Association of MTR , MTRR and MTHFR gene polymorphisms in Non-Syndromic Cleft Lip and Palate in Paediatric patients in Northern India

Non syndromic cleft lip and palate includes a wide spectrum of clinical variability from a simple unilateral lip scar to bilateral cleft lip and cleft palate. Folic acid can prevent the neural tube defects such as cleft lip and palate. Polymorphisms of the folate pathway genes causes disturbed activity of the key enzymes of folate metabolism which may be a contributing factor for the development of cleft lip and palate. Main enzymes of folate pathway such as methionine synthase, methionine synthase reductase and methylene tetrahydrofolate reductase are encoded by MTR, MTRR and MTHFR genes respectively. This research evaluates the association of MTR, MTRR and MTHFR gene polymorphisms in non-syndromic cleft lip and palate in northern Indian children.


Introduction
Cleft lip or palate (CL/P) is a common human congenital defect promptly recognized at birth. Despite the variability driven by socioeconomic status and ethnic background, the worldwide prevalence of CL/P is often cited as 1:700 live births [1]. Cleft lip is consistently more common in males at a 2:1 ratio, in contrast to cleft palate which has a similar ratio in favor of females. Unilateral cleft lip shows a 2:1 left predominance [2]. There is geographical variation in the prevalence of cleft in the world. Cleft lip and palate results from failure of fusion of the maxillary processes or palatal shelves, which occur between the 4 th and 12 th weeks of embryogenesis. Maternal alcohol intake or exposure to tobacco and several chemicals, such as retinoic acid and folate antagonists (e.g., valproic acid), among others, has been shown to be teratogenic, thus representing risk factors to embryos during the first trimester of pregnancy [3]. The involvement of the MTR, MTRR, and MTHFR enzymes in folate metabolism and methyl group metabolism makes these enzymes crucial for the maintenance of proper DNA methylation and nucleic acid synthesis. MTR is encoded by the MTR gene and is responsible for the regeneration of methionine from homocysteine and MTR gene mutations may contribute to various diseases, including cardiovascular diseases, cancers, birth defects and congenital anomalies [4].

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Folic acid supplemented periconceptionally in the mother appears to dramatically reduce the frequency of neural tube defects. However, the mechanism underlying this beneficial effect remains unclear. Genes involved in cellular folate transportation may be prime candidates for folate regulated NTDs. If vitamin supplements namely folic acid and cobalamins were not taken during early pregnancy the risk for CLP could be tripled [5]. In periconceptional folic acid deficiency, the MTHFR C677T thermally labile variant could lead to a risk of CLP that was increased by 10 times [3]. Extensive studies have explored the relationships of gene polymorphisms related to the folic acid/homocysteine metabolic pathway with NSCLP and the results have revealed that the relationships of genetic mutations with NSCLP susceptibility vary between different populations. Conflicting results regarding the MTR A2756G polymorphism and the risk of NSCLP have been reported among Chinese and Indian population. It necessitates the further studies among Indian and Chinese cleft lip and palate population to find the genetic association [6].
Maternal folate deficiency may be a contributing factor to cleft aetiology when maternal family history is observed and offer a possible explanation for the conflicting results of previous studies investigating MTHFR genotype in relation to CLP. Such results would also suggest the importance of collecting parental and grandparental data when evaluating parental effects. A significantly higher mutation frequency of MTHFR in mothers of children with CLP. Thus, the important of periconceptional folate intake were emphasized in these studies and its deficiencies could lead to CLP [7]. So, we correlated the maternal folate intake with genetic polymorphisms of non-syndromic cleft lip and palate in this study. A meta-analysis indicated that the MTRR A66G polymorphism but not the MTR A2756G polymorphism, may increase the maternal risk for neural tube defects among Caucasians [8].
A case control study in Chinese population suggests that MTR A2756G, MTRR A66G, and MTHFR C677T gene polymorphisms were associated with the risk of NSCLP. The MTR A2756G, MTRR A66G, MTHFR C677T and MTHFR A1298C polymorphisms were assessed by polymerase chain reaction-restriction fragment length polymorphism [9]. In addition, a recent study reported that the MTRR A66G polymorphism but not the MTR A2756G polymorphism may contribute to NSCLP in Indian populations [10]. The MTHFR C677T polymorphism is associated with the risk of NSCLP but not with other clefts in the craniofacial region of south Indian population. The TT genotype increased the risk of NSCLP and CT genotype showed a decrease in the risk [11]. These conflicting results in different population suggests further studies in specific population to analyse the prevalence and association of gene polymorphisms in NSCLP. Consanguineous marriage and recurrent abortions in mother are potential risk factors for the development of congenital disorders. In this study we analysed the MTR A2756G, MTRR A66G, MTHFR C677T and A1298C gene polymorphisms in north Indian population to check the prevalence and their association in non-syndromic cleft lip and palate with the history of folate intake, consanguinity and recurrent abortion.

