- Email: [email protected]
Association between MTHFD1 polymorphisms and neural tube defect susceptibility
Journal of the Neurological Sciences 348 (2015) 188–194
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Association between MTHFD1 polymorphisms and neural tube defect susceptibility Jingjing Meng a, Lei Han b, Bo Zhuang c,⁎ a b c
Neonatal Ward, Jining No. 1 People's Hospital, Jining 272011, China Department of PICU, The Affiliated Hospital of Jining Medical College, Jining 272000, China Department of Pediatric Surgery, Jining No. 1 People's Hospital, Jining 272011, China
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 20 November 2014 Accepted 1 December 2014 Available online 6 December 2014 Keywords: Neural tube defects Methylenetetrahydrofolate dehydrogenase (MTHFD1) 1958GNA Polymorphism Folate metabolism Association
a b s t r a c t Objectives: Neural tube defect (NTD) is a common disease among neonates with multiplex symptom and complex origins, and the exact mechanism of NTD has not been definitely elucidated. Nevertheless, it is hypothesized that NTD risk can be prevented by periconceptional folic acid in folate metabolism. The methylenetetrahydrofolate dehydrogenase (MTHFD1) gene has been proved to play an important role in folate metabolism, which was strongly associated with the high risk for NTD. We focused on three folate metabolism-related single-nucleotide polymorphisms (SNPs) on the MTHFD1 gene to evaluate the associations between MTHFD1 polymorphisms and NTD susceptibility. Methods: We genotyped blood samples from 222 specimens (including 122 NTD-affected infants and 100 healthy controls) in a case–control study. We investigated the association between NTD and three selected tag-SNPs on MTHFD1 gene: 401A NG (rs1950902), 2305C N T (rs17857382) and 1958G NA (rs2236225) by the SNapShot method. These SNPs were identified by Haploview 4.2 software with HapMap databases, and then these associations were evaluated by the Mann–Whitney test, one-way analysis of variance (ANOVA) and chi-square test. Furthermore, a meta-analysis of the association between MTHFD1 1958GN A and NTD risk was also performed. Results: In our study, an increased risk of NTD was observed for 1958GN A of MTHFD1 (AA vs. GG: OR = 2.63, 95% CI = 2.61–5.70; AA vs. GG + GA: OR = 2.10, 95% CI = 1.07–4.14; A vs. G: OR = 1.62, 95% CI = 1.11–2.36). However, the other two SNPs (401A NG and 2305CN T) displayed no statistically significant association with NTD risk. The overall result of the meta-analysis indicated that the 1958G NA variant might not be a genetic susceptible factor for the Caucasian population. Conclusions: Our analysis implicated that MTHFD1 1958GNA was significantly associated with the susceptibility of NTD in a Chinese population. In addition, the AA homozygote carriers were more likely to suffer NTD, compared with the others with GA or GG genotypes. Validation of the risk effect and functional impact of this polymorphism is needed in future investigations. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Neural tube defect (NTD) is among the most common and deadly human congenital malformations at birth and manifested as a wide range of phenotypes, primarily including anencephaly, spina bifida and encephalocele which result from failure of the neural tube to close properly in the developing brain or lower spine [1,2]. The incidence rate of NTD is about 0.2% worldwide, but this rate varies in different races and sections with a conspicuous diversity which may be a result of genetic diversity [3]. Some previous studies demonstrated that the etiology of NTD is implicated with several factors, like gene–environment
⁎ Corresponding author at: Department of Pediatric Surgery, Jining No. 1 People's Hospital, No. 6 Jiankang Road, Shandong Province 272011, China. E-mail address: [email protected] (B. Zhuang).
http://dx.doi.org/10.1016/j.jns.2014.12.001 0022-510X/© 2014 Elsevier B.V. All rights reserved.
interactions and complex genetic factors [4–7]. These unclear factors would lie in the increased recurrence risk in siblings of individuals with NTD, the increased risk for NTD among other relatives, and the increased risk for birth defects overall among both close and distant relatives [8]. Recent studies also revealed that the NTD incidence and overall birth defect risk are higher in maternal relatives than in paternal relatives [9,10]. Though the exact mechanism of NTD has not been completely elucidated, it is hypothesized that genes coding folatedependent enzymes might have an impact on NTD risk. Epidemiological studies have revealed that periconceptional vitamin supplementation with folic acid substantially lowers the percentage of women with NTD-affected pregnancies [11]. Thus, elucidation of the mechanisms by which folate reduces the risk of NTD, currently focused on candidate genes that are involved in folate-related pathways could be informative for developing improved prevention strategies and reducing the global burden of NTD [12].
J. Meng et al. / Journal of the Neurological Sciences 348 (2015) 188–194
Methylenetetrahydrofolate dehydrogenase (MTHFD1) is a nicotinamide adenine dinucleotide phosphate (NADP)-dependent trifunctional cytoplasmic enzyme (often referred to as “C1-THF synthase”), acting as 10-formyl, 5,10-methenyl, and 5,10-methylene derivatives [13]. This folate-dependent enzyme has been proved to play an important role in folate metabolism, which is an attractive candidate gene associated with the high risk of NTD, but these associations remain controversial since several studies suggest no associations. Hence, in our study, some potential SNPs in the MTHFD1 gene were identified from public databases, and through analysis we finally choose 401AN G, 2305CN T and 1958GNA as representative SNPs to investigate their associations with NTD risk. Considering the limitation of an individual study with a small sample size, a further meta-analysis with public data for the purpose of getting an exhaustive conclusion of empirical evidences to prove the association between MTHFD1 1958GN A and NTD risk was also performed. Elucidation of the interaction might contribute to ameliorate the diagnosis and prognosis of NTD. 2. Material and methods 2.1. Study population Our study was conducted in strict accordance with the protocol approved by the Jining No. 1 People's Hospital, and a written informed consent must be provided by each participant (or their guardian). We recruited 222 specimens in total from Jining No. 1 People's Hospital. Among them, 122 subjects were confirmed as NTD patients; the other 100 subjects were healthy infants used as normal controls in this study. The neonates of NTD cases within our samples consisted of 62 females and 60 males, while the control group consisted of 47 females and 53 males as outlined in Table 2. We collected 5 ml venous blood sample from each participant after their guardians provided a written informed consent. All the samples and complete follow-up data were available for each specimen (more clinical characteristics are presented in Table 2).
189
Table 2 Comparison of neural tube defect patients and controls by selective characteristics. Clinical characteristics Sex Male Female Maternal age ≤20 20–30 ≥30 Gestational weeks ≤20 20–25 ≥25 Use of folic acid supplements Yes No Daily dietary intake of folates Q1 Q2–Q4 Overall intake of folates Lowa Highb
NTD patients (N = 122)
Normal controls (N = 100)
P-value
60 (49.2%) 62 (50.8%)
53 (53.0%) 47 (47.0%)
0.571
22 (18.0%) 92 (75.4%) 8 (6.6%)
23 (23.0%) 68 (68.0%) 9 (9.0%)
0.469
76 (62.3%) 31 (25.4%) 15 (12.3%)
59 (59.0%) 25 (25.0%) 16 (16.0%)
0.725
66 (54.1%) 56 (45.9%)
61 (61.0%) 39 (39.0%)
0.301
30 (24.6%) 92 (75.4%)
26 (26.0%) 74 (74.0%)
0.810
16 (13.1%) 106 (86.9%)
12 (12.0%) 88 (88.0%)
0.803
NTD, neural tube defect. a Women in the lowest quartile of maternal folate intake (Q1 ≤ 309.49 μg/100 g/day) who did not take supplemental folic acid in the periconceptional period. b All other combinations of folate intake (Q2, Q3 or Q4) and folic acid supplementation (yes/no).
56 μC for 30 s and then 72 μC for 40 s [14]. After amplification, the pyrosequencing primer was used to extend the PCR product by adding nucleotides in a specific sequential order based on the known sequence [8] as described previously. Appropriate controls were included in all assays and at least 10% of samples were repeatedly genotyped with N 99% agreement. We use the signal peaks' resulting pyrogram to determine the genotype. The primers and conditions of all assays are available upon request.
