Investigations of the genomic region that contains the clf1 mutation, a causal gene in multifactorial cleft lip and palate in mice

BACKGROUND: Human nonsyndromic cleft lip and palate, CL (P), is genetically complex, with one contributing gene on chromosome 17q. A potentially homologous gene, clf1 on distal chromosome 11, is part of the digenic cause of the 10-30% CL (P) in the A/WySn mouse strain. Here we report our progress toward identifying the clf1 mutation. METHODS: Transcription from all of the known and predicted genes in the 1. 5-Mb candidate region was examined in A/WySn and control (AXB-4/Pgn) ED10-11 embryo heads. The marker haplotype for 28 inbred strains across the clf1 region was obtained. The entire transcripts of Wnt9b and Wnt3 in A/WySn were sequenced. Using long PCR, the genomic region from Wnt3 throughWnt9b was screened in A/WySn for an inserted retrotransposon. RESULTS: Gosr2, Wnt9b, Wnt3, Nsf, Arf2, Crhr1, Mapt, Cdc27, Myl4, Itgb3, chr11₂0. 152, chr11₂0. 154, chr11₂0. 155, and chr11₂0. 156 are expressed in ED10-11 heads. None is absent or detectably reduced in A/WySn. The ancestral pre-clf1 mutation haplotype was found in CBA/J mice. By a test-cross, CBA/J was confirmed to lack the clf1 mutation. Three single-nucleotide variants in A/WySn (vs. C57BL/6J) were found in each of the 3' untranslated regions (3'UTRs) of Wnt3 and of Wnt9b, respectively; their presence in CBA/J shows that none are the clf1 mutation. An inserted intracisternal A particle (IAP) retrotransposon located 6. 6 kb from the 3' end of Wnt9b was found in A/WySn and in all clf1 strains tested. This IAP is absent in C57BL/6J and CBA/J. CONCLUSIONS: The clf1 mutation is a genomic alteration present in A/WySn and absent in the ancestral chromosomal segment in CBA/J. The IAP retrotransposon insertion near Wnt9b in A/WySn fits this criterion; we predict that interference with Wnt9b function by this IAP is the clf1 mutation.