Patient selection
Patients visiting the outpatient department of Paediatric and Preventive Dentistry, department of Plastic Surgery, department of Obstetrics and Gynaecology and department of Paediatrics, King George's Medical University were screened. The patients were assessed for eligibility as per the inclusion and exclusion criteria.

c)
Patients who have undergone surgical correction of nonsyndromic cleft lip, cleft palate and cleft lip with palate were also included in this study.

Exclusion criteria
a) Cleft lip and palate associated with other congenital disorders or congenital malformations, such as neural tube defects or congenital heart diseases with concomitant cleft lip and palate, kabuki make-up syndrome, Van der Woude syndrome, Meckel syndrome, or velocardiofacial syndrome. b) Non syndromic cleft lip and palate patients with associated hypertension, coronary heart disease, or other important organ diseases.

c)
Patients with history of systemically debilitating diseases like uncontrolled diabetes, immunosuppression, severe asthma etc.

Investigative procedure
a) Informed consent is taken from the parents or guardians of the patients for participation into the study. The detailed history is taken from the patient/ parents with demographic details.
b) The history includes familial association of cleft lip and palate, religion, region where they belong to, consanguineous marriage, history of any drug intake, recurrent abortions, trauma to mother during pregnancy, thyroid disorder in mother, maternal folic acid deficiency, and folic acid supplement intake.  b) The genotyping was carried out in patients suffering from Cleft Lip with or Without Cleft Palate. A total 4 SNPs were analysed (Table 1). All MTR, MTRR, and MTHFR Gene polymorphisms were analyzed for their correlation with Cleft Lip with or Without Cleft Palate. PCR was performed in a gradient thermocycler using thin walled 0.2 ml PCR tubes. ii. PCR based amplification assay: a) The polymorphisms of the MTR, MTRR, MTHFR gene were genotyped using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. PCR was carried out on a Biometra T Robot Thermocycler (PCR) (LabRepCo, LLC. USA) using the respective primers.
b) The MTR A2756G, MTRR A66G, MTHFR C677T and A1298C genotypes is sequenced and analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The PCR-amplified products were digested overnight at 37ºC with specific restriction enzymes (HaeIII for MTR A2756G, NdeI for MTRR A66G, Hinf for MTHFR C677T, and MboII for MTHFR A1298C) in a total volume of 20 mL containing 17 mL PCR-amplified products, ddH2O, 2 mL 10· reaction buffer and 1 mL restriction enzyme (10 U/mL). Six percent polyacrylamide gel electrophoresis was used to separate the enzyme-digested products. The genotypes were determined according to the enzyme map.
c) Gel electrophoresis pictures of PCR based amplification assay for genotypes MTR A2756G, MTRR A66G, MTHFR C677T and MTHFR A1298C were shown in Figure 3-6 respectively.

