Maternal and infant MTHFR gene polymorphisms and non-syndromic oral cleft susceptibility: An updated meta-analysis

Journal Pre-proof Maternal and infant MTHFR gene polymorphisms and non-syndromic oral cleft susceptibility: An updated meta-analysis Bhaskar V.K.S. Lakkakula, Shubham Sengupta, Jayant Agrawal, Shivani Singh, Prachi Mendhey, Pratik Jangde, Anurag Sharma, Prashant Anand Pande, Payal Krishan, Priyanka Shukla, Saimila Momin, Ganji Purnachandra Nagaraju, Smaranika Pattnaik

PII:

S1359-5113(19)31143-2

DOI:

https://doi.org/10.1016/j.procbio.2019.10.010

Reference:

PRBI 11798

To appear in:

Process Biochemistry

Received Date:

29 July 2019

Revised Date:

3 October 2019

Accepted Date:

10 October 2019

Please cite this article as: Lakkakula BVKS, Sengupta S, Agrawal J, Singh S, Mendhey P, Jangde P, Sharma A, Pande PA, Krishan P, Shukla P, Momin S, Nagaraju GP, Pattnaik S, Maternal and infant MTHFR gene polymorphisms and non-syndromic oral cleft susceptibility: An updated meta-analysis, Process Biochemistry (2019), doi: https://doi.org/10.1016/j.procbio.2019.10.010

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Maternal and infant MTHFR gene polymorphisms and non-syndromic oral cleft susceptibility: An updated meta-analysis Bhaskar VKS Lakkakula1, Shubham Sengupta2, Jayant Agrawal2, Shivani Singh2, Prachi Mendhey2, Pratik Jangde2, Anurag Sharma2, Prashant Anand Pande21, Payal Krishan2, Priyanka Shukla2, Saimila Momin3, Ganji Purnachandra Nagaraju3, Smaranika Pattnaik4* 1

Sickle Cell Institute Chhattisgarh, Raipur, Chhattisgarh, 492001, India Department of Life Sciences, School of Sciences, ITM University, Raipur, Chhattisgarh, 492001, India 3 Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA. 4 Department of Biotechnology and Bioinformatics, Sambalpur University

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Keywords: Orofacial cleft, NSCLP, Folate, MTHFR, C677T, A1298C, Polymorphism,

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Meta-analysis

Short title: Updated meta-analysis of NSCLP and MTHFR polymorphisms

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# All authors equally contributed Highlights

Folate plays a major role in nucleotide synthesis and DNA methylation pathways

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Periconceptional folic acid supplementation contributes by decreasing the incidence of NSCLP

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MTHFR is a crucial regulatory enzyme necessary for folate metabolism

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MTHFR C677T and A1298C polymorphisms results in the synthesis of a thermolabile form of MTHFR

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*Address for Correspondence Smaranika Pattnaik Department of Biotechnology and Bioinformatics Sambalpur University Jyoti Vihar, Burla Sambalpur- 768019 Odisha- India

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Graphical Abstract

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Highlights  Folate plays a major role in nucleotide synthesis and DNA methylation pathways 

Periconceptional folic acid supplementation decreases the incidence of oral clefts

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MTHFR is a crucial regulatory enzyme necessary for folate metabolism

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MTHFR polymorphisms results in the synthesis of a thermolabile form of MTHFR

Abstract Non-syndromic cleft lip with or without cleft palate (NSCLP) is a common craniofacial malformation. Irregular folate metabolism plays a significant role in the etiopathology of

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NSCLP. In this study, we aim to examine the association of the maternal and cleft child methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms (C677T and A1298C) with nonsyndromic cleft lip with or without cleft palate (NSCLP) by carefully evaluating

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established studies. The meta-analysis includes 39 studies that focused on MTHFR C677T and A1298C polymorphisms in cleft children or cleft children’s mothers. All statistical data

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underwent random or fixed effects model with an odds ratio and 95% confidence intervals as

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effect measures and was preformed using a web tool MetaGenyo. Statistical analyses showed that the MTHFR C677T is significantly associated with the increased risk of NSCLP in

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children but not in the mothers. In contrast to this, there is no evidence for association between MTHFR A1298C and NSCLP risk in both children and the mothers. Furthermore, there is no evidence for publication bias for both MTHFR C677T and A1298C polymorphisms in cleft children as well as the mothers of cleft children. In conclusion, we

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determined that there is a strong association between the MTHFR C677T polymorphism and NSCLP.