2.2. DNA methods 2.3. Statistical analysis The whole genomic DNA was extracted from blood samples of each specimen using Clotspin Baskets per manufacturer's protocol (Qiagen, Valencia, CA). Quantification was conducted by spectrophotometry (260 nm) and all samples were normalized to 10 ng/μl. For PCR reactions, 2 μl extracted genomic DNA (40 μl) was used [8]. In this study, SNapShot assay was used to genotype DNA samples for MTHFD1 401A NG, 2305C NT and 1958G NA according to the manufacturer's instructions (Applied Biosystems). The primers to amplify different fragments containing each SNP as shown in Table 2 were designed using PSQ Assay Design Software Version 1.0.6. Briefly, the amplification program was performed using the 9800 Fast PCR System according to manufacturer's suggested protocol (all the assays were performed in a 20 μl reaction mix containing 0.5 μM each of forward and reverse primers and Applied Biosystems GeneAmp® Fast PCR Master Mix), at the start of 95 μC for 2 min, followed by 33 cycles of 94 μC for 20 s,
We processed the data with Microsoft Excel in the first place, and then all statistical analyses were performed using SPSS 20.0 software. To verify the data quality, we implement a chi-square goodness-of-fit test to find out whether the variants among subjects have any significant variation from the Hardy–Weinberg equilibrium (HWE). The associations were evaluated by the Mann–Whitney test, one-way analysis of variance (ANOVA) and chi-square test. Genotypes were analyzed using an additive model and dichotomized using a recessive model; both were in accordance with previous studies [15]. A meta-analysis of the association between the MTHFD1 1958G N A polymorphism and NTD risk was conducted using the pooled odds ratios (ORs) with their corresponding 95% confidence intervals (CIs) under five genetic models. A two-tailed P value less than 0.05 was considered to be statistically significant for all analyses.
Table 1 Primers of MTHFD1 gene polymorphisms for PCR amplification. SNP
Chr pos
Gen pos
Alias name
Primers for PCR amplification
Restriction enzyme
rs1950902
64415662
Exon 5
401ANG
AluI
rs17857382
64449470
Exon 20
2305CNT
rs2236225
64442127
Exon 19
1958GNA
F: 5′-CAATCCTCCCACCTCTGC-3′ R: 3′-GAATGGGTAGGTGTAGGA-5′ F: 5′-TTTATAGGACGGATACAGAGT-3′ R: 3′-TGTTCGTGGGTCGTCGAA-5′ F: 5′-TAATCACGAGGATAAGAGCA-3′ R: 3′-CTTTGGTCCTGTGGCACT-5′
SNP, single-nucleotide polymorphism; F, forward; and R, reverse.
HinfI EcoRII
190
J. Meng et al. / Journal of the Neurological Sciences 348 (2015) 188–194
Fig. 1. Linkage disequilibrium of the five tag-SNPs.
in the use of folic acid supplements, the daily dietary intake of folates and the overall intake of folates variable across the two groups were more subtle, show no noteworthy difference (P N 0.05) respectively (more clinical characteristic data were summarized in Table 2).
3. Result 3.1. Study characteristics In this study, we obtained blood samples from 122 subjects diagnosed with NTD (consisted of 60 males and 62 females) and 100 normal controls (consisted of 53 males and 47 females) enrolled from healthy neonates in the department of gynecology and obstetrics of Jining No. 1 People's Hospital, respectively. As showed in Table 2, the mean age of the maternal relatives ranged from 20 to 30; our data showed that 92 mothers in the NTD group and 68 mothers in normal controls were within this age group. Other specimens not belonging to this age group were separated into two groups: one was mothers below 20years-old (22 of the NTD patients and 23 of the normal controls), and the other one was mothers above 30-years-old (8 of the NTD patients and 9 of the normal controls). Despite the differences among age and sex, there is no significant difference observed among all the specimens (P N 0.05).We also considered other possible influential factors such as gestational weeks, use of folic acid supplements and folate intake. The gestational weeks were shorter (less than 20 weeks) in the NTD infants' mothers (62.3%) than the normal infants' mothers (59.0%); however, the P value of 0.725 was not statistically significant. Also, the differences
3.2. SNP selection In our study, we retrieved twelve polymorphisms on MTHFD1 from unrelated Chinese in Jining individuals in public genotype databases (HapMap). The pairwise option of the Haploview 4.2 software was used to identify the tag-SNPs and an r2 of 0.8 was selected as a threshold for the analyses [14]. Finally, we selected three of them to represent all SNPs on MTHFD1, as they were supposed to result in translational modification and therefore may alter enzyme function [9]. They were 401A NG, 2305C NT and 1958G NA; the linkage disequilibrium plot (generated by the Haploview 4.2 software) among all the twelve SNPs on the MTHFD1 gene was shown in Fig. 1. The relative locations of the three suitable tag-SNPs on the MTHFD1 gene were presented in Fig. 2. The positions of the three tag-SNPs were on exon 5, exon 19 and exon 20 regions, separately. Primer design and PCR application were shown in Table 1. rs17857382
Ex1 Ex2
Ex3Ex4 Ex5
Ex6 Ex7
Ex8 Ex9 Ex10 Ex11Ex13
Ex12
5’UTR
Ex14 Ex16 Ex18
Ex15 Ex17
rs1950902 Fig. 2. Genetic location of the selected five tag-SNPs.
Ex19
Ex20
rs2236225
Ex21
3‘UTR
J. Meng et al. / Journal of the Neurological Sciences 348 (2015) 188–194 Table 3 Associations between three common polymorphisms of MTHFD1 gene and the risk of neural tube defects. Genotype
NTD patients (N = 122)
Healthy controls (N = 100)
OR (95% CI)
rs1950902 (GNA) GG 39 GA 60 AA 23 GA+AA 83 GG+GA 99 AA 23 G allele 138 A allele 106
32 48 20 68 80 20 112 88
Ref. 1.03 (0.56–1.87) 0.94 (0.44–2.02) 1.00 (0.57–1.77) Ref. 0.93 (0.48–1.81) Ref. 0.98 (0.67–1.43)
rs17857382 (CNT) CC 71 CT 40 TT 11 CT+TT 51 CC+CT 111 TT 11 C allele 182 T allele 62
53 41 6 47 94 6 147 53
Ref. 0.73 (0.41–1.28) 1.37 (0.48–3.94) 0.81 (0.48–1.38) Ref. 1.55 (0.55–4.36) Ref. 0.94 (0.62–1.45)
rs2236225 (GNA) GG 31 GA 58 AA 33 GA+AA 91 GG+GA 89 AA 33 G allele 120 A allele 124
37 48 15 63 85 15 122 78
Ref. 1.44 (0.78–2.66) 2.63 (2.61–5.70) 1.72 (0.97–3.07) Ref. 2.10 (1.07–4.14) Ref. 1.62 (1.11–2.36)
χ2
P value
0.007 0.022 b0.001
0.934 0.881 0.996
0.046
0.830
0.014
0.906
1.224 0.341 0.602
0.269 0.560 0.438
0.707
0.400
0.068
0.794
1.382 6.103 3.474
0.240 0.014 0.062
4.708
0.030
6.192
0.013
NTD, neural tube defect; OR, odds ratio; CI, confidence interval. Bold entries indicate P value less than 0.05.
3.3. Associations between MTHFD1 polymorphisms and the risk of NTD The risks of the three SNPs on MTHFD1 were examined from each specimen with OR (95% CI) and P value (the detailed associations among three common polymorphisms of MTHFD1 gene and the risk of neural tube defects were shown in Table 3). As a result, the SNPs of r2305C N T and 401A NG have no statistically significant association
191
with NTD, their ORs were relatively close to 1.0 and no significant valuable data was provided through our study between the two groups, with all their P values above 0.05, respectively. The two polymorphisms were unable to differentiate NTD from the normal controls. However, we found that 1958GN A in the exon 19 region of MTHFD1 was significantly associated with NTD risk. Compared with the homozygote GG genotype subjects, the AA genotype showed a significant 2.63-fold increased risk of NTD (OR = 2.63, 95% CI = 2.61–5.70, P = 0.014). A significant positive correlation was also noticed in the recessive model (AA vs. GG+GA, OR = 2.10, 95% CI = 1.07–4.14, P = 0.030). Similarly, in allele frequency distributions, a significant difference between the two groups was found as well (OR = 1.62, 95% CI = 1.11–2.36, P = 0.013). By contrast, no significant difference was observed regarding GA frequencies in either dominant model or heterozygote model (P = 0.062, P = 0.240, respectively). All these data of result were illustrated in Table 3.
