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Nonsyndromic cleft lip with or without cleft palate is a common birth defect with substantial clinical and social impact, and whose causes include both genetic and environmental factors. Folate and homocysteine (Hcy) metabolism have been indicated to play a role in the etiology of NSCLP.
Objective: The aim of this study was to determine the prevalence of NOS3 27-bp VNTR and to evaluate whether this polymorphism contributed to the risk of NSCLP.
Material and methods: We studied the 27 base pair tandem repeat polymorphism in intron 4 of the endothelial nitric oxide synthase (NOS3) gene in 230 unrelated individuals belong to 7 Indian populations along with 141 NSCLP cases and 142 unrelated controls. The genotyping was performed by polymerase chain reaction and electrophoresis. The data were statistically analysed using the χ2-test.
Results: The NOS3 27-bp VNTR 4a allele is present in six of the seven populations analyzed and allele frequencies range from 6.8% in Sugali to 23.5% in Madiga populations. NOS3 showed protective association with predisposition towards NSCLP for the heterozygous (4b/a) genotypes (4b/b vs. 4b/a: OR =0.58, 95% CI =0.34 to 0.99, p=0.044).
Conclusions: The current study suggests significant differences in the frequency of the NOS3 VNTR allele across the populations. There is protective association between NOS3 27-bp VNTR polymorphism and NSCLP in the Indian population.
Our perception on the etiology of nonsyndromic cleft lip with or without cleft palate is relatively poor because it is a complex multi-factorial trait, exhibiting varying levels of penetrance, gender disparities and environmental overlays involved in its pathogenesis . Nonsyndromic cleft lip and palate (NSCLP) is multifactorial trait and may be influenced by the additive effects of several genes corresponding to the pathways that regulate transcription factors, growth factors, cell signaling, folate and detoxification metabolisms [2, 3]. Several studies have shown a strong association between homocysteine and nitric oxide (NO) synthesis [4-6]. Methionine synthase converts cellular homocysteine to methionine and is a major enzyme in the biosynthetic pathways for folates, S-adenosylmethionine and biological methylations. Nitric oxide-induced inactivation of methionine synthase alters the levels of homocysteine  and could therefore provide a connection between the plasma homocysteine and orofacial clefts. The gene coding for the nitric oxide synthase (NOS3) is located on chromosome 7q36, spans 21 kb, and contains 26 exons that encode 1203 amino acid, 133-kDa protein. The exon 7 Glu298Asp missense variant and the intron-4 27-base pair (bp) variable number of tandem repeat (VNTR) polymorphism of NOS3 are postulated to be associated with altered eNOS function, leading to impair NO synthesis [8, 9].
Since its identification several studies have been designed to evaluate associations between NOS3 intron4b/a genotypes and essential hypertension , coronary risk , osteomyelitis , sepsis , diabetic retinopathy  but the results were inconsistent because of phenotypic heterogeneity, population stratification and the interaction other risk factors. As the elevated homocysteine levels are involved in the impaired palatogenesis, the present study was aimed to investigate the association between cleft lip and palate and NOS3 intron4b/a genotypes. The ethnic variations in the NOS3 27-bp VNTR in South Indian populations was also examined.
MATERIALS AND METHODS:
A total of 230 unrelated individuals belong to 7 Indian populations and additional 141 cases with NSCLP and 142 unrelated control individuals without clefts were included in this study. The populations samples included from seven ethnic groups of south India were apparently normal healthy volunteers. The subjects with nonsyndromic clefts and unrelated control individuals without clefts or family history of clefting, were ascertained at Sri Ramachandra cleft and craniofacial centre, Sri Ramachandra University, Chennai, India. To determine their individual cleft status all the subjects with clefts were examined clinically twice by two surgeons independently and also through their medical records. All the subjects in the case group are isolated nonsyndromic clefts, and none of them is presented with other congenital malformations or major developmental disorders. The study was approved by the Institutional Ethics Committee of Sri Ramachandra University, Chennai, India. As many of the children are under 15 years of age, consent was requested from their parents. Three ml of blood sample was collected from all the participants after obtaining the informed consent. Genomic DNA of the above samples was isolated by standard protocols with phenol-chloroform extraction and ethanol precipitation . Genotyping for NOS3 27-bp VNTR polymorphism was performed following published PCR-based methods . Amplifications were performed in a 10-µl volume containing 2x Ampliqon master mix, 1 pmol of forward primer, one pmol of reverse primer, and 40 ng of genomic DNA. All PCR products were examined by agarose gel electrophoresis, and the genotypes were scored by two researchers independently to minimize error. Allele frequencies were determined by direct counting of alleles at each locus. The genotype distribution for each site in each group was evaluated for Hardy-Weinberg's equilibrium (HWE). The observed and expected genotype distributions and allele frequencies were computed using the HWSIM program . The association between NOS3 27-bp VNTR polymorphism and NSCLP was analysed using χ2-test. Odds ratios and 95% CI were calculated. All statistical analyses were performed with SPSS statistical software version 17.0 (SPSS Inc, Chicago, Illinois) for Windows.