Keywords: Orofacial cleft, NSCLP, Folate, MTHFR, C677T, A1298C, Polymorphism, Meta-analysis

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Introduction Non-syndromic cleft lip with or without cleft palate (NSCLP) is a common craniofacial malformation caused by multiple genes or by an interplay of genetic and environmental factors [1]. The incidence of orofacial clefting depends on a variety of factors, which include and are not limited to: geographical region, gender ethnicity and even socioeconomic background. The variable expression, genetic heterogeneity and low penetrance of genes in the orofacial cleft can have a critical implication in genetic analysis [2]. It has been hypothesised that a decrease in folate during the periconceptional period can manifest as

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birth defects, leading to negative consequences in new-borns [3, 4]. Many recent population studies in humans have suggested that periconceptional folic acid supplementation can decrease the incidence of NSCLP [5]. Since the publication of these studies, a considerable

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amount of new evidence pertaining to the function of folic acid in NSCLP has been published

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[6].

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Folates are cofactors that are necessary for the maintenance and stability of the genome; specifically folates provide one-carbon moieties for the nucleotide synthesis pathway [7].

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Furthermore, folates act as a co-substrate for DNA methyltransferases that perform DNA methylation. As DNA methylation plays a key role in embryonic development, aberrant epigenetic mechanisms adversely influence orofacial development and cause OFCs [8]. Epigenome-wide association studies (EWAS) reveal that within distinct orofacial cleft

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subtypes, there are methylation profiles specific for each subtype [9].

Methylenetetrahydrofolate reductase (MTHFR) is an important regulatory enzyme in folate metabolism. The gene encoding the MTHFR enzyme is harbored on chromosome 1p36.3 and is known to have C677T and A1298C functional polymorphisms that affect the folate status.

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The C677T polymorphism converts alanine to valine at codon 222 (A222V; rs1801133), which leads to the formation of a thermolabile MTHFR enzyme [10]. Further computational modeling reveals that the C677T mutation leads to major conformational changes within the tertiary structure of MTHFR, resulting in a significant reduction in its FAD-binding affinity [11]. On the other hand, the A1298C polymorphism converts glutamine to alanine at position 429 (Glu429Ala; rs1801131) and alters the specific activity of the enzyme but to a lesser extent than the MTHFR C677T polymorphism [12]. It has been suggested that decreased levels of folate associated with MTHFR polymorphisms could potentially be the cause of oral

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clefts. Several studies have examined the association between MTHFR C677T and A1298C polymorphisms and NSCLP risk; however, findings have been inconsistent [6]. The aim of this current meta-analysis is to quantitatively summarize the association of the maternal and

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cleft child MTHFR gene C677T and A1298C polymorphisms with NSCLP.

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Materials and Methods

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Literature search

A comprehensive search in PubMed, ScienceDirect and Google Scholar was conducted to

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collect copious and detailed published studies related to MTHFR gene and NSCLP. The keywords such as ‘nonsyndromic oral clefts’, ‘NSCLP’, ‘MTHFR’, ‘C677T (rs1801133) polymorphism’ and ‘A1298C (rs1801131) polymorphism’ were used in various combinations and without any restrictions in order to obtain all relevant studies; the last search was

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conducted on 30th June 2019. Furthermore, additional papers were hand-searched in reference lists. After the initial screening, a full text of all relevant papers was obtained and further filtered in order to fit into the inclusion criteria: 1) all the studies had a similar purpose of examining the association of either maternal or infant MTHFR polymorphisms with NSCLP, 2) investigations were prospective case-control studies and 3) studies had enough information

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to calculate odds ratio. The major exclusion criteria include: (1) studies that did not fit the inclusion criteria, (2) no control group, and (3) non-availability of genotype frequencies.