3.4. Meta-analysis of the association between 1958GN A and NTD risk In our meta-analysis, we extracted data from 9 relevant articles [1,8–10,15–19]; they were all selected through a pre-specified search strategy. Quality assessment for the overall studies was shown with a bar graph according to the Newcastle–Ottawa Scale tool in Fig. 3. These articles provided data of 7749 Caucasians in total (3411 were NTD cases and 4338 were healthy controls) for our analysis. The overall details of the 9 included studies are revealed in Table 4. We obtain 10 eligible studies in our meta-analysis (our study was also included) for 1958G NA polymorphism on MTHFD1. The fixed effect models were used in overall analysis when the P-value of chi-squared test is over 0.05 and the I-square is below 50%, which means that the heterogeneity was not significant. On the contrary, the random effect models were performed when we detected significant between-study heterogeneity. Through all the genetic model analysis, we found that the 1958G N A polymorphism positive correlation with NTD susceptibility was only significant in Asian population analysis, while the overall results were not significant. We also observed that some of the associations in other studies were significant based on the Caucasian population, merely the association was not robust enough to affect the overall result.
Study controls for maternal conditions Study controls for demographic character Selection of controls Same method of ascertainment for cases Representativeness of the cases Non-response rate Is the case definition adequate Definition of controls Ascertainment of exposure 0
20
40
60 percent No
Fig. 3. Overall quality assessment of included studies.
Yes
80
100
192
J. Meng et al. / Journal of the Neurological Sciences 348 (2015) 188–194
Table 4 Main characteristics of studies in this meta-analysis. First author
Fisk Green R Marini NJ Shaw GM Carroll N van der Linden IJ Parle-McDermott A De Marco P Brody LC Hol FA Our study
Year
2013 2011 2009 2009 2007 2006 2006 2002 1998 2014
Country
Racial descent
Ireland USA USA Ireland Netherlands Ireland Italy Ireland Netherlands China
Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Asian
Sample size Case
Control
13 241 359 500 216 548 375 1056 103 122
309 239 259 446 460 770 523 997 335 100
Detection method
PCR–RFLP TaqMan PCR–RFLP PCR–RFLP PCR–RFLP PCR–RFLP PCR–RFLP PCR–RFLP PCR–SSCP PCR–SSCP
NTD cases
Healthy controls
GG
GA
AA
MAF
GG
GA
AA
MAF
2 50 113 144 31 50 25 101 32 31
8 86 179 254 58 84 74 178 55 58
3 36 67 102 14 42 43 57 16 33
0.538 0.459 0.436 0.458 0.417 0.477 0.563 0.435 0.422 0.508
113 49 95 123 71 209 143 283 100 37
148 69 135 244 98 411 251 526 172 48
48 20 55 79 34 150 129 188 63 15
0.395 0.395 0.430 0.451 0.409 0.462 0.487 0.452 0.445 0.390
MAF, minor allele frequency.
4. Discussion The aim of our study was to investigate the associations between MTHFD1 polymorphisms and NTD susceptibility in a Chinese population. Hence, we focused on the associations between NTD and the three folate metabolism related SNPs on MTHFD1: 401AN G, 2305CNT and 1958G N A. The level of NTD risk was evaluated in the current case–control study. As a result, our evidence verified that the genetic variation of MTHFD1 1958G NA was associated with an increased risk of NTD. Furthermore, the subsequent analysis implied that parents with the MTHFD1 1958G NA A allele (especially AA genotype) were more likely to have a NTD neonate. The other polymorphisms in our assays failed to show a significant association on the NTD susceptibility. NTD is attributed to a multifactorial etiology, including genetic and environmental factors [17,20,21]. Thus, the embryonic effects and maternal effects should be considered, as well as the potential interactions of them. However, the exact mechanism of NTD has not been completely elucidated. Thus, it is hypothesized that polymorphisms within folate-dependent enzymes might have an impact on NTD risk [22]. In
some recent researches, up-regulation of MTHFD1 was reported to be involved in the folate metabolic pathway and was also a significant prognostic marker and it was also found to associate with an increased risk of NTD [10,18,19]. MTHFD1 is a nicotinamide adenine dinucleotide phosphate (NADP)dependent trifunctional cytoplasmic enzyme (often referred to as “C1THF synthase”). Furthermore, MTHFD1 SNP 1958GN A is a functional exonic SNP that has been studied in several populations, and the previous studies that indicate the genetic variants of MTHFD1 can result in some kind of diseases [3,23–30]. In Carroll N's analysis, he revealed that MTHFD1 SNP 1958G N A (R653Q) has a highly significant association with NTD risk in both case (genotype and allele frequencies) and maternal (allele frequencies only) groups [16]. And in the study of Ridgely F.G., the cause of NTD was considered to be consistent with both genetic and epigenetic mechanisms contributing to the increased risk of NTD and overall birth defect risk in the maternal lineage which had a higher number of genotypes associated with lower folate metabolism than paternal relatives [31]. Likewise in Parle-McDermott A's study, he verified that some SNPs on MTHFD1 may be subjected to selection pressure in
Fig. 4. Potential mechanisms of MTHFD1 polymorphisms in the etiopathogenesis of NTD.
J. Meng et al. / Journal of the Neurological Sciences 348 (2015) 188–194
the past while the other known variants in this block are within intronic regions that are unlikely to contain regulatory sequences and are not obvious candidates for a disease association [10]. Based on all the previous studies and our analysis, we focused on three SNP locations on MTHFD1. We found that the polymorphisms in MTHFD1 gene may influence cellular folate metabolism by lowering folate status or shifts in cellular folate distribution. In these processes, the main role for MTHFD1 provides 10-formyl THF and 5,10-methylene THF for purine and pyrimidine synthesis. There are two ways for 10-formyl THF synthesis, synthesize directly from formate and THF through the synthetase activity and synthesize from 5,10-methenyl THF through the cyclohydrolase activity, which is channeled from 5,10-methylene THF via the dehydrogenase activity. MTHFD1 plays an indirect role in homocysteine metabolism by providing some of the 5,10-methylene THF pool, but the major source of this is still SHMT. Besides, the enzymes MTHFR and MS are directly involved in homocysteine metabolism. Thus, the polymorphisms in MTHFD1 were hypothesized to influence the risk of NTD through two potential mechanisms as showed in Fig. 4. One is to reduce the availability of folates for the TS reaction and DNA synthesis by debasing the availability of folates or redistributing folates toward homocysteine remethylation, which also results in uracil misincorporation into DNA, possibly leading to double-strand breaks and chromosomal damage. The other one is to reduce the availability of folate for the methionine cycle resulting in reduced transmethylation capacity and DNA hypomethylation/demethylation. DNA hypomethylation and uracil misincorporation are considered as two important factors in NTD. Through our meta-analysis, we verified that the association between MTHFD1 1958GN A polymorphism and NTD risk was significant only in Asians, while the result showed no significance in the Caucasian population. Nevertheless, the reason was still controversial, some researches showed that it relied on a combination of differences in polymorphism distributions with non-genetic factors, as well as the interactions with environmental and social factors, which also depends on the variability of races, because genetic factors may vary among populations. Besides, the numbers of studies and sample size in Asian research were not ample to evaluate the association, while we also observed some significant association in the Caucasian population, but the overall results remain insignificant. Hence, to further substantiate the effect of race difference in this SNP on NTD susceptibility, larger scale studies and unified selection criteria are needed. Similarly, our study was limited in its effect estimation owing to small sample sizes for some comparisons. Moreover, the results in our study were just based on the Chinese which may not be applicable to other ethnicities. Besides, except the influence of folate acid intake and gestational weeks, other pathological characteristics of NTD, environmental factors as well as the interaction with individual genetic background which are closely related to the pathogenesis of NTD were not assessed in this study, with regard to some limitations of our assays. Further studies of the risk effects and the functional impact of this polymorphism in further validation are needed, and new research on the association between NTD and other SNPs on MTHFD1 is also recommended. 5. Conclusion In summary, we get the conclusion that SNP MTHFD1 1958GN A is significantly associated with an increasing risk of NTD susceptibility in the Chinese. Furthermore, neonates who harbor the AA homozygote of the MTHFD1 1958GN A are more likely to suffer NTD, compared to the others with GA or GG. Through our meta-analysis, the AA genotype also had a high correlation with NTD in the Asian population. This implies that the examination for AA genotype of this SNP beforehand may prevent the birth of NTD neonates who do not survive it. More detailed investigations and further large-scale studies on genetic functions to provide more conclusive and accurate evidence are required in the future.
