TYMS gene 5′- and 3′-untranslated region polymorphisms and risk of non-syndromic cleft lip and palate in an Indian population

Dear Editor: Increased homocysteine levels due to vitamin B6 or B12 deficiency or genetic defects in folate pathway genes are associated with an increased incidence of non-syndromic cleft lip with or without cleft palate (NSCLP) [1] . Thymidylate synthase (TS) is a folatedependent enzyme that catalyzes methylation of 29-deoxyuridine-59-monophosphate (dUMP) to 29-deoxythymidine-59-monophosphate (dTMP), a rate-limiting step in DNA synthesis, for which 5,10-methylene-tetrahydrofolate (CH2-THF) is the methyl donor. TS competes with 5,10-methylenetetrahydrofolate reductase (MTHFR) for the availability of CH2-THF. The TYMS gene is located on chromosome 18p11.32 and is about 30 kb in length with 7 exons [2] . Two most extensively studied TYMS variants are located in the promoter enhancer region of the 59-untranslated region (UTR) and 39-UTR. The VNTR polymorphism (rs45445694) is located in 59-UTR, consisting of 2 or 3 tandem repeats of 28 bp (2R or 3R). A 6-bp insertion and deletion polymorphism (indel) has been identified in the 39-UTR (rs16430) of the TYMS [3] . These 2 polymorphisms have been extensively studied for association with cleft lip and palate [4] . Although the TYMS plays a critical role in fetal development, so far it has not yet been reported to be associated with NSCLP in the Indian population. In this study, we investigated the effects of TYMS functional variants (rs45445694 and rs16430) on the risks of NSCLP in a southern Indian population. The study was carried out in 283 ethnically matched unrelated subjects, including 142 unrelated NSCLP patients (123 with cleft lip and palate and 19 with cleft palate only) and 141 healthy controls without family history of cleft. Subjects with malformation syndromes and major developmental disorders were excluded. The study was approved by the local institutional ethics committee and written informed consent was obtained from all the participants. Peripheral blood (3 mL) was collected from each subject and DNA was obtained using a standard procedure. TYMS 6-bp indel genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method [5] . TYMS 59-UTR VNTR was genotyped according to the PCR method [6] . Allele frequencies were estimated by the gene counting method. Hardy-Weinberg equilibrium (HWE) was performed to assess the cases and control groups using chi-square test. Association between two TS SNPs and different cleft phenotypes (NSCLP, CLP and cleft palate only (CPO)) was analyzed by x 2 -test. Odds ratio and 95% confidence inter-