Results
A total of 164 patients of both genders were included in the study. Table 2 shows age distribution of patients and parents. Mean age of the patients enrolled in this study was 11.85 ± 3.99 years which ranges from 4 months of age to 15 years of age. Mean age of fathers and mothers of the patients included in the study were 43.97 ± 7.16 years and 37.46 ± 6.55 years respectively.  Figure 7 show the percentage of different types of cleft lip and/or palate patients in the total study population. In the total of 164 patients, cleft lip patients were the least prevalence with 1.8% (n=3) and cleft lip and palate patients were the most prevalence with 90.9% (n=149). Unilateral cleft lip and palate of the left side is the most common among type in this population (56.7%, n=93). 11.1% (n=7) patients have the positive family history with their siblings affected with cleft lip and palate. Mothers of the patients have the positive history of recurrent abortion of 9.8% (n=16). 73.2% (n=120) of mothers did not take any form of folic acid and iron supplement during their pregnancy.  Figure 8 show the genotype and allele prevalence of genes studied. AA genotype is more prevalent in MTR A2756G gene (62.2%, n=102) and MTRR A66G gene (58.5%, n=96).TT genotype is more prevalent in MTHFR C677T gene (78.0%, n=128). MTHFR A1298C gene shows more AC genotype prevalence (51.2%, n=84).       Figure 9 show the significant association of MTHFR A1298C polymorphism and gestational folic acid intake (p< 0.001) with AC genotype predominance. MTR A2756G, MTRR A66G, MTHFR C677T gene polymorphism are not associated with gestational folic acid intake. Table 7 shows the prevalence of various types of clefts with gene polymorphisms. MTR A2756G gene polymorphism is associated with cleft lip and palate (p<0.001) with AA genotype predominance. MTRR A66G gene polymorphisms is also associated (p=0.047) with cleft lip and palate. Table 8 shows the association of gene polymorphisms with history of recurrent abortions in mother. MTHFR C677T gene polymorphism shows the significant (p=0.010) association with non-syndromic cleft lip and palate, while other gene polymorphisms are not statistically significant with cleft lip and palate when compared with recurrent abortions (p>0.3). Table 9 shows the prevalence of family history of consanguinity in study population. 34.1% (n=56)  Figure 10 show the association of cleft lip and palate gene polymorphisms with the consanguinity. MTR A2756G gene polymorphism is significant association with the consanguinity (p=0.032) and AA genotype predominance in noted MTR A2756G gene polymorphism. MTHFR C677T gene polymorphism is also associated with the consanguinity (p=0.049).

Discussion
Oro facial cleft is the most common developmental anomaly of the head and neck region and second most common congenital anomaly in the body next to congenital heart diseases [12]. Cleft lip and palate cause negative impact on psychosocial life of the children and their families. Cleft lip and palate affect phonation, auditory ability and oro facial development. The etiology of the disease is very complex that is the genetic and environmental interactions. Folic acid is essential to prevent the neural tube defects. Key enzymes of folic acid pathway are maintained by MTR, MTRR, MTHFR genes. Many studies have found the association of these gene polymorphisms with non-syndromic cleft lip and palate, but there are population-based variations in the association [9]. Our study focuses genetic polymorphisms of folic acid pathway genes MTR, MTRR, MTHFR in north Indian population at a tertiary level of care institute of Uttar Pradesh. The incidence and geographical distribution of clefts varies across the world. This may be due to variation in the prevalence of birth and under reporting. According to a study, 3 children with cleft are born in every hour and a total of 26,950 new cases are reported from India every year [7].
The present study demonstrates the prevalence of cleft lip is 1.8% and cleft lip and palate is 90.9%, isolated cleft palate is 7.3% which coincides with the previous study by Menezes LM

