677T polymorphism, the following primers were used: Forward primer, 5'-TGAAGGAGAAGGTGTCTGCGGGA-3' and reverse primer, 5'-AGGACGGTGCGGTGAGAGTG-3' (15). DNA (200 ng) was used as a template, and each primer set (0.2 µM final concentration) was treated in 10 µl of reaction solution containing 0.25 U Takara Ex Taq® (Takara Bio), dNTPs (0.4 mM each), and reaction buffer containing 4 mM Mg2+. After preheating for 5 min at 94˚C, the samples were denatured for 1 min at 94˚C followed by amplification at 64˚C for 1 min and extension at 72˚C for 1 min; the reaction was operated for 30 cycles following extension at 72˚C for 5 min.

For the detection of the A1298C polymorphism, the following primers were used: Forward primer, 5'-CTTTGGGGAGCTGAAGGACTACTAC-3' and reverse primer: 5'-CACTTTGTGACCATTCCGGTTTG-3' (16). We used 200 ng of DNA as a template, and each primer set (0.2 µM final concentration) was treated in 10 µl of reaction solution containing 0.25 U Takara Ex Taq®, dNTPs (0.4 mM each), and reaction buffer containing 4 mM Mg2+. After preheating at 94˚C for 5 min, the samples were denatured at 94˚C for 1 min followed by amplification at 57˚C for 1 min and extension at 72˚C for 1 min; the reaction was repeated for 30 cycles following extension at 72˚C.

The PCR products amplified by the two primer sets were digested by restriction enzymes for the detection of polymorphisms. For C677T detection, the PCR products were a 198-bp fragment. Conversion of C to T creates an HinfI restriction site, the 198 bp fragment was digested into 175-bp and 23-bp fragments (Fig. 1A). For the A1298C detection, the PCR products were 163 bp and MboII digested the fragment into 56-bp, 31-bp, 30-bp, 28-bp and 18-bp fragments. Conversion of A to C abolishes the MboII restriction site, it digests into 84, 31, 30, 18 bp fragments (Fig. 1B). All the subject genotypes were determined by PCR-RFLP and the allele and genotype frequencies were determined. With regard to the C677T polymorphism, the wild-type 677th base of the MTHFR gene is referred to as CC (677CC), the heterozygous type as CT (677CT), and the recessive type as TT (677TT). With regard to the A1298C polymorphism, the wild-type is referred to as AA (1298AA), the heterozygous type as AC (1298AC), and the recessive type as CC (1298CC).

Transmission disequilibrium test (TDT)

We examined the over-transmission of C677T and A1298C from heterozygous parents to their children with CL/P (patients) by conducting a transmission disequilibrium test (TDT). The family-based association test (FBAT) developed by Laird and Lange was conducted as previously described (17).

Haplotype analysis

Since the transmission of an allele from parents to their children is known to differ from family to family, we investigated the linkage disequilibrium of the subjects' haplotypes. In linkage disequilibrium test, D, D' and r, coefficient of linkage disequilibrium, were calculated. D indicates the coefficient of linkage disequilibrium, D' is the normalized coefficient of linkage disequilibrium, r is the coefficient of correlation. SNPstats, a web tool for SNP analysis, (https://snpstats.net) was used to estimate the haplotypes from genotype frequencies and to further analyze the linkage disequilibrium and haplotype association with CL/P (18).

Statistical analysis

We tested the Hardy-Weinberg equilibrium (HWE) for each patient, the patient's father and mother, and the controls with the use of the R package ‘HardyWeinberg’ (19,20). Fisher's exact test was used for the statistical analyses. The results of the TDT were analyzed using McNemar's test, and haplotypes were analyzed with the use of SNPstats software. We calculated the odds ratios (ORs) with confidence intervals (CIs) to assess the associations of genotypes. The strength of the association was tested under genetic models comparing the allele, genotype, and dominant (TT+CT vs. CC for C677T; CC+AC vs. AA for A1298C) or recessive (CC+CT vs. TT for C677T; AA+AC vs. CC for A1298C) models, respectively. A P-value <0.05 was indicative of a statistically significant difference.