193
Conflict of interest None.
References [1] Marini NJ, Hoffmann TJ, Lammer EJ, Hardin J, Lazaruk K, Stein JB, et al. A genetic signature of spina bifida risk from pathway-informed comprehensive gene-variant analysis. PLoS One 2011;6:e28408. [2] Ouyang S, Liu Z, Li Y, Ma F, Wu J. Cystathionine beta-synthase 844ins68 polymorphism is unrelated to susceptibility to neural tube defects. Gene 2014;535:119–23. [3] Sutherland HG, Hermile H, Sanche R, Menon S, Lea RA, Haupt LM, et al. Association study of MTHFD1 coding polymorphisms R134K and R653Q with migraine susceptibility. Headache 2014;54:1506–14. [4] Bi XH, Zhao HL, Zhang ZX, Liu Q, Zhang JW. Association analysis of CbetaS 844ins68 and MTHFD1 G1958A polymorphisms with Alzheimer's disease in Chinese. J Neural Transm (Vienna, Austria: 1996) 2010;117:499–503. [5] Christensen KE, Rohlicek CV, Andelfinger GU, Michaud J, Bigras JL, Richter A, et al. The MTHFD1 p.Arg653Gln variant alters enzyme function and increases risk for congenital heart defects. Hum Mutat 2009;30:212–20. [6] Crisan TO, Trifa A, Farcas M, Militaru M, Netea M, Pop I, et al. The MTHFD1 c.1958 GNA polymorphism and recurrent spontaneous abortions. J Matern Fetal Neonatal Med 2011;24:189–92. [7] Field MS, Kamynina E, Agunloye OC, Liebenthal RP, Lamarre SG, Brosnan ME, et al. Nuclear enrichment of folate cofactors and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) protect de novo thymidylate biosynthesis during folate deficiency. J Biol Chem 2014;289:29642–50. [8] Fisk Green R, Byrne J, Crider KS, Gallagher M, Koontz D, Berry RJ. Folate-related gene variants in Irish families affected by neural tube defects. Front Genet 2013;4:223. [9] Brody LC, Conley M, Cox C, Kirke PN, McKeever MP, Mills JL, et al. A polymorphism, R653Q, in the trifunctional enzyme methylenetetrahydrofolate dehydrogenase/ methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthetase is a maternal genetic risk factor for neural tube defects: report of the Birth Defects Research Group. Am J Hum Genet 2002;71:1207–15. [10] Parle-McDermott A, Kirke PN, Mills JL, Molloy AM, Cox C, O'Leary VB, et al. Confirmation of the R653Q polymorphism of the trifunctional C1-synthase enzyme as a maternal risk for neural tube defects in the Irish population. Eur J Hum Genet 2006;14:768–72. [11] Smithells RW, Sheppard S, Schorah CJ. Vitamin deficiencies and neural tube defects. Arch Dis Child 1976;51:944–50. [12] Etheredge AJ, Finnell RH, Carmichael SL, Lammer EJ, Zhu H, Mitchell LE, et al. Maternal and infant gene–folate interactions and the risk of neural tube defects. Am J Med Genet A 2012;158A:2439–46. [13] Hum DW, Bell AW, Rozen R, MacKenzie RE. Primary structure of a human trifunctional enzyme. Isolation of a cDNA encoding methylenetetrahydrofolate dehydrogenase–methenyltetrahydrofolate cyclohydrolase–formyltetrahydrofolate synthetase. J Biol Chem 1988;263:15946–50. [14] Cao Q, Wang J, Zhang M, Li P, Qian J, Zhang S, et al. Genetic variants in RKIP are associated with clear cell renal cell carcinoma risk in a Chinese population. PLoS One 2014;9:e109285. [15] De Marco P, Merello E, Calevo MG, Mascelli S, Raso A, Cama A, et al. Evaluation of a methylenetetrahydrofolate-dehydrogenase 1958GNA polymorphism for neural tube defect risk. J Hum Genet 2006;51:98–103. [16] Carroll N, Pangilinan F, Molloy AM, Troendle J, Mills JL, Kirke PN, et al. Analysis of the MTHFD1 promoter and risk of neural tube defects. Hum Genet 2009;125: 247–56. [17] Hol FA, van der Put NM, Geurds MP, Heil SG, Trijbels FJ, Hamel BC, et al. Molecular genetic analysis of the gene encoding the trifunctional enzyme MTHFD (methylenetetrahydrofolate-dehydrogenase, methenyltetrahydrofolatecyclohydrolase, formyltetrahydrofolate synthetase) in patients with neural tube defects. Clin Genet 1998;53:119–25. [18] Shaw GM, Lu W, Zhu H, Yang W, Briggs FB, Carmichael SL, et al. 118 SNPs of folaterelated genes and risks of spina bifida and conotruncal heart defects. BMC Med Genet 2009;10:49. [19] van der Linden IJ, Heil SG, Kouwenberg IC, den Heijer M, Blom HJ. The methylenetetrahydrofolate dehydrogenase (MTHFD1) 1958GNA variant is not associated with spina bifida risk in the Dutch population. Clin Genet 2007;72:599–600. [20] Greene ND, Stanier P, Copp AJ. Genetics of human neural tube defects. Hum Mol Genet 2009;18:R113–29. [21] Shields DC, Kirke PN, Mills JL, Ramsbottom D, Molloy AM, Burke H, et al. The “thermolabile” variant of methylenetetrahydrofolate reductase and neural tube defects: an evaluation of genetic risk and the relative importance of the genotypes of the embryo and the mother. Am J Hum Genet 1999;64:1045–55. [22] Pangilinan F, Molloy AM, Mills JL, Troendle JF, Parle-McDermott A, Signore C, et al. Evaluation of common genetic variants in 82 candidate genes as risk factors for neural tube defects. BMC Med Genet 2012;13:62. [23] Christensen KE, Dahhou M, Kramer MS, Rozen R. The MTHFD1 1958GNA variant is associated with elevated C-reactive protein and body mass index in Canadian women from a premature birth cohort. Mol Genet Metab 2014;111:390–2. [24] Cui Y, Jing Y, Sun Z. Lack of association between MTHFD1 G401A polymorphism and ovarian cancer susceptibility. Tumour Biol 2014;35:3385–9. [25] da Silva LM, Galbiatti AL, Ruiz MT, Raposo LS, Maniglia JV, Pavarino EC, et al. MTHFD1 G1958A, BHMT G742A, TC2 C776G and TC2 A67G polymorphisms and head and neck squamous cell carcinoma risk. Mol Biol Rep 2012;39:887–93.
194
J. Meng et al. / Journal of the Neurological Sciences 348 (2015) 188–194
[26] Garcia-Gonzalez I, Solis-Cardenas AD, Flores-Ocampo JA, Alejos-Mex R, HerreraSanchez LF, Gonzalez-Herrera LJ. G894T (NOS3) and G1958A (MTHFD1) gene polymorphisms and risk of ischemic heart disease in Yucatan, Mexico. Clin Investig Arterioscler 2014 [Epub ahead of print]. [27] Jiang J, Zhang Y, Wei L, Sun Z, Liu Z. Association between MTHFD1 G1958A polymorphism and neural tube defects susceptibility: a meta-analysis. PLoS One 2014;9: e101169. [28] Keller MD, Ganesh J, Heltzer M, Paessler M, Bergqvist AG, Baluarte HJ, et al. Severe combined immunodeficiency resulting from mutations in MTHFD1. Pediatrics 2013;131:e629–34.