Advances in Dentistry & Oral Health
et al. and a study conducted in Japan [13]. Whereas in a study conducted in south Indian population, the frequency of Cleft lip only was more than Cleft lip with palate and isolated cleft palate and in Republic of Korea, cleft palate was more common than the cleft lip with palate [14]. These findings suggest that there is a demographic variation in the frequency of different phenotypes. Studies show different sex distribution also in the prevalence of clefts where studies in various countries observed a male predominance [7]. Our study demonstrated Cleft lip with palate to be the predominant type of orofacial clefts and a high male to female sex ratio among CPL (1.1:1) and CL patients (1.5:1). In contrast, the ratio of cleft palate was more frequent in females with a high female: male ratio (1.2:1). These results are similar with the previous study conducted by Diwana et al. [15]. It was found that unilateral clefts were more common than bilateral clefts. Our study also demonstrated left-sided clefts to be more common than right sided clefts. In this study, unilateral cleft lip and palate is more prevalent (62.8%) and the unilateral cleft lip and palate of left side is more common (56.1%). The reason for the left side laterality is not proven.
Previous studies showed that there is a definite influence of consanguinity in the occurrence of clefts as it is suggested that this association is probably related with a recessive genetic component and environmental factors. In the present study, the consanguinity was positive in around 34.10% (n=56). The findings of the study were similar to the studies done by Dwidedi et al. [15] in a tertiary hospital in Dehradun and other studies conducted in Saudi Arabia and Iran where the consanguinity was positive in around 48.9%, 54.4%, and 61.6%, respectively [15]. Neela PK et al. [16] concluded that the consanguinity is the major risk factor for the development of cleft lip and palate. In our study approximately 60% of the clefts born to consanguineous parents are seen in males in present study the association of consanguinity with MTR A2756G polymorphism with statistically significant value (p=0.032). MTHFR C677T polymorphism is also significant when the consanguinity is considered (p=0.049).
One of the contributing factors for the occurrence of MTHFR gene polymorphisms may be recurrent abortions. MTHFR C677T gene polymorphism is found to be associated with the parent mothers who had the history of recurrent abortions(p<0.010) in our study. It has been previously reported that taking folic acid supplementation at early pregnancy stage reduces the cleft palate risk and peri conceptional usage of folate reduced the miscarriage risk and prevents the occurrence of birth defects [17]. But the effect of folic acid, a cofactor for MTHFR enzyme action is protective in some populations Mossey et al. [18] but ineffective in some others van Rooji et al. [19]. Lopez cartes et al. [20] found that these polymorphisms alter homocysteine levels and reduce enzymatic activity, which may lead to DNA damage associated with folate deficiency that may further contribute to the development of non-syndromic cleft lip and palate. In our study out of 164 patients, mothers of 120 (73.2%) patients did not take any folic acid and iron supplements during the pregnancy period. This may be a cause of polymorphism of MTHFR A1298C gene (p<0.001) and depressed action of methylene tetra hydro folate reductase enzyme leading to clefts though it was found to be statistically insignificant. This may be due to less sample size and possible other environment-gene interactions.
MTR A2756G gene polymorphism is highly associated with all clinical types of non-syndromic cleft lip and palate patients of the study population (p<0.001) which was similar to studies by Denis aslar et al. and Wang et al. who found that MTR A2756G gene polymoprphism is highly associated with non-syndromic cleft lip in Turkish population and with cleft lip and palate in Chinese population respectively while no statistically significant correlation was found in the study conducted in Brazil [9,21,22]. In the present study there is suggested geographical variation and undiscovered gene -environment interaction may be attributing to MTR A2756G gene polymorphism in Indian population. In our study population the MTRR A66G is also associated with cleft lip and palate prevalence (p<0.047). The results coincide with the previous studies by Wang et al.9 in Chinese population and by Vandana Rai et al. [23] in Eastern India (2019). In contrast study conducted by Nasri K et al. [24] shows MTRR A66G is not associated with non-syndromic cleft lip and palate. There is geographical variation in the prevalence of MTRR A66G polymorphism.
The present study correlates the environmental factors such as maternal folic acid intake, thyroid disorder in mother, maternal alcohol consumption and maternal drug intake etc., but there are some limitations to the study. First, the limited sample size of this study may have biased the associations of the MTR A2756G, MTRR A66G, MTHFR C677T and MTHFR A1298C gene polymorphisms with the non-syndromic cleft lip and palate risk. Second, this study did not consider the possibility of linkage disequilibrium between SNP-SNP interactions. These limitations could be overcome with more specific studies with larger samples and with more advanced molecular genetics technology. Future studies will uncover additional mechanistic insights and potential gene-environment interactions of the MTR A2756G, MTRR A66G, MTHFR C677T and MTHFR A1298C gene polymorphisms with non-syndromic cleft lip and palate.

Conclusion
Based on the observations and findings of the present study, the following conclusions were drawn: i.
Unilateral cleft lip and palate of left side were found 56% that was more prevalent in paediatric patients in northern India among cleft lip and palate patients. ii.
Unilateral cleft lip and palate of right side were found 6.1%, unilateral cleft lip 1.8%, bilateral cleft lip with palate 28% and isolated cleft palate were found 7.3% in paediatric patients in northern India among cleft lip and palate patients. iii. MTR A2756G and MTRR A66G gene polymorphisms were found associated with non-syndromic cleft lip and palate patients of northern India. iv. MTHFR A1298C gene polymorphism was found associated with no maternal folate intake by mothers of nonsyndromic cleft lip and palate patients.

v.
MTR A2756G and MTHFR C677T gene polymorphisms were found associated with non-syndromic cleft lip and palate with consanguineous parents.
vi. MTHFR C677T gene polymorphism was found associated with history of recurrent abortion in mothers of non-syndromic cleft lip and palate.
However, further studies are needed to validate the findings and to explore possible gene-environmental interactions.