Results

Polymorphism at base pair (bp) 677 of the MTHFR gene

The genotype frequency of the MTHFR C677T polymorphism of the patients, patients' fathers and mothers, and controls were in agreement with the HWE: P=0.89, P=0.55, P=0.17, and P=0.60, respectively. There were no significant differences in the allele frequencies of C and T at bp 677 of the MTHFR gene between the patients and controls. The genotype frequencies also showed no significant differences between the patients and controls (Table II). The dominant or recessive genetic model for C677T was tested to investigate the association of genotypes and CL/P, although the results revealed no association (Table II).

Table II

Polymorphisms of MTHFR C677T.

Table II

Polymorphisms of MTHFR C677T.

 Odds ratio
GroupPolymorphismCases (n=240) n (%)Controls (n=68) n (%)OR95% CIP-value
PatientGenotype     
       CC89 (37.1)27 (39.7)1.000  
       CT116 (48.3)34 (50.0)1.0350.582-1.8410.907
       TT35 (14.6)7 (10.3)1.5170.605-3.8010.372
 Allele     
       C294 (61.3)88 (64.7)1.000  
       T186 (38.8)48 (35.3)1.1600.780-1.7250.464
 Genetic model     
       Dominant151 (62.9)41 (60.3)1.1170.643-1.9400.694
       Recessive205 (85.4)61 (89.7)0.6720.284-1.5890.363
Type of cleft      
     CLAGenotype     
       CC32 (43.8)27 (39.7)1.000  
       CT32 (43.8)34 (50.0)0.7940.393-1.6050.521
       TT9 (12.4)7 (10.3)1.0850.357-3.3000.886
 Allele     
       C95 (66.0)88 (64.7)1.000  
       T49 (34.0)48 (35.3)0.9460.578-1.5470.824
 Genetic model     
       Dominant41 (56.2)41 (60.3)0.8440.431-1.6500.619
       Recessive64 (87.7)25 (78.1)1.9910.669-5.9250.210
     CLPGenotype     
       CC41 (33.1)27 (39.7)1.000  
       CT61 (49.2)34 (50.0)1.1810.622-2.2450.610
       TT22 (17.7)7 (10.3)2.0700.777-5.5120.141
 Allele     
       C143 (57.7)88 (64.7)1.000  
       T105 (42.3)48 (35.3)1.3460.873-2.0750.177
 Genetic model     
       Dominant83 (66.9)41 (60.3)1.3330.722-2.4610.357
       Recessive102 (82.3)61 (89.7)0.5320.215-1.3190.168
     CLA+CLPGenotype     
       CC73 (37.1)27 (39.7)1.000  
       CT93 (47.2)34 (50.0)1.0120.560-1.8270.969
       TT31 (15.7)7 (10.3)1.6380.645-4.1580.296
 Allele     
       C239 (60.7)88 (64.7)1.000  
       T155 (39.3)48 (35.3)1.1890.793-1.7840.403
 Genetic model     
       Dominant124 (62.9)41 (60.3)1.1190.636-1.9690.697
       Recessive166 (84.3)61 (89.7)0.6140.257-1.4680.270
     CPGenotype     
       CC16 (37.2)27 (39.7)1.000  
       CT23 (53.5)34 (50.0)1.1420.506-2.5760.750
       TT4 (9.30)7 (10.3)0.9640.244-3.8150.959
 Allele     
       C55 (64.0)88 (64.7)1.000  
       T31 (36.0)48 (35.3)1.0330.588-1.8150.909
 Genetic model     
       Dominant27 (62.8)41 (60.3)1.1110.506-2.4400.793
       Recessive39 (90.7)61 (89.7)1.1190.307-4.0750.865
Parents      
     FatherGenotype     
       CC31 (30.1)10 (34.5)1.000  
       CT54 (52.4)15 (51.7)1.1610.466-2.8960.748
       TT18 (17.5)4 (13.8)1.4520.397-5.310.572
 Allele     
       C116 (56.3)35 (60.3)1.000  
       T90 (43.7)23 (39.7)1.1810.652-2.1380.583
 Genetic model     
       Dominant72 (69.9)19 (65.5)1.2220.51-2.9290.652
       Recessive85 (82.5)25 (86.2)0.7560.234-2.4380.638
     MotherGenotype     
       CC56 (36.6)17 (43.6)1.000  
       CT80 (52.3)19 (48.7)1.2780.611-2.6740.514
       TT17 (11.1)3 (7.70)1.7200.45-6.5830.424
 Allele     
       C192 (62.7)53 (67.9)1.000  
       T114 (37.3)25 (32.1)1.2590.742-2.1360.393
 Genetic model     
       Dominant97 (63.4)22 (56.4)1.3380.656-2.7310.422
       Recessive136 (88.9)36 (92.3)0.6670.185-2.4010.533

[i] MTHFR, 5,10-methylentetrahydrofolate reductase; CLA, cleft lip; CLP, cleft lip palate; CP, cleft palate.