[29] Lorenc A, Seiemak-Mrozikiewicz A, Barlik M, Wolski H, Drews K. The role of 401aNG polymorphism of methylenetetrahydrofolate dehydrogenase gene (MTHFD1) in fetal hypotrophy. Ginekol Pol 2014;85:494–9. [30] Murthy J, Gurramkonda VB, Lakkakula BV. Significant association of MTHFD1 1958GbA single nucleotide polymorphism with nonsyndromic cleft lip and palate in Indian population. Med Oral Patol Oral Cir Bucal 2014;19:e616–21. [31] Bacon-Baguley T, Kudryk BJ, Walz DA. Thrombospondin interaction with fibrinogen. Evidence for binding to the A alpha- and B beta-chains of fibrinogen. J Biol Chem 1987;262:1927–30.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Association between MTHFD1 polymorphisms and neural tube defect susceptibility Jingjing Meng a, Lei Han b, Bo Zhuang c,⁎ a b c
Neonatal Ward, Jining No. 1 People's Hospital, Jining 272011, China Department of PICU, The Affiliated Hospital of Jining Medical College, Jining 272000, China Department of Pediatric Surgery, Jining No. 1 People's Hospital, Jining 272011, China
a r t i c l e
i n f o
Article history: Received 6 March 2014 Received in revised form 20 November 2014 Accepted 1 December 2014 Available online 6 December 2014 Keywords: Neural tube defects Methylenetetrahydrofolate dehydrogenase (MTHFD1) 1958GNA Polymorphism Folate metabolism Association
a b s t r a c t Objectives: Neural tube defect (NTD) is a common disease among neonates with multiplex symptom and complex origins, and the exact mechanism of NTD has not been definitely elucidated. Nevertheless, it is hypothesized that NTD risk can be prevented by periconceptional folic acid in folate metabolism. The methylenetetrahydrofolate dehydrogenase (MTHFD1) gene has been proved to play an important role in folate metabolism, which was strongly associated with the high risk for NTD. We focused on three folate metabolism-related single-nucleotide polymorphisms (SNPs) on the MTHFD1 gene to evaluate the associations between MTHFD1 polymorphisms and NTD susceptibility. Methods: We genotyped blood samples from 222 specimens (including 122 NTD-affected infants and 100 healthy controls) in a case–control study. We investigated the association between NTD and three selected tag-SNPs on MTHFD1 gene: 401A NG (rs1950902), 2305C N T (rs17857382) and 1958G NA (rs2236225) by the SNapShot method. These SNPs were identified by Haploview 4.2 software with HapMap databases, and then these associations were evaluated by the Mann–Whitney test, one-way analysis of variance (ANOVA) and chi-square test. Furthermore, a meta-analysis of the association between MTHFD1 1958GN A and NTD risk was also performed. Results: In our study, an increased risk of NTD was observed for 1958GN A of MTHFD1 (AA vs. GG: OR = 2.63, 95% CI = 2.61–5.70; AA vs. GG + GA: OR = 2.10, 95% CI = 1.07–4.14; A vs. G: OR = 1.62, 95% CI = 1.11–2.36). However, the other two SNPs (401A NG and 2305CN T) displayed no statistically significant association with NTD risk. The overall result of the meta-analysis indicated that the 1958G NA variant might not be a genetic susceptible factor for the Caucasian population. Conclusions: Our analysis implicated that MTHFD1 1958GNA was significantly associated with the susceptibility of NTD in a Chinese population. In addition, the AA homozygote carriers were more likely to suffer NTD, compared with the others with GA or GG genotypes. Validation of the risk effect and functional impact of this polymorphism is needed in future investigations. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Neural tube defect (NTD) is among the most common and deadly human congenital malformations at birth and manifested as a wide range of phenotypes, primarily including anencephaly, spina bifida and encephalocele which result from failure of the neural tube to close properly in the developing brain or lower spine [1,2]. The incidence rate of NTD is about 0.2% worldwide, but this rate varies in different races and sections with a conspicuous diversity which may be a result of genetic diversity [3]. Some previous studies demonstrated that the etiology of NTD is implicated with several factors, like gene–environment
⁎ Corresponding author at: Department of Pediatric Surgery, Jining No. 1 People's Hospital, No. 6 Jiankang Road, Shandong Province 272011, China. E-mail address: [email protected] (B. Zhuang).
http://dx.doi.org/10.1016/j.jns.2014.12.001 0022-510X/© 2014 Elsevier B.V. All rights reserved.
interactions and complex genetic factors [4–7]. These unclear factors would lie in the increased recurrence risk in siblings of individuals with NTD, the increased risk for NTD among other relatives, and the increased risk for birth defects overall among both close and distant relatives [8]. Recent studies also revealed that the NTD incidence and overall birth defect risk are higher in maternal relatives than in paternal relatives [9,10]. Though the exact mechanism of NTD has not been completely elucidated, it is hypothesized that genes coding folatedependent enzymes might have an impact on NTD risk. Epidemiological studies have revealed that periconceptional vitamin supplementation with folic acid substantially lowers the percentage of women with NTD-affected pregnancies [11]. Thus, elucidation of the mechanisms by which folate reduces the risk of NTD, currently focused on candidate genes that are involved in folate-related pathways could be informative for developing improved prevention strategies and reducing the global burden of NTD [12].
J. Meng et al. / Journal of the Neurological Sciences 348 (2015) 188–194
Methylenetetrahydrofolate dehydrogenase (MTHFD1) is a nicotinamide adenine dinucleotide phosphate (NADP)-dependent trifunctional cytoplasmic enzyme (often referred to as “C1-THF synthase”), acting as 10-formyl, 5,10-methenyl, and 5,10-methylene derivatives [13]. This folate-dependent enzyme has been proved to play an important role in folate metabolism, which is an attractive candidate gene associated with the high risk of NTD, but these associations remain controversial since several studies suggest no associations. Hence, in our study, some potential SNPs in the MTHFD1 gene were identified from public databases, and through analysis we finally choose 401AN G, 2305CN T and 1958GNA as representative SNPs to investigate their associations with NTD risk. Considering the limitation of an individual study with a small sample size, a further meta-analysis with public data for the purpose of getting an exhaustive conclusion of empirical evidences to prove the association between MTHFD1 1958GN A and NTD risk was also performed. Elucidation of the interaction might contribute to ameliorate the diagnosis and prognosis of NTD. 2. Material and methods 2.1. Study population Our study was conducted in strict accordance with the protocol approved by the Jining No. 1 People's Hospital, and a written informed consent must be provided by each participant (or their guardian). We recruited 222 specimens in total from Jining No. 1 People's Hospital. Among them, 122 subjects were confirmed as NTD patients; the other 100 subjects were healthy infants used as normal controls in this study. The neonates of NTD cases within our samples consisted of 62 females and 60 males, while the control group consisted of 47 females and 53 males as outlined in Table 2. We collected 5 ml venous blood sample from each participant after their guardians provided a written informed consent. All the samples and complete follow-up data were available for each specimen (more clinical characteristics are presented in Table 2).
189
Table 2 Comparison of neural tube defect patients and controls by selective characteristics. Clinical characteristics Sex Male Female Maternal age ≤20 20–30 ≥30 Gestational weeks ≤20 20–25 ≥25 Use of folic acid supplements Yes No Daily dietary intake of folates Q1 Q2–Q4 Overall intake of folates Lowa Highb
NTD patients (N = 122)
Normal controls (N = 100)
P-value
60 (49.2%) 62 (50.8%)
53 (53.0%) 47 (47.0%)
0.571
22 (18.0%) 92 (75.4%) 8 (6.6%)
23 (23.0%) 68 (68.0%) 9 (9.0%)
0.469
76 (62.3%) 31 (25.4%) 15 (12.3%)
59 (59.0%) 25 (25.0%) 16 (16.0%)
0.725
66 (54.1%) 56 (45.9%)
61 (61.0%) 39 (39.0%)
0.301
30 (24.6%) 92 (75.4%)
26 (26.0%) 74 (74.0%)
0.810
16 (13.1%) 106 (86.9%)
12 (12.0%) 88 (88.0%)
0.803
NTD, neural tube defect. a Women in the lowest quartile of maternal folate intake (Q1 ≤ 309.49 μg/100 g/day) who did not take supplemental folic acid in the periconceptional period. b All other combinations of folate intake (Q2, Q3 or Q4) and folic acid supplementation (yes/no).
56 μC for 30 s and then 72 μC for 40 s [14]. After amplification, the pyrosequencing primer was used to extend the PCR product by adding nucleotides in a specific sequential order based on the known sequence [8] as described previously. Appropriate controls were included in all assays and at least 10% of samples were repeatedly genotyped with N 99% agreement. We use the signal peaks' resulting pyrogram to determine the genotype. The primers and conditions of all assays are available upon request.
