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, chr11_20.152, chr11_20.154, chr11_20.155, and chr11_20.156 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.
Juriloff
Diana M
DM
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada. juriloff@interchange.ubc.ca
Harris
Muriel J
MJ
Dewell
Sarah L
SL
Brown
Carolyn J
CJ
Mager
Dixie L
DL
Gagnier
Liane
L
Mah
Diana G
DG
D000818:Animals; D002874:Chromosome Mapping; D033481:Chromosomes, Mammalian / Q000235:genetics*; D002971:Cleft Lip / Q000235:genetics*; D002972:Cleft Palate / Q000235:genetics*; D003433:Crosses, Genetic; D018507:Gene Expression Regulation, Developmental; D008040:Genetic Linkage; D008445:Maxillofacial Development / Q000235:genetics; D051379:Mice; D008817:Mice, Mutant Strains; D009154:Mutation; D040641:Quantitative Trait Loci / Q000235:genetics; D014158:Transcription, Genetic