Polymorphism at base pair (bp) 1298 of the MTHFR gene

The genotype frequency of MTHFR A1298C polymorphism of the patients, patients' fathers and mothers, and controls were in agreement with the HWE: P=1.00, P=0.40, P=0.79, and P=1.00, respectively. There were no significant differences in the allele frequencies of A and C at bp 1298 of the MTHFR gene between the patients and controls. The genotype frequency also showed no significant difference between the patients and controls (Table III). The dominant or recessive genetic model for A1298C was tested to investigate the association of genotypes and CL/P, although the results revealed no association (Table III).

Table III

Polymorphism of the MTHFR A1298C.

Table III

Polymorphism of the MTHFR A1298C.

 Odds ratio
GroupPolymorphismCases (n=240) n (%)Controls (n=68) n (%)OR95% CIP-value
PatientGenotype     
       AA155 (64.6)43 (63.2)1.000  
       AC77 (32.1)22 (32.4)0.9710.552-1.3820.921
       CC8 (3.30)3 (4.40)0.7400.223-2.0500.665
 Allele     
       A232 (73.2)65 (72.2)1.000  
       C85 (26.8)25 (27.8)0.9270.603-1.2880.753
 Genetic model     
       Dominant85 (35.4)25 (36.8)0.9430.557-1.3570.838
       Recessive232 (96.7)65 (95.6)1.3380.293-2.2740.672
Type of cleft      
     CLAGenotype     
       AA51 (69.9)43 (63.2)1.000  
       AC21 (28.8)22 (32.4)0.7660.430-1.4370.474
       CC1 (1.40)3 (4.40)0.5620.124-2.5330.533
 Allele     
       A72 (76.6)65 (72.2)1.000  
       C22 (23.4)25 (27.8)0.7590.485-1.3360.369
 Genetic model     
       Dominant22 (30.1)25 (36.8)0.7420.435-1.4060.404
       Recessive71 (97.3)65 (95.6)1.6380.201-3.0740.592
     CLPGenotype     
       AA73 (58.9)43 (63.2)1.000  
       AC45 (36.3)22 (32.4)1.2050.575-1.4830.564
       CC6 (4.80)3 (4.40)0.3560.142-1.9300.165
 Allele     
       A118 (69.8)65 (72.2)1.000  
       C51 (30.2)25 (27.8)1.1510.638-1.3700.589
 Genetic model     
       Dominant51 (41.1)25 (36.8)1.2020.589-1.4610.554
       Recessive118 (95.2)65 (95.6)0.9080.232-2.1910.894
     CLA+CLPGenotype     
       AA124 (62.9)43 (63.2)1.000  
       AC65 (33.0)22 (32.4)1.0250.557-1.4070.936
       CC8 (4.10)3 (4.40)0.9250.245-2.1410.911
 Allele     
       A189 (72.1)65 (72.2)1.000  
       C73 (27.9)25 (27.8)0.9980.617-1.3130.994
 Genetic model     
       Dominant73 (37.1)25 (36.8)1.0130.568-1.3850.966
       Recessive189 (95.9)65 (95.6)1.0900.267-2.1900.900
     CPGenotype     
       AA31 (72.1)43 (63.2)1.000  
       AC11 (25.6)22 (32.4)0.6940.362-1.5170.402
       CC1 (2.30)3 (4.40)0.4620.071-3.1940.504
 Allele     
       A42 (77.8)65 (72.2)1.000  
       C12 (22.2)25 (27.8)1.3690.513-1.6720.443
 Genetic model     
       Dominant12 (27.9)25 (36.8)0.6660.366-1.4800.335
       Recessive42 (97.7)65 (95.6)1.9380.134-4.1530.566
Parents      
     FatherGenotype     
       AA63 (61.2)18 (62.1)1.000  
       AC33 (32.0)10 (34.5)0.9430.404-1.6270.896
       CC7 (6.80)1 (3.40)2.0000.156-3.8670.522
 Allele     
       A158 (76.7)46 (79.3)1.000  
       C48 (23.3)12 (20.7)1.1330.517-1.5330.731
 Genetic model     
       Dominant40 (38.8)11 (37.9)1.0390.435-1.6280.930
       Recessive96 (93.2)28 (96.6)0.4900.087-2.9280.504
     MotherGenotype     
       AA104(68)25 (64.1)1.000  
       AC44 (28.8)12 (30.8)0.8810.437-1.5120.749
       CC5 (3.30)2 (5.10)0.6010.147-2.3720.553
 Allele     
       A252 (82.4)62 (79.5)1.000  
       C54 (17.6)16 (20.5)0.8300.495-1.3740.558
 Genetic model     
       Dominant49 (32.0)14 (35.9)0.8410.444-1.4690.646
       Recessive148 (96.7)37 (94.9)1.6000.229-2.8250.580