Data extraction All the authors carefully extracted genotype data for the MTHFR polymorphisms independently. The data on the following categories were considered: author, publication date, country in which the study was conducted, ethnicity of the study population, number of genotyped cleft and control subjects and number of each genotype for both polymorphisms

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(C677T and A1298C). The extracted data was then tabulated. As the Hardy-Weinberg equilibrium (HWE) is a surrogate for assessing study quality, genotypes of the control groups were checked for HWE. This meta-analysis evaluates the association of C677T and A1298C

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polymorphisms with NSCLP risk as per the guidelines issued in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [13]. The process for

Statistical analysis

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literature search and selection of papers eligible for meta-analysis is shown in figure 1.

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The strength of the associations between MTHFR C677T or A1298C polymorphism and NSCLP risk was assessed by calculating the crude odds ratios (OR) and corresponding 95% confidence interval (CI) limits for each study. We choose OR as the effect size to find NSCLP risk associated with rare genotypes (CT and TT for C677T; AC and CC for A1298C).

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Heterogeneity between studies was assessed using the Cochran's Q test [14] and by measuring inconsistency value I2 [15]. Both fixed and random effect models were used for analysis and high-resolution forest plots were prepared to depict both OR and 95% CI limits. To test the potential publication bias, Begg's funnel plot and Egger's test were used.

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Publication bias was presented using the funnel plot of precision by the log of the odds ratio. All meta-analyses were conducted using a web tool MetaGenyo [16].

Results Selection of studies Based on the inclusion and exclusion criteria, a total of 39 papers were selected for the extraction of MTHFR C677T and A1298C genotypes data on cleft children or cleft children’s mothers. Among them, MTHFR C677T genotypes of the children and the mothers were extracted respectively from thirty-five [17-51] and eleven papers [19, 23, 28, 32, 34, 42, 49, 52-55]. The genotype information on MTHFR A1298C polymorphism of the children and the mothers were extracted respectively from eighteen [18, 22-25, 30, 31, 33-38, 40, 42, 46,

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47, 49] and nine papers [18, 23, 25, 30, 33, 34, 42, 49, 55]. The main characteristics of the selected studies for MTHFR C677T and A1298C polymorphisms are summarized in supplementary table 1 and table 2, respectively. In order to assess the cause of heterogeneity, we conducted a subgroup analysis stratified by ethnicity. The results suggested that ethnicity

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is a contributing factor to the substantial heterogeneity between the studies of cleft children. The MTHFR A1298C polymorphism studies in Caucasian cleft children showed no

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significant heterogeneity (CC+AC versus AA: Pheterogeneity = 0.082, I-squared = 0.43) (Table 1). MTHFR C677T and A1298C variant allele frequencies in both NSCLP and the

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respectively.

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control groups of the selected studies are depicted in supplementary figure 1 and figure 2,

Heterogeneity test

For MTHFR C677T, the heterogeneity test showed a true heterogeneity between studies of

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cleft children (TT+CT versus CC: Pheterogeneity < 0.001, I-squared = 0.61) but no significant heterogeneity was found between studies of cleft children’s mothers (TT+CT versus CC: Pheterogeneity = 0.145, I-squared = 0.32) (Table 1). The A1298C polymorphism studies also showed significant heterogeneity between studies of cleft children (CC+AC versus AA: Pheterogeneity < 0.001, I-squared = 0.62), but not for the mothers of cleft

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children (CC+AC versus AA: Pheterogeneity = 0.430, I-squared = 0.005) (Table 1).

MTHFR C677T polymorphism and NSCLP susceptibility MTHFR C677T polymorphism and NSCLP susceptibility in cleft children (Random effect model) and the mothers of cleft children (Fixed effect model) were shown in table 1 and figure 2A and B. Meta-analysis of all thirty-five studies on cleft children showed that the mutant allele is significantly associated with NSCLP (T versus C: OR = 1.14, 95% CI = 1.041.26, P = 0.007) (Table 1). Furthermore, the pooled odds ratio for mutant genotypes in the

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dominant model showed increased risk of NSCLP in cleft children (TT+CT versus CC: OR = 1.12, 95% CI = 0.98-1.28, P = 0.097). The sub-group analysis by ethnicity also provided evidence for the increased risk of NSCLP in both Asian and Caucasian cleft children (Table

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1). In the mothers of the cleft children, the MTHFR C677T polymorphism T allele or mutant

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genotypes does not increase the risk for NSCLP in the fixed effect model (T versus C: OR = 1.05, 95% CI = 0.87-1.28, P = 0.600; TT+CT versus CC: OR = 0.93, 95% CI = 0.78-1.11, P

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= 0.421). A further subgroup analysis in the Asian and Caucasian group showed that the maternal MTHFR C677T variant allele or genotypes did not reveal any significant association

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with NSCLP risk.

