GWAS Follow-up Studies: NTN1 and NOG, New Candidates for NSCL/P

Objective: The orofacial defect cleft lip with or without palate (CL/P), is a common congenital anomaly with environmental and genetic contributors. Several new non-syndromic CL/P (NSCL/P) candidate genes, including NTN1 and NOG, have been identified in the past two years by genome-wide association studies (GWAS). The goal of this study was to replicate the GWAS findings for NTN1 (significant for European and Asian populations) and NOG (significant for Europeans only) and to sequence these genes for possibly etiologic mutations.

Method: Significant SNPs in NTN1 and NOG were genotyped in DNA from 275 Iowan NSCL/P case-parent-trios and 220 Filipino NSCL/P multiplex-families, and analyzed by the Transmission-Disequilibrium Test (TDT) using a family-based association test (FBAT). Direct sequencing was performed for the coding exons of both genes in 304 individuals from Iowa (217 cases, 84 controls) and 180 individuals from the Philippines (89 cases, 91 controls).

Results: Initial FBAT statistics replicated previous results. A p-value of 6 x 10^-3 was obtained for rs9788972 (NTN1) for the Filipinos while the Iowan trios reached near-significance for this marker (p=0.073). In addition, rs227731 (NOG) also reached near-significance (p=0.094) for the Filipinos. Fourteen new mutations were found in NTN1 (5 missense, 2 synonymous, and 7 non-coding). Eight new mutations were found in NOG, all non-coding. Of the missense mutations in NTN1, Y162C and G440S are predicted to be probably damaging by PolyPhen-2 prediction algorithm.

Conclusion: Preliminary statistics replicated the GWAS results while more definitive results are still pending. The discovery of rare variants in these two genes within affected individuals, suggests their potential role in the etiology of NSCL/P, especially the two new missense mutations in NTN1 predicted to have damaging functional effects. Additional investigations are needed to further analyze these genes and the role of their variants.