AbstractBACKGROUND: Nonsyndromic cleft lip (CL) with or without cleft palate (CLP) is a common human birth defect with complex genetic etiology. One of the unidentified genes maps to chromosome 17q21. A mouse strain, A/WySn, has CLP with complex genetic etiology that models the human defect, and 1 of its causative genes, clf1, maps to a region homologous to human 17q21. Extensive studies of the candidate region pointed to a novel insertion of an IAP transposon 3′ from the gene Wnt9b as the clf1 mutation. Independently a recessive knockout mutation of Wnt9b (Wnt9b−) was reported to cause a lethal syndrome that includes some CLP. METHODS: A standard genetic test of allelism between clf1 and the Wnt9b− mutation was done. A total of 83 F1 embryos at gestation day 14 (GD 14) from Wnt9b−/+ males crossed with A/WySn females, and 79 BC1 GD 14 embryos from F1 Wnt9b−/clf1 males back‐crossed to A/WySn females were observed for CL. Embryo genotypes at clf1 and Wnt9b were obtained from DNA markers. Genotypes for a second unlinked modifier locus from A/WySn, clf2, were similarly obtained. RESULTS: The compound mutant embryos (Wnt9b−/clf1) had high frequencies of CL: 27% in the F1 and 63% in the BC1. The clf2 modifier gene was found to have 3 alleles segregating in this study and to strongly influence the penetrance of CL in the compound mutant. CONCLUSIONS: The noncomplementation of clf1 and Wnt9b− confirms that clf1 is a mutation of the Wnt9b gene. The homologous human WNT9B gene and 3′ conserved noncoding region should be examined for a role in human nonsyndromic CLP. Birth Defects Research (Part A) 76:574–579, 2006. © 2006 Wiley–Liss, Inc.