MTHFR A1298C polymorphism and NSCLP susceptibility MTHFR A1298C polymorphism and NSCLP susceptibility in cleft children (Random effect

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model) and the mothers of cleft children (Fixed effect model) are shown in table 1 and figure 3A and B. Meta-analysis of the MTHFR A1298C polymorphism in cleft children, under both the dominant and allelic models, demonstrated no significant association between NSCLP and the MTHFR A1298C polymorphism (Table 1). Furthermore, the subgroup analysis by ethnicity also exhibited no significant association between the MTHFR A1298C

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polymorphism and NSCLP in Asian and Caucasian populations (Table 1). In the mothers of cleft children, the MTHFR A1298C polymorphism C allele or mutant genotypes does not increase the risk for the NSCLP (fixed effect model) (C versus A: OR = 0.94, 95% CI = 0.83-1.07, P = 0.338; CC+AC versus AA: OR = 0.95, 95% CI = 0.82-1.11, P = 0.532). However, a subgroup analysis by ethnicity revealed that the MTHFR 1298C allele or genotypes showed a trend of decreased risk of NSCLP in Asians (C versus A: OR = 0.69, 95% CI = 0.32-1.48, P = 0.345; CC+AC versus AA: OR = 0.59, 95% CI = 0.23-1.47, P =

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0.255).

Sensitivity analysis and publication bias

The sensitivity analysis demonstrated that the omission of each study did not significantly

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affect the pooled OR of both MTHFR C677T and A1298C variants, which indicated that the results of the meta-analysis is statistically robust. The Begg and Mazumdar rank correlation

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test (dominant model) showed no evidence of publication bias for MTHFR C677T and

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A1298C polymorphisms (Figure 4). In all comparisons, the funnel plots’ shape is symmetrical, suggesting that there is no significant evidence for publication bias for these

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polymorphisms (Figure 4). Furthermore, Egger’s test provided statistical evidence for the funnel plot symmetry for both MTHFR C677T and A1298C polymorphisms in cleft children as well as in the mothers of cleft children (Figure 4).

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Discussion

Summary of the previous meta-analysis conducted for NSCLP and MTHFR polymorphisms is documented in Table 2. Our meta-analysis reveals that the mutant allele of MTHFR C677T is significantly associated with the increased risk of NSCLP children but not in the mothers. In contrast to this, there is no evidence for association between MTHFR A1298C and NSCLP

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risk in both children and the mothers. Meta-analysis of studies on MTHFR C677T and A1298C involving children reveals significant heterogeneity between studies. However, no heterogeneity between studies was found in the meta-analysis of studies involving the mothers with MTHFR C677T and A1298C. Furthermore, there is no evidence for publication bias for both MTHFR C677T and A1298C polymorphisms in cleft children as well as the mothers of cleft children.

MTHFR C677T substitution has been studied in different ethnicities for its possible

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association with NSCLP. An initial study was conducted by Tolarova et al., in which frequent occurrences of 677 TT homozygotes were observed in Argentinean CL/P patients than in controls [18]. Later on, several studies showed significant associations of MTHFR mutant

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genotypes with NSCLP in different populations [26, 31, 35, 37, 44]. In contrast to this, there are several studies focusing on MTHFR C677T and NSCLP risk that have failed to show

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significant association between them in the context of geographical locations [17, 19, 22, 27-

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29, 33, 38, 56-58]. Furthermore, some studies showed that the maternal MTHFR genotype (677 T) could be a predisposition to CL/P [21, 23, 43, 59-61]. Results of our meta-analysis

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are consistent with some of the previous meta-analyses (Table 2) that report increased risk of NSCLP with the MTHFR C677T polymorphism [62-64]. In contrast to this, other metaanalyses revealed no association between the MTHFR C677T polymorphism and NSCLP risk [65, 66]. The difference in the results could be due to variation in the allele frequency of

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C677T polymorphism in different geographical locations. This genetic heterogeneity was shown in Zhu et al., in which the C677T was moderately associated with NSCLP in families from northern China; however, no association between C677T and NSCLP was found in families from southern China [67].

