Objective: To evaluate the association of transforming growth factor β3 ( TGFβ3), muscle segment homeobox 1 ( MSX1), Metalloproteinases 3 ( MMP3), and MMP9 genes as candidates for nonsyndromic cleft lip and/or palate in an Indian population.
Design: Case-control association study, mutational screening, and functional evaluation of obtained mutations.
Setting: Mutational screening of the developmental genes, TGFβ3 and MSX1, along with functional evaluation and association of promoter region SNPs-one each in MMP3 and MMP9.
Patients, participants: Two hundred forty five NSCL±P cases from G. S. Memorial Plastic Surgery Hospital and Trauma Center, Varanasi and 201 healthy controls without a family history of congenital malformations from nearby schools, primary health centers, and the university hospital.
Main outcome measure(s): Sequencing, SSCP, and PCR-RFLP were used for candidate gene screening. MatInspector and electrophoretic mobility shift assay (EMSA) were used to check the differential transcription factor binding of the variants at promoter region. Luciferase assay was used to test the transcriptional potential of the variant, and evaluation of the alternative splice site was carried out using exon-trapping experiment.
Results: Metalloproteinases3 -1171 5A/6A was associated with NSCL±P, whereas MMP9 -1562 C/T did not show association. A rare variant in the promoter region of TGFβ3 (rs117462711) creates a differential binding site, confirmed by EMSA. Luciferase assay showed 3.7-fold increased expression level in mutant construct. A synonymous change in MSX1 (rs34165410) showed association with NSCL±P, which may create an alternative splice site or lead to low codon usage. Exon-trapping experiment failed to confirm alternative splicing, indicating low codon usage frequency of the mutant affecting the gene function.
Conclusions: TGFβ3, MSX1, and MMP3 are candidates for NSCL±P.
Keywords: association; case–control study; synonymous mutation.