[i] MTHFR, 5,10-methylentetrahydrofolate reductase; CLA, cleft lip; CLP, cleft lip palate; CP, cleft palate.

Polymorphism of the MTHFR gene in each cleft type

Since the onset mechanism of each cleft type differs, we compared the polymorphisms of the MTHFR gene in the patients with cleft lip, those with cleft lip with palate, and those with cleft palate only. Our analyses revealed that there were no significant differences in the allele frequency or genotype frequencies at either bp 677 or bp 1,298 of the MTHFR gene (Tables II and III). The dominant or recessive genetic model also did not reveal an association to each cleft type.

TDT

We investigated 28 families in which both parents had heterozygous C677T polymorphism (677CT) and nine families with heterozygous A1298C polymorphism (1298AC). No significant transmission disequilibrium was revealed in 677CT or 1298AC by the TDT results (Table IV).

Table IV

Results of the transmission disequilibrium test (TDT) of MTHFR C677T and A1298C polymorphisms.

Table IV

Results of the transmission disequilibrium test (TDT) of MTHFR C677T and A1298C polymorphisms.

AlleleTransmittedNot transmittedP-value
677C33230.181
677T2333 
1298A8100.637
1298C108 

[i] MTHFR, 5,10-methylentetrahydrofolate reductase.

Haplotype analysis

The haplotype analysis showed no linkage disequilibrium between C677T and A1298C (Table V). The haplotype estimation determined three types: C_A, T_A, and C_C. The frequency of haplotypes was similar to those reported in the Japanese population from the 1000 Genome Project: C_A, 44.2%; T_A, 38.0%; C_C, 17.8%; T_C, not detected in the Japanese population (21). We detected a rare haplotype (T_C) in one patient. No significant between-group differences were observed except for the rare case of T_C (Table V).

Table V

Haplotype analysis results for MTHFR 677 and 1,298 polymorphisms.

Table V

Haplotype analysis results for MTHFR 677 and 1,298 polymorphisms.

Linkage disequilibrium analysis
 C677TA1298C   
C677T-D=-0.0663   
  D'=0.8814   
  r=-0.3428   
A1298C--   
Haplotype frequency estimation (n=308) and association analysis
HaplotypeTotalControlsPatientsOR (95% CI)P-value
C_A0.43160.44120.42871.00 
T_A0.37090.35290.37541.09 (0.71-1.070)0.69
C_C0.18910.20590.18380.93 (0.55-1.57)0.79
T_C0.008900.012138636069.94<0.0001 (38636069.93-38636069.95)

[i] D, coefficient of linkage disequilibrium; D', normalized coefficient of linkage disequilibrium; r, coefficient of correlation. In comparison of the odds ratio, the most frequent haplotype C_A was used as reference. MTHFR, 5,10-methylentetrahydrofolate reductase.

Discussion

As one of the most common congenital abnormalities, cleft lip with or without cleft palate (CL/P) is a multifactorial genetic disorder that involves multiple genetic and environmental factors. Folic acid deficiency in mothers causes developmental defects in human fetuses and increases the risk of a malformed fetus, and folic acid supplementation has been reported to prevent the onset of cleft palate (6,7). Folic acid is a member of the vitamin B family that is involved in the synthesis of DNA, RNA, and amino acids. Folic acid is also involved in the reaction in which homocysteine is methylated and converted to methionine (8). In addition, folic acid is thought to play important roles in organogenesis (22).