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Numerous studies have been conducted to investigate the association between the MTHFR A1298C locus and NSCLP and have resulted in conflicting findings [22, 31, 33, 35, 60, 68]. Our findings from the current meta-analyses are consistent with the findings of the previous meta-analyses, in which no association between the MTHFR A1298C and NSCLP risk was inferred [62, 65, 69, 70]. The loss in MTHFR activity was found in combined heterozygotes of the C677T and A1298C polymorphisms, but the effect of compound genotypes on the risk of NSCLP was not included in a majority of studies. Analysis linkage disequilibrium between the C677T and A1298C polymorphisms in the Indian population exhibited complete absence

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of LD between these two polymorphisms [38]. In contrast to the results of the Indian population, strong linkage disequilibrium between these polymorphisms was noted in

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Swedish and German populations [71, 72].

However, several investigators have shown that the periconceptional folic acid

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supplementation leads to a reduction in the incidence of NSCL/P in the many populations

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that they studied [73-75]. Additionally, some investigators have suggested that the genetic contribution to orofacial clefts is independent of pathways by which folates provide

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protection from NSCL/P [76]. This current meta-analysis has both methodological strengths and limitations. The main strength of this paper is the inclusion of multiple studies. However, the limitation of our study is not assessing the environmental interventions or the maternal folate status, which are known to influence the association of MTHFR polymorphisms and

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NSCLP risk; we did not include such information due to data deficiencies. In conclusion, we suggest that the MTHFR C677T polymorphism is associated with the NSCLP risk.

Conflicts of interest: There are no conflicts of interests. Authors’ Contribution: B.V.K.S.L., S.M. A.N., G.P.N., and S.P., designed the study, proposed the search terms, managed the work, and reviewed data extraction, wrote the manuscript and approved the final version. S.S., J.A., S.S., P.M., P.J., A.S., P.A.P., P.K., and

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P.S., searched the databases and participated in title and abstract screening, full-text screening, data extraction and analyzed the data. All authors have critically reviewed the manuscript and finally approved the manuscript.

Ethical considerations The authors of this manuscript declare that they all have followed the ethical requirements for this communication. Also, ethical issues (including plagiarism, data fabrication, double publication) have been completely observed by the authors.

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Funding/Support None.

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Figure legends Figure 1: Flow chart of literature retrieval and selection for quantitative analysis. Figure 2: ORs and 95 % CI limits of the individual studies for the association between the MTHFR C677T polymorphism and NSCLP. A. NSCLP children MTHFR C677T vs. Control children MTHFR C677T. B. NSCLP maternal MTHFR C677T vs. Control maternal MTHFR C677T. Figure 3: ORs and 95 % CI limits of the individual studies for the association between the MTHFR C677T polymorphism and NSCLP. A. NSCLP children MTHFR A1298C vs.

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Control children MTHFR A1298C. B. NSCLP maternal MTHFR A1298C vs. Control maternal MTHFR A1298C.

Figure 4: Eggers funnel plots for MTHFR C677T and A1298C polymorphisms in dominant

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genetic model.

For each study standard error of the log odds ratio [OR]) is plotted against each study’s effect

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estimate (OR). A. NSCLP children MTHFR C677T vs. Control children MTHFR C677T; B.

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NSCLP maternal MTHFR C677T vs. Control maternal MTHFR C677T; C. NSCLP children MTHFR A1298C vs. Control children MTHFR A1298C; D. NSCLP maternal MTHFR

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A1298C vs. Control maternal MTHFR A1298C.