2.2. DNA methods 2.3. Statistical analysis The whole genomic DNA was extracted from blood samples of each specimen using Clotspin Baskets per manufacturer's protocol (Qiagen, Valencia, CA). Quantification was conducted by spectrophotometry (260 nm) and all samples were normalized to 10 ng/μl. For PCR reactions, 2 μl extracted genomic DNA (40 μl) was used [8]. In this study, SNapShot assay was used to genotype DNA samples for MTHFD1 401A NG, 2305C NT and 1958G NA according to the manufacturer's instructions (Applied Biosystems). The primers to amplify different fragments containing each SNP as shown in Table 2 were designed using PSQ Assay Design Software Version 1.0.6. Briefly, the amplification program was performed using the 9800 Fast PCR System according to manufacturer's suggested protocol (all the assays were performed in a 20 μl reaction mix containing 0.5 μM each of forward and reverse primers and Applied Biosystems GeneAmp® Fast PCR Master Mix), at the start of 95 μC for 2 min, followed by 33 cycles of 94 μC for 20 s,
We processed the data with Microsoft Excel in the first place, and then all statistical analyses were performed using SPSS 20.0 software. To verify the data quality, we implement a chi-square goodness-of-fit test to find out whether the variants among subjects have any significant variation from the Hardy–Weinberg equilibrium (HWE). The associations were evaluated by the Mann–Whitney test, one-way analysis of variance (ANOVA) and chi-square test. Genotypes were analyzed using an additive model and dichotomized using a recessive model; both were in accordance with previous studies [15]. A meta-analysis of the association between the MTHFD1 1958G N A polymorphism and NTD risk was conducted using the pooled odds ratios (ORs) with their corresponding 95% confidence intervals (CIs) under five genetic models. A two-tailed P value less than 0.05 was considered to be statistically significant for all analyses.
Table 1 Primers of MTHFD1 gene polymorphisms for PCR amplification. SNP
Chr pos
Gen pos
Alias name
Primers for PCR amplification
Restriction enzyme
rs1950902
64415662
Exon 5
401ANG
AluI
rs17857382
64449470
Exon 20
2305CNT
rs2236225
64442127
Exon 19
1958GNA
F: 5′-CAATCCTCCCACCTCTGC-3′ R: 3′-GAATGGGTAGGTGTAGGA-5′ F: 5′-TTTATAGGACGGATACAGAGT-3′ R: 3′-TGTTCGTGGGTCGTCGAA-5′ F: 5′-TAATCACGAGGATAAGAGCA-3′ R: 3′-CTTTGGTCCTGTGGCACT-5′
SNP, single-nucleotide polymorphism; F, forward; and R, reverse.
HinfI EcoRII
190
J. Meng et al. / Journal of the Neurological Sciences 348 (2015) 188–194
Fig. 1. Linkage disequilibrium of the five tag-SNPs.
in the use of folic acid supplements, the daily dietary intake of folates and the overall intake of folates variable across the two groups were more subtle, show no noteworthy difference (P N 0.05) respectively (more clinical characteristic data were summarized in Table 2).
3. Result 3.1. Study characteristics In this study, we obtained blood samples from 122 subjects diagnosed with NTD (consisted of 60 males and 62 females) and 100 normal controls (consisted of 53 males and 47 females) enrolled from healthy neonates in the department of gynecology and obstetrics of Jining No. 1 People's Hospital, respectively. As showed in Table 2, the mean age of the maternal relatives ranged from 20 to 30; our data showed that 92 mothers in the NTD group and 68 mothers in normal controls were within this age group. Other specimens not belonging to this age group were separated into two groups: one was mothers below 20years-old (22 of the NTD patients and 23 of the normal controls), and the other one was mothers above 30-years-old (8 of the NTD patients and 9 of the normal controls). Despite the differences among age and sex, there is no significant difference observed among all the specimens (P N 0.05).We also considered other possible influential factors such as gestational weeks, use of folic acid supplements and folate intake. The gestational weeks were shorter (less than 20 weeks) in the NTD infants' mothers (62.3%) than the normal infants' mothers (59.0%); however, the P value of 0.725 was not statistically significant. Also, the differences
3.2. SNP selection In our study, we retrieved twelve polymorphisms on MTHFD1 from unrelated Chinese in Jining individuals in public genotype databases (HapMap). The pairwise option of the Haploview 4.2 software was used to identify the tag-SNPs and an r2 of 0.8 was selected as a threshold for the analyses [14]. Finally, we selected three of them to represent all SNPs on MTHFD1, as they were supposed to result in translational modification and therefore may alter enzyme function [9]. They were 401A NG, 2305C NT and 1958G NA; the linkage disequilibrium plot (generated by the Haploview 4.2 software) among all the twelve SNPs on the MTHFD1 gene was shown in Fig. 1. The relative locations of the three suitable tag-SNPs on the MTHFD1 gene were presented in Fig. 2. The positions of the three tag-SNPs were on exon 5, exon 19 and exon 20 regions, separately. Primer design and PCR application were shown in Table 1. rs17857382
Ex1 Ex2
Ex3Ex4 Ex5
Ex6 Ex7
Ex8 Ex9 Ex10 Ex11Ex13
Ex12
5’UTR
Ex14 Ex16 Ex18
Ex15 Ex17
rs1950902 Fig. 2. Genetic location of the selected five tag-SNPs.
Ex19
Ex20
rs2236225
Ex21
3‘UTR
J. Meng et al. / Journal of the Neurological Sciences 348 (2015) 188–194 Table 3 Associations between three common polymorphisms of MTHFD1 gene and the risk of neural tube defects. Genotype
NTD patients (N = 122)
Healthy controls (N = 100)
OR (95% CI)
rs1950902 (GNA) GG 39 GA 60 AA 23 GA+AA 83 GG+GA 99 AA 23 G allele 138 A allele 106
32 48 20 68 80 20 112 88
Ref. 1.03 (0.56–1.87) 0.94 (0.44–2.02) 1.00 (0.57–1.77) Ref. 0.93 (0.48–1.81) Ref. 0.98 (0.67–1.43)
rs17857382 (CNT) CC 71 CT 40 TT 11 CT+TT 51 CC+CT 111 TT 11 C allele 182 T allele 62
53 41 6 47 94 6 147 53
Ref. 0.73 (0.41–1.28) 1.37 (0.48–3.94) 0.81 (0.48–1.38) Ref. 1.55 (0.55–4.36) Ref. 0.94 (0.62–1.45)
rs2236225 (GNA) GG 31 GA 58 AA 33 GA+AA 91 GG+GA 89 AA 33 G allele 120 A allele 124
37 48 15 63 85 15 122 78
Ref. 1.44 (0.78–2.66) 2.63 (2.61–5.70) 1.72 (0.97–3.07) Ref. 2.10 (1.07–4.14) Ref. 1.62 (1.11–2.36)
χ2
P value
0.007 0.022 b0.001
0.934 0.881 0.996
0.046
0.830
0.014
0.906
1.224 0.341 0.602
0.269 0.560 0.438
0.707
0.400
0.068
0.794
1.382 6.103 3.474
0.240 0.014 0.062
4.708
0.030
6.192
0.013
NTD, neural tube defect; OR, odds ratio; CI, confidence interval. Bold entries indicate P value less than 0.05.
3.3. Associations between MTHFD1 polymorphisms and the risk of NTD The risks of the three SNPs on MTHFD1 were examined from each specimen with OR (95% CI) and P value (the detailed associations among three common polymorphisms of MTHFD1 gene and the risk of neural tube defects were shown in Table 3). As a result, the SNPs of r2305C N T and 401A NG have no statistically significant association
191
with NTD, their ORs were relatively close to 1.0 and no significant valuable data was provided through our study between the two groups, with all their P values above 0.05, respectively. The two polymorphisms were unable to differentiate NTD from the normal controls. However, we found that 1958GN A in the exon 19 region of MTHFD1 was significantly associated with NTD risk. Compared with the homozygote GG genotype subjects, the AA genotype showed a significant 2.63-fold increased risk of NTD (OR = 2.63, 95% CI = 2.61–5.70, P = 0.014). A significant positive correlation was also noticed in the recessive model (AA vs. GG+GA, OR = 2.10, 95% CI = 1.07–4.14, P = 0.030). Similarly, in allele frequency distributions, a significant difference between the two groups was found as well (OR = 1.62, 95% CI = 1.11–2.36, P = 0.013). By contrast, no significant difference was observed regarding GA frequencies in either dominant model or heterozygote model (P = 0.062, P = 0.240, respectively). All these data of result were illustrated in Table 3.
