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Towards finding the genes that cause cleft lip in a multifactorial mouse model Dewell, Sarah Lynne

Abstract

Nonsyndromic cleft lip (with or without cleft palate) is one of the most common birth defects. It occurs in approximately one in a thousand live births, although the frequency varies with geographical location, ethnic background and socioeconomic status. Cleft lip is a multifactorial threshold trait whose etiology includes genetic, environmental and chance factors. Despite many studies on human populations, no candidate gene has been shown conclusively to be involved in the risk of developing the defect. There is also no consensus on the role of any specific environmental factors that have been proposed to increase the risk of cleft lip. The "A" strains of inbred mice are the only known mouse strains with nonsyndromic cleft lip. Cleft lip is genetically complex in the "A" strains, thus providing a good model of the human condition. The mouse model used in this study was A/WySn, which has a frequency of cleft lip of 20-30%. Risk of cleft lip in A/WySn is caused by the combined effects of a recessive mutation (clf1), a semi-dominant mutation (Clf2), and a genetic maternal effect. The recessive mutation in the gene clf1 has been mapped to a 2 cM region on mid-distal mouse chromosome 11. A new backcross panel was generated for this project from the initial cross of A/WySn to AXB-4/Pgn. 70 cleft lip embryos were generated, and polymorphic markers were used to look for recombinants between clf1 and the markers, to confirm the boundaries of the clf1 candidate interval and to try to reduce the size of the interval. Five recombinants were found that confirmed the lower breakpoint of the candidate interval. In addition, using new polymorphic markers from within genes, the new panel placed several genes inside or outside the interval. In the second part of this project, the expression of all of the known candidate genes was examined in adult testis tissue and in Day 10-11 embryo heads. The expression of candidate genes was first examined in adult testis tissue, where a variant was seen in the gene Crhr. This gene was then studied in greater detail. Next, because the critical time of lip formation is Day 10 - Day 11 in the mouse, RT-PCR expression of 9 known genes (Arf2, Itgb3, Crhr, Gosr2, Wnt3, Wnt15, Mapt, Myla, Nsf) and 1 putative gene (KIAA1267) in the clf1 candidate interval was examined in embryonic heads from Day 10 and Day 11 embryos. All 9 known genes are expressed at the critical time in development, while the predicted gene is not. In the third part of this project, 6-9 polymorphic markers were used to compare the "A" strain haplotype in the clfl candidate interval with 37 other inbred strains. The data indicate that the "A" strain shares its haplotype with only one other strain, CBA/J. This finding will be useful in confirming a putative clf1 mutation, when found. In the fourth part of this project, one region of the mouse genome was examined for a third locus contributing to the risk of cleft lip in A/WySn. The region was examined in the new backcross panel for non-random segregation of polymorphic markers; however, no evidence of an association with cleft lip was seen.

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