Population specific frequencies and counts of NOS3 27-bp VNTR genotypes among different populations were shown in Table 1. The NOS3 4a allele is present in six of the seven populations analyzed and allele frequencies range from 6.8% in Sugali to 23.5% in Madiga populations. Genotype distributions are following Hardy Weinberg equation in all populations. The NOS3 4a allele frequencies that collected from various studies conducted on several human populations were presented in table 1. Data showed significant differences in the frequency of the NOS3 4a allele across the populations. The NOS3 27-bp VNTR genotypes are following the Hardy-Weinberg equilibrium in control group as well as in NSCLP group (Table 2). The NOS3 VNTR mutation was found in 49 of 141 control subjects and in the NSCLP group 35 of 142 subjects. The NOS3 4a allele frequencies are 18.4% and 13.7% respectively for control and NSCLP group. Analysis pertaining to the assessment of risks associated with individual mutant genotypes of the NOS3 VNTR with regards to risk of NSCLP depicted protective association with predisposition towards NSCLP for the heterozygous genotypes (4b/a) (4b/b vs. 4b/a: OR =0.58, 95% CI =0.34 to 0.99, p=0.044). The NOS3 VNTR did not show significant association with NSCLP at allele level (4b vs. 4a: OR = 0.70, 95% CI =0.45-1.11, p = 0.127) (Table 2).
An analysis of 7 populations has shown significant difference in allele frequencies among populations. The NOS3 27-bp VNTR 4 repeat allele occurs more frequently among Africans than the Europeans and other populations . The current study suggests that NOS3 27-bp VNTR is associated with nonsyndromic cleft lip with or without cleft palate in the Indian population.
Several polymorphisms in the NOS3 gene may be associated with reduced eNOS activity and basal NO production . The 27-bp VNTR has been associated with variations in plasma levels of nitric oxide and its metabolites [20, 21]. The association between eNOS mRNA levels and the 4-repaet allele appeared to be dose-dependent . The 27-bp VNTR plays a cis-acting role in NOS3 promoter activity and inhibits eNOS expression [23, 24]. Endothelial cells containing the 4-repeat (4a) allele produce a higher level of eNOS mRNA compared with cells containing more common 5-repeat (4b) allele . Till date NOS3 27-bp VNTR was not studied for cleft lip and palate, but several other polymorphisms were analysed to assess the association between NSCLP and NOS3 gene. Analysis of NOS3 -922A>G homozygotes showed a 60 percent increased risk of cleft lip and palate but not the cleft palate only . In non-Hispanic white group the interaction between CRISPLD2 and NOS3 polymorphisms altered the transmission of NOS3 alleles and exhibited association with NSCLP . The interaction between NOS3 and other folate gene polymorphisms in risk of NSCLP varies in Hispanic and non-Hispanic populations . Unfortunately our study has not included the other polymorphism of NOS3 genes to validate the above findings.
In conclusion, our results support the involvement of NOS3 27-bp VNTR polymorphisms in NSCLP. Although, limited data that available on folate and homocysteine metabolism would not support their direct role in NSCLP pathogenesis. As maternal hyperhomocysteinemia is a key risk factor in the aetiology of oral clefts and folate supplementation is the most important matter in prevention of oral clefts, it is very important to study interaction of folic acid or 5-MTHF in production of nitric oxide from endothelial nitric oxide synthase.
The authors declare that they have no competing interests.
LVKSB: designed research and wrote the manuscript; JM, GV, LS, PRM and MR: conducted experiments and analyzed data; LVKSB: had primary responsibility for the final content; and all authors: read and approved the final manuscript.
L.V.K.S. Bhaskar acknowledges funding from the Indian Council of Medical Research (ICMR), Government of India (Project Ref. No. 56/15/2007-BMS).