3.4. Meta-analysis of the association between 1958GN A and NTD risk In our meta-analysis, we extracted data from 9 relevant articles [1,8–10,15–19]; they were all selected through a pre-specified search strategy. Quality assessment for the overall studies was shown with a bar graph according to the Newcastle–Ottawa Scale tool in Fig. 3. These articles provided data of 7749 Caucasians in total (3411 were NTD cases and 4338 were healthy controls) for our analysis. The overall details of the 9 included studies are revealed in Table 4. We obtain 10 eligible studies in our meta-analysis (our study was also included) for 1958G NA polymorphism on MTHFD1. The fixed effect models were used in overall analysis when the P-value of chi-squared test is over 0.05 and the I-square is below 50%, which means that the heterogeneity was not significant. On the contrary, the random effect models were performed when we detected significant between-study heterogeneity. Through all the genetic model analysis, we found that the 1958G N A polymorphism positive correlation with NTD susceptibility was only significant in Asian population analysis, while the overall results were not significant. We also observed that some of the associations in other studies were significant based on the Caucasian population, merely the association was not robust enough to affect the overall result.
Study controls for maternal conditions Study controls for demographic character Selection of controls Same method of ascertainment for cases Representativeness of the cases Non-response rate Is the case definition adequate Definition of controls Ascertainment of exposure 0
20
40
60 percent No
Fig. 3. Overall quality assessment of included studies.
Yes
80
100
192
J. Meng et al. / Journal of the Neurological Sciences 348 (2015) 188–194
Table 4 Main characteristics of studies in this meta-analysis. First author
Fisk Green R Marini NJ Shaw GM Carroll N van der Linden IJ Parle-McDermott A De Marco P Brody LC Hol FA Our study
Year
2013 2011 2009 2009 2007 2006 2006 2002 1998 2014
Country
Racial descent
Ireland USA USA Ireland Netherlands Ireland Italy Ireland Netherlands China
Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Asian
Sample size Case
Control
13 241 359 500 216 548 375 1056 103 122
309 239 259 446 460 770 523 997 335 100
Detection method
PCR–RFLP TaqMan PCR–RFLP PCR–RFLP PCR–RFLP PCR–RFLP PCR–RFLP PCR–RFLP PCR–SSCP PCR–SSCP
NTD cases
Healthy controls
GG
GA
AA
MAF
GG
GA
AA
MAF
2 50 113 144 31 50 25 101 32 31
8 86 179 254 58 84 74 178 55 58
3 36 67 102 14 42 43 57 16 33
0.538 0.459 0.436 0.458 0.417 0.477 0.563 0.435 0.422 0.508
113 49 95 123 71 209 143 283 100 37
148 69 135 244 98 411 251 526 172 48
48 20 55 79 34 150 129 188 63 15
0.395 0.395 0.430 0.451 0.409 0.462 0.487 0.452 0.445 0.390
MAF, minor allele frequency.
4. Discussion The aim of our study was to investigate the associations between MTHFD1 polymorphisms and NTD susceptibility in a Chinese population. Hence, we focused on the associations between NTD and the three folate metabolism related SNPs on MTHFD1: 401AN G, 2305CNT and 1958G N A. The level of NTD risk was evaluated in the current case–control study. As a result, our evidence verified that the genetic variation of MTHFD1 1958G NA was associated with an increased risk of NTD. Furthermore, the subsequent analysis implied that parents with the MTHFD1 1958G NA A allele (especially AA genotype) were more likely to have a NTD neonate. The other polymorphisms in our assays failed to show a significant association on the NTD susceptibility. NTD is attributed to a multifactorial etiology, including genetic and environmental factors [17,20,21]. Thus, the embryonic effects and maternal effects should be considered, as well as the potential interactions of them. However, the exact mechanism of NTD has not been completely elucidated. Thus, it is hypothesized that polymorphisms within folate-dependent enzymes might have an impact on NTD risk [22]. In
some recent researches, up-regulation of MTHFD1 was reported to be involved in the folate metabolic pathway and was also a significant prognostic marker and it was also found to associate with an increased risk of NTD [10,18,19]. MTHFD1 is a nicotinamide adenine dinucleotide phosphate (NADP)dependent trifunctional cytoplasmic enzyme (often referred to as “C1THF synthase”). Furthermore, MTHFD1 SNP 1958GN A is a functional exonic SNP that has been studied in several populations, and the previous studies that indicate the genetic variants of MTHFD1 can result in some kind of diseases [3,23–30]. In Carroll N's analysis, he revealed that MTHFD1 SNP 1958G N A (R653Q) has a highly significant association with NTD risk in both case (genotype and allele frequencies) and maternal (allele frequencies only) groups [16]. And in the study of Ridgely F.G., the cause of NTD was considered to be consistent with both genetic and epigenetic mechanisms contributing to the increased risk of NTD and overall birth defect risk in the maternal lineage which had a higher number of genotypes associated with lower folate metabolism than paternal relatives [31]. Likewise in Parle-McDermott A's study, he verified that some SNPs on MTHFD1 may be subjected to selection pressure in
Fig. 4. Potential mechanisms of MTHFD1 polymorphisms in the etiopathogenesis of NTD.
J. Meng et al. / Journal of the Neurological Sciences 348 (2015) 188–194
the past while the other known variants in this block are within intronic regions that are unlikely to contain regulatory sequences and are not obvious candidates for a disease association [10]. Based on all the previous studies and our analysis, we focused on three SNP locations on MTHFD1. We found that the polymorphisms in MTHFD1 gene may influence cellular folate metabolism by lowering folate status or shifts in cellular folate distribution. In these processes, the main role for MTHFD1 provides 10-formyl THF and 5,10-methylene THF for purine and pyrimidine synthesis. There are two ways for 10-formyl THF synthesis, synthesize directly from formate and THF through the synthetase activity and synthesize from 5,10-methenyl THF through the cyclohydrolase activity, which is channeled from 5,10-methylene THF via the dehydrogenase activity. MTHFD1 plays an indirect role in homocysteine metabolism by providing some of the 5,10-methylene THF pool, but the major source of this is still SHMT. Besides, the enzymes MTHFR and MS are directly involved in homocysteine metabolism. Thus, the polymorphisms in MTHFD1 were hypothesized to influence the risk of NTD through two potential mechanisms as showed in Fig. 4. One is to reduce the availability of folates for the TS reaction and DNA synthesis by debasing the availability of folates or redistributing folates toward homocysteine remethylation, which also results in uracil misincorporation into DNA, possibly leading to double-strand breaks and chromosomal damage. The other one is to reduce the availability of folate for the methionine cycle resulting in reduced transmethylation capacity and DNA hypomethylation/demethylation. DNA hypomethylation and uracil misincorporation are considered as two important factors in NTD. Through our meta-analysis, we verified that the association between MTHFD1 1958GN A polymorphism and NTD risk was significant only in Asians, while the result showed no significance in the Caucasian population. Nevertheless, the reason was still controversial, some researches showed that it relied on a combination of differences in polymorphism distributions with non-genetic factors, as well as the interactions with environmental and social factors, which also depends on the variability of races, because genetic factors may vary among populations. Besides, the numbers of studies and sample size in Asian research were not ample to evaluate the association, while we also observed some significant association in the Caucasian population, but the overall results remain insignificant. Hence, to further substantiate the effect of race difference in this SNP on NTD susceptibility, larger scale studies and unified selection criteria are needed. Similarly, our study was limited in its effect estimation owing to small sample sizes for some comparisons. Moreover, the results in our study were just based on the Chinese which may not be applicable to other ethnicities. Besides, except the influence of folate acid intake and gestational weeks, other pathological characteristics of NTD, environmental factors as well as the interaction with individual genetic background which are closely related to the pathogenesis of NTD were not assessed in this study, with regard to some limitations of our assays. Further studies of the risk effects and the functional impact of this polymorphism in further validation are needed, and new research on the association between NTD and other SNPs on MTHFD1 is also recommended. 5. Conclusion In summary, we get the conclusion that SNP MTHFD1 1958GN A is significantly associated with an increasing risk of NTD susceptibility in the Chinese. Furthermore, neonates who harbor the AA homozygote of the MTHFD1 1958GN A are more likely to suffer NTD, compared to the others with GA or GG. Through our meta-analysis, the AA genotype also had a high correlation with NTD in the Asian population. This implies that the examination for AA genotype of this SNP beforehand may prevent the birth of NTD neonates who do not survive it. More detailed investigations and further large-scale studies on genetic functions to provide more conclusive and accurate evidence are required in the future.