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Fig 1

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Fig 2

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Fig 3

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Fig 4

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Table 1: The association of maternal and children MTHFR gene polymorphisms and non-syndromic cleft lip and palate in different genetic models. NSCLP children vs. Control children (REM) By ethnicity Caucasia Overall Asian n

11

3

8

0.61 <0.001 1.12 (0.991.26)

0.53 0.019 1.05 (0.871.28)

0.81 0.005 1.22

0.26 0.225 1.01 (0.881.17)

0.60 0.001 1.07 (0.921.26) Caucasia n

0.32 0.145 0.93 (0.781.11)

0.63 0.067 1.09

Overall

Asian

0.23 0.247 0.91 (0.751.11) Caucasia n

9

9

1

8

0.49 0.048 0.92 (0.771.10)

0.0 0.568 0.94 (0.831.07)

NA NA 0.69

0.0 0.531 0.95 (0.841.08)

0.43 0.082 0.92 (0.741.15)

0.005 0.430 0.95 (0.821.11)

NA NA 0.59

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(0.59- 2.53)

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MTHFR 677C>T Number of studies 35 16 Allele contrast (T vs. C) I2 0.61 0.63 PHeterogeneity <0.001 <0.001 OR 1.14 1.18 (1.0495% CI 1.26) (0.99- 1.39) Dominant model (TT+CT vs. CC) I2 0.61 0.61 PHeterogeneity <0.001 0.001 OR 1.12 1.19 (0.9895% CI 1.28) (0.94- 1.49) MTHFR Overall Asian 1298A>C Number of studies 18 9 Allele contrast (C vs. A) I2 0.63 0.72 PHeterogeneity <0.001 <0.001 OR 1.01 1.12 (0.8895% CI 1.17) (0.89- 1.41) Dominant model (CC+AC vs. AA) I2 0.62 0.73 PHeterogeneity <0.001 <0.001 OR 1.01 1.14 (0.8595% CI 1.22) (0.84- 1.55)

NSCLP mothers vs. Control mothers (FEM) Overall By ethnicity Caucasia Asian n

(0.49- 2.44)

(0.32- 1.48)

(0.23- 1.47)

0.0 0.435 0.97 (0.831.13)

REM: Random effect model; FEM: fixed effect model.

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Table 2: Summary of previous meta-analyses conducted for MTHFR polymorphisms and NSCLP. Race

No. of studie s

Heter Publ ogenei icati ty on Bias

Inference on association between MTHFR polymorphism and NSCLP

MTHFR C677T Verkleij-Hagoort et al. 2007 [40]

Caucasians

8

No

Yes

8†

No

Yes

8

No

No

6†

No

No

Asian

4

Yes

No¥

Caucasian

8

No

Mixed

6

Yes

Assis Machado et al. 2018 [38]

Caucasian

3

No

Yes

Not associated with NSCLP risk in children Maternal MTHFR 677 is not associated with NSCLP risk Significantly increased NSCLP risk Maternal MTHFR 677 significantly increased NSCLP risk Not associated with NSCLP risk in children Associated with NSCLP risk in children Not associated with NSCLP risk in children Associated with NSCLP risk

Rai 2018 [39]

Caucasian

16

Yes

No

Asian

6

Yes

No

5

No

Yes

5†

No

Yes

Caucasians

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MTHFR A1298C Verkleij-Hagoort et al. 2007 [40]

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Pan et al. 2015 [41]

Asians

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Zhao et al. 2014 [37]

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Meta-analysis

Mixed

11

No

No

Rai et al. 2014 [44]

Mixed

10†

No

No

Zhao et al. 2014 [37]

Asians

6

Yes

No

3†

Yes

No

Asian

2

No

No¥

Caucasian

4

No

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Rai et al 2014 [45]

Pan et al. 2015 [41]

Associated with increased NSCLP risk Associated with increased NSCLP risk Not associated with NSCLP risk Maternal MTHFR 1298 is not associated with NSCLP risk Not associated with NSCLP risk Maternal MTHFR 1298 is not associated with NSCLP risk Not associated with NSCLP risk Maternal MTHFR 1298 is not associated with NSCLP risk Not associated with NSCLP risk Not associated with NSCLP risk

Mixed 1 No Not associated with NSCLP risk † studies related to maternal genotype; ¥ publication bias in pooled studies

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