1Professor & Head, Department of Paediatric and Preventive Dentistry, King George’s Medical University, India
12Professor, Department of Plastic Surgery, King George’s Medical University, India
1Professor, Department of Obstetrics and Gynaecology, King George’s Medical University, India
1Professor, Department of Paediatrics, King George’s Medical University, India
1Professor, Department of Paediatric and Preventive Dentistry, King George’s Medical University, India
1Assistant Professor, Department of Cytogenetics, King George’s Medical University, India
1Junior Resident, Department of Paediatric and Preventive Dentistry, King George’s Medical University, India
Submission: February 02, 2021; Published: March 03, 2021
*Corresponding author: Aravindhan Arumugam, Department of Paediatric and Preventive Dentistry, King George’s Medical University, India
How to cite this article: Rakesh Kumar Chak, Veerendra Prasad, Nisha Singh, Chandrakanta, Richa Khanna, Nitu Nigam, Aravindhan Arumugam.
Association of MTR, MTRR and MTHFR gene polymorphisms in Non-Syndromic Cleft Lip and Palate in Paediatric patients in Northern India. Adv Dent &
Oral Health. 2021; 14(1): 555877. DOI: 10.19080/ADOH.2021.14.555877
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.
Keywords: Non syndromic; Cleft lip and palate; MTR; MTRR; MTHFR; Gene polymorphisms; folate
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 . 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 . 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 4th and 12th 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 . 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 . 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 . In
periconceptional folic acid deficiency, the MTHFR C677T thermally
labile variant could lead to a risk of CLP that was increased by
10 times . 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 .
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 . 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 .
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 . In addition, a recent study reported
that the MTRR A66G polymorphism but not the MTR A2756G
polymorphism may contribute to NSCLP in Indian populations
. 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 . 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.
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.
a) Patients in the age group 0-15 years.
b) Patients with non-syndromic cleft lip, cleft palate and
cleft lip with palate were included in this study.
c) Patients who have undergone surgical correction of nonsyndromic
cleft lip, cleft palate and cleft lip with palate were
also included in this study.
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.
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
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
Sample collection and DNA isolation
ii. Gene polymorphism analysis by PCR- RFLP procedure
iii. Data collection and statistical analysis
i. Sample collection and DNA isolation:
a) 2ml of blood samples were collected by venepuncture
in EDTA tubes (Figure 1 & 2) and DNA will be extracted from
whole blood using the DNA extraction kit according to the
manufacturers’ instructions. (QiAgen DNA extraction kit).
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
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.
Table 3 shows gender distribution of patients of this study with
the equal distribution of male and female study population (50%
male[n=82] and 50% female[n=82]). Table 4 & 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. Table 5 & 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).
Table 6 & 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) patients had the consanguineous parents of total 164 patients.
Table 10 & 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).
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 . 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 . 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 .
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 et al. and a study conducted in Japan . 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 . 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 . 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. . 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.  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 . Neela PK et al. 
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 .
But the effect of folic acid, a cofactor for MTHFR enzyme action is
protective in some populations Mossey et al.  but ineffective
in some others van Rooji et al. . Lopez cartes et al.  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.  in Eastern India (2019). In contrast study
conducted by Nasri K et al.  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
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.
Wang Wei, Jiao, Hui X, Ping X, Yu X, et al. (2016) MTR, MTRR, and MTHFR Gene Polymorphisms and Susceptibility to Nonsyndromic Cleft Lip with or Without Cleft Palate. J Genetic testing and molecular biomarkers 10: 1089.
Jose BA, Mokhasi V, Subramani SA, Shashirekha M (2020) MTHFR C677T Polymorphism and risk of nonsyndromic cleft in craniofacial region in a South Indian Population. J anatomical society of India 5:10-13.