Epigenetic mechanism causes Wnt9b deficiency and nonsyndromic cleft lip and palate in the A/WySn mouse strain.

BACKGROUND:The heritable multifactorial etiology of human nonsyndromic cleft lip with or without cleft palate (CL ± P) is not understood. CL ± P occurs in 15% of neonates in the homozygous A/WySn mouse strain, with a multifactorial genetic etiology, the clf1 and clf2 variant genes. Clf1 acts as a mutant allele of Wnt9b but its coding sequence is normal. An IAP (intracisternal A particle) retrotransposon inserted near the Wnt9b gene is associated with clf1. METHODS:Transcription of noncoding sequence between the IAP and the Wnt9b gene was examined in A/WySn embryos. The levels of Wnt9b transcript and of an "IAP antisense" transcript initiated in the IAP and extending into the noncoding interval were assayed in A/WySn and C57BL/6J whole embryos or heads across embryonic days 8 to 12. Methylation of the 5' LTR of the IAP was examined in E12 A/WySn embryo heads. RESULTS:Mean Wnt9b transcript levels were lower in A/WySn than in C57BL/6J at all ages examined and lower in CL ± P embryos than in their normal littermates. The "IAP antisense" transcript was found in all A/WySn embryos and was highest in CL ± P embryos. The IAP at Wnt9b was generally unmethylated in CL ± P embryos and approximately 50% methylated in normal littermates. CONCLUSION:The clf1 mutation in A/WySn is a "metastable epiallele", in which stochastic deficiency in some individuals of DNA methylation of a retrotransposon uniquely inserted near the Wnt9b gene allows transcriptional activity of the retrotransposon and interference with transcription from Wnt9b. Methylation of metastable epialleles should be investigated in human nonsyndromic CL ± P.

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