193
Conflict of interest None.
References [1] Marini NJ, Hoffmann TJ, Lammer EJ, Hardin J, Lazaruk K, Stein JB, et al. A genetic signature of spina bifida risk from pathway-informed comprehensive gene-variant analysis. PLoS One 2011;6:e28408. [2] Ouyang S, Liu Z, Li Y, Ma F, Wu J. Cystathionine beta-synthase 844ins68 polymorphism is unrelated to susceptibility to neural tube defects. Gene 2014;535:119–23. [3] Sutherland HG, Hermile H, Sanche R, Menon S, Lea RA, Haupt LM, et al. Association study of MTHFD1 coding polymorphisms R134K and R653Q with migraine susceptibility. Headache 2014;54:1506–14. [4] Bi XH, Zhao HL, Zhang ZX, Liu Q, Zhang JW. Association analysis of CbetaS 844ins68 and MTHFD1 G1958A polymorphisms with Alzheimer's disease in Chinese. J Neural Transm (Vienna, Austria: 1996) 2010;117:499–503. [5] Christensen KE, Rohlicek CV, Andelfinger GU, Michaud J, Bigras JL, Richter A, et al. The MTHFD1 p.Arg653Gln variant alters enzyme function and increases risk for congenital heart defects. Hum Mutat 2009;30:212–20. [6] Crisan TO, Trifa A, Farcas M, Militaru M, Netea M, Pop I, et al. The MTHFD1 c.1958 GNA polymorphism and recurrent spontaneous abortions. J Matern Fetal Neonatal Med 2011;24:189–92. [7] Field MS, Kamynina E, Agunloye OC, Liebenthal RP, Lamarre SG, Brosnan ME, et al. Nuclear enrichment of folate cofactors and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) protect de novo thymidylate biosynthesis during folate deficiency. J Biol Chem 2014;289:29642–50. [8] Fisk Green R, Byrne J, Crider KS, Gallagher M, Koontz D, Berry RJ. Folate-related gene variants in Irish families affected by neural tube defects. Front Genet 2013;4:223. [9] Brody LC, Conley M, Cox C, Kirke PN, McKeever MP, Mills JL, et al. A polymorphism, R653Q, in the trifunctional enzyme methylenetetrahydrofolate dehydrogenase/ methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthetase is a maternal genetic risk factor for neural tube defects: report of the Birth Defects Research Group. Am J Hum Genet 2002;71:1207–15. [10] Parle-McDermott A, Kirke PN, Mills JL, Molloy AM, Cox C, O'Leary VB, et al. Confirmation of the R653Q polymorphism of the trifunctional C1-synthase enzyme as a maternal risk for neural tube defects in the Irish population. Eur J Hum Genet 2006;14:768–72. [11] Smithells RW, Sheppard S, Schorah CJ. Vitamin deficiencies and neural tube defects. Arch Dis Child 1976;51:944–50. [12] Etheredge AJ, Finnell RH, Carmichael SL, Lammer EJ, Zhu H, Mitchell LE, et al. Maternal and infant gene–folate interactions and the risk of neural tube defects. Am J Med Genet A 2012;158A:2439–46. [13] Hum DW, Bell AW, Rozen R, MacKenzie RE. Primary structure of a human trifunctional enzyme. Isolation of a cDNA encoding methylenetetrahydrofolate dehydrogenase–methenyltetrahydrofolate cyclohydrolase–formyltetrahydrofolate synthetase. J Biol Chem 1988;263:15946–50. [14] Cao Q, Wang J, Zhang M, Li P, Qian J, Zhang S, et al. Genetic variants in RKIP are associated with clear cell renal cell carcinoma risk in a Chinese population. PLoS One 2014;9:e109285. [15] De Marco P, Merello E, Calevo MG, Mascelli S, Raso A, Cama A, et al. Evaluation of a methylenetetrahydrofolate-dehydrogenase 1958GNA polymorphism for neural tube defect risk. J Hum Genet 2006;51:98–103. [16] Carroll N, Pangilinan F, Molloy AM, Troendle J, Mills JL, Kirke PN, et al. Analysis of the MTHFD1 promoter and risk of neural tube defects. Hum Genet 2009;125: 247–56. [17] Hol FA, van der Put NM, Geurds MP, Heil SG, Trijbels FJ, Hamel BC, et al. Molecular genetic analysis of the gene encoding the trifunctional enzyme MTHFD (methylenetetrahydrofolate-dehydrogenase, methenyltetrahydrofolatecyclohydrolase, formyltetrahydrofolate synthetase) in patients with neural tube defects. Clin Genet 1998;53:119–25. [18] Shaw GM, Lu W, Zhu H, Yang W, Briggs FB, Carmichael SL, et al. 118 SNPs of folaterelated genes and risks of spina bifida and conotruncal heart defects. BMC Med Genet 2009;10:49. [19] van der Linden IJ, Heil SG, Kouwenberg IC, den Heijer M, Blom HJ. The methylenetetrahydrofolate dehydrogenase (MTHFD1) 1958GNA variant is not associated with spina bifida risk in the Dutch population. Clin Genet 2007;72:599–600. [20] Greene ND, Stanier P, Copp AJ. Genetics of human neural tube defects. Hum Mol Genet 2009;18:R113–29. [21] Shields DC, Kirke PN, Mills JL, Ramsbottom D, Molloy AM, Burke H, et al. The “thermolabile” variant of methylenetetrahydrofolate reductase and neural tube defects: an evaluation of genetic risk and the relative importance of the genotypes of the embryo and the mother. Am J Hum Genet 1999;64:1045–55. [22] Pangilinan F, Molloy AM, Mills JL, Troendle JF, Parle-McDermott A, Signore C, et al. Evaluation of common genetic variants in 82 candidate genes as risk factors for neural tube defects. BMC Med Genet 2012;13:62. [23] Christensen KE, Dahhou M, Kramer MS, Rozen R. The MTHFD1 1958GNA variant is associated with elevated C-reactive protein and body mass index in Canadian women from a premature birth cohort. Mol Genet Metab 2014;111:390–2. [24] Cui Y, Jing Y, Sun Z. Lack of association between MTHFD1 G401A polymorphism and ovarian cancer susceptibility. Tumour Biol 2014;35:3385–9. [25] da Silva LM, Galbiatti AL, Ruiz MT, Raposo LS, Maniglia JV, Pavarino EC, et al. MTHFD1 G1958A, BHMT G742A, TC2 C776G and TC2 A67G polymorphisms and head and neck squamous cell carcinoma risk. Mol Biol Rep 2012;39:887–93.
194
J. Meng et al. / Journal of the Neurological Sciences 348 (2015) 188–194
[26] Garcia-Gonzalez I, Solis-Cardenas AD, Flores-Ocampo JA, Alejos-Mex R, HerreraSanchez LF, Gonzalez-Herrera LJ. G894T (NOS3) and G1958A (MTHFD1) gene polymorphisms and risk of ischemic heart disease in Yucatan, Mexico. Clin Investig Arterioscler 2014 [Epub ahead of print]. [27] Jiang J, Zhang Y, Wei L, Sun Z, Liu Z. Association between MTHFD1 G1958A polymorphism and neural tube defects susceptibility: a meta-analysis. PLoS One 2014;9: e101169. [28] Keller MD, Ganesh J, Heltzer M, Paessler M, Bergqvist AG, Baluarte HJ, et al. Severe combined immunodeficiency resulting from mutations in MTHFD1. Pediatrics 2013;131:e629–34.
[29] Lorenc A, Seiemak-Mrozikiewicz A, Barlik M, Wolski H, Drews K. The role of 401aNG polymorphism of methylenetetrahydrofolate dehydrogenase gene (MTHFD1) in fetal hypotrophy. Ginekol Pol 2014;85:494–9. [30] Murthy J, Gurramkonda VB, Lakkakula BV. Significant association of MTHFD1 1958GbA single nucleotide polymorphism with nonsyndromic cleft lip and palate in Indian population. Med Oral Patol Oral Cir Bucal 2014;19:e616–21. [31] Bacon-Baguley T, Kudryk BJ, Walz DA. Thrombospondin interaction with fibrinogen. Evidence for binding to the A alpha- and B beta-chains of fibrinogen. J Biol Chem 1987;262:1927–30.