Single-nucleotide polymorphisms of C677T and A1298C in MTHFR were shown to change the serum folic acid concentration (13). Genotype 677TT or 1298CC would decrease the enzyme activity of MTHFR and lower the production of 5-methyl tetrahydrofolic acid (5-methyl THF). The 5-methyl THF regulates the methylation of homocysteine to methionine as a coenzyme, and a decrease in 5-methyl THF disturbs this process and increases the serum level of homocysteine. Hyperhomocysteinemia is known to be involved in neural tube defects by affecting neural crest cells. It is believed that in such a scenario, mesenchymal cells derived from neural crest cells would also be affected, thus ultimately resulting in CL/P (9,11,22). To date, there have been no studies investigating MTHFR C677T polymorphism in relation to cleft types in Japanese subjects. The present study is the first to analyze MTHFR A1298C polymorphism together with MTHFR C677T polymorphism in Japanese patients with CL/P.

Contrary to our expectations, there was no significant difference in A1298C or in C677T of the MTHFR gene among the patients with any cleft type and the healthy control subjects. In addition, there was no significant transmission disequilibrium in either 677CT or 1298AC. The haplotype analysis also revealed no significant difference. Tolarova and Harris reported that MTHFR C677T polymorphism is involved in the onset of CL/P, and Martinelli et al noted that they observed no notable differences of MTHFR C677T polymorphism between patient and parent groups, but they did observe marked differences in the allele frequency between the mothers and the control group, and the frequency of T allele was high (10,23). In their study of mothers with low folic acid intake during pregnancy, van Rooij et al reported that the onset of CL/P was correlated with MTHFR C677T and A1298C polymorphisms (24). However, Shaw et al observed no correlations between C677T polymorphism and the development of cleft lip or cleft lip and palate (11), and Kanno et al documented no correlation between MTHFR C677T polymorphism and the development of CL/P in a Japanese series (25). According to a study by Pezzetti et al, MTHFR C677T polymorphism was correlated with the development of cleft lip with or without cleft palate, while A1298C polymorphism was not (4). With these conflicting results, no conclusions can be drawn regarding the relationship between the development of CL/P and MTHFR C677T or A1298C polymorphisms.

During the years 2007 to 2020, there were 15 meta-analyses concerning MTHFR C677T and A1298C polymorphisms and CL/P. Among them, 12 studies concerned C677T; nine studies suggested an increased risk for CL/P (26-34), whereas the other three studies denied the association (35-37). Most studies have thus suggested that the C677T polymorphism is likely to increase the risk for CL/P, which disagrees with our findings. Regarding the A11298C polymorphism, there are seven studies and six of them suggested no association with CL/P (27,29,34,36,38,39), which is consistent with our findings. There is a single study that suggested an association between A1298C and CL/P (30). Most of the studies mentioned the heterogeneity of the association due to the different ethnicities of the subjects. They also noted that environmental factors such as alcohol consumption, smoking habits, and maternal folic acid supplementation are important (37,38,40). According to the meta-analyses conducted to date, in Asians, C677T T allele or recessive genotype TT tends to be a risk for CL/P.

Ethnic differences in the distribution of MTHFR C677T and A1298C polymorphisms have been reported (13,41). Yoshida reported that the distribution of polymorphisms in Vietnamese and Mongolians differed from that in other Asians (41), and there are differences in distribution between our findings in Japanese and the frequency of MTHFR C677T polymorphism in Vietnamese. We also collected other cohorts by searching PubMed. We identified 21 matches reporting the prevalence of MTHFR C677T and A1298C in various ethnic populations, and there were some differences in allele frequencies. For example, genotype CC at bp 677 is likely to be dominant in Moroccan patients (42) whereas genotype TT in 677 is dominant in Chinese patients and Iranian patients (43,44). Some ethnicities even showed higher frequencies of a functionally superior genotype of MTHFR C677T and A1298C in the patient groups, which is the opposite of our hypothesis. This discrepancy may be due in part to the multiplicity of the enzymes, including MTHFR, that regulate the serum level of folic acid. In addition, it is known that folic acid nutritional supplementation can overcome the decreased activity of, at least, MTHFR. There is also a possibility that if the mother's folic acid serum level is sufficient, the child may overcome the recessive genotype.

Concerning the nutritional status of Japanese women, it has been reported that although their daily intake of folic acid is not sufficient, their serum concentrations are higher compared to women in other countries due to the tendency of Japanese women's dietary behavior during pregnancy (45). They tend to adopt healthy behaviors such as eating more colored vegetables instead of meat, which results in their consumption of folic acid as a highly bioavailable natural food (46). In the earlier study (45), even without a folic acid-fortified diet, most of the subjects exhibited a folic acid concentration that was sufficient to prevent neural tube defects.

A few studies have investigated gene-environment interactions; one study conducted in Caucasians demonstrated a reduced effect of maternal folic acid supplementation on the development of CL/P, although there was no association with C677T genetic status (37). Although, there have been no investigations of gene-environment interactions, especially maternal folic acid supplementation, in Asian populations including Japanese. The gene-environment interactions in these populations remain to be elucidated in future studies.

It is possible that our present sample size was not large enough to observe the association, and we therefore combined the patients examined by Tohoku University in a previous Japanese cohort with our present results in order to increase the sample number (26). We observed no significant difference in the allele frequencies or dominant genetic model frequency (C vs. T, case n=411, control n=317, odds ratio=0.954, 95% CI: 0.845-1.076, P-value=0.694; dominant model, cases n=205, control n=162, odds ratio=0.984, 95% CI: 0.830-1.168, P-value=0.926).

Concerning the meta-analysis data, C677T mutation might be a risk for CL/P development in Asian population. However, we could not observe such an association neither C677T nor A1298C in the Japanese population. It is likely that MTHFR C677T and A1298C polymorphisms have less impact on CL/P development in Japanese populations. Part of the reason for this may be because of the typical diet consumed by pregnant Japanese women, which may be the reason that we did not observe significant differences in the present case-control study. In this context, it is necessary to investigate both serum levels of vitamins, other major environmental factors including folic acid supplementation, smoking, alcohol consumption, and genetic factors at the same time points in order to determine whether decreased folic acid metabolism and/or gene polymorphisms of metabolic enzymes are risk factors for the development of CL/P.

Acknowledgements

We express our deepest gratitude to Dr Yutaka Imai for encouraging us to conduct this study, and to Dr Kazumoto Kimura of the Medical Information Center for his guidance concerning the statistical analysis.

Funding

No funding was received.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

CK and WY performed and analyzed the RFLP-PCR. YK and CK interpreted the data and were the major contributors to the manuscript's writing, and they contributed equally to this work. NN and HK contributed to the presentation of the data and the manuscript's revision. All authors read and approved the final manuscript.

Ethics approval and consent to participate

This study was approved by both the Clinical Research Ethics Committee of Dokkyo University Hospital (approval no. R-33-22J), and the Research Ethics Committee of Aichi-Gakuin University School of Dentistry (approval no. 55). The consent of the patients and controls for publication was obtained before their participation in the study.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Komiyama Y, Koshiji C, Yoshida W, Natsume N and Kawamata H: 5,10‑Methylenetetrahydrofolate reductase (<em>MTHFR</em>) C677T/A1298C polymorphisms in patients with nonsyndromic cleft lip and palate. Biomed Rep 13: 57, 2020
APA
Komiyama, Y., Koshiji, C., Yoshida, W., Natsume, N., & Kawamata, H. (2020). 5,10‑Methylenetetrahydrofolate reductase (<em>MTHFR</em>) C677T/A1298C polymorphisms in patients with nonsyndromic cleft lip and palate. Biomedical Reports, 13, 57. https://doi.org/10.3892/br.2020.1364
MLA
Komiyama, Y., Koshiji, C., Yoshida, W., Natsume, N., Kawamata, H."5,10‑Methylenetetrahydrofolate reductase (<em>MTHFR</em>) C677T/A1298C polymorphisms in patients with nonsyndromic cleft lip and palate". Biomedical Reports 13.6 (2020): 57.
Chicago
Komiyama, Y., Koshiji, C., Yoshida, W., Natsume, N., Kawamata, H."5,10‑Methylenetetrahydrofolate reductase (<em>MTHFR</em>) C677T/A1298C polymorphisms in patients with nonsyndromic cleft lip and palate". Biomedical Reports 13, no. 6 (2020): 57. https://doi.org/10.3892/br.2020.1364