Alpha Omega Alpha Honor Medical Society

2010 Research Abstract

Genetic defects causing non-syndromic cleft lip and palate in CL/Fr mice

Investigator: Felix H. T. Lui, University of Hawaii at Manoa John A. Burns School of Medicine
Mentor: Scott Lozanoff, PhD, Department of Anatomy, Biochemistry and Physiology, University of Hawaii School of Medicine

Birth defects are the leading cause of infant mortality in Hawaii. In Hawaii, the incidence of oral facial clefts is 1 in 524 births, with higher incidence among Hawaiians, Samoans, Filipinos, Japanese, Chinese, Koreans and Vietnamese. However, little evidence exists concerning the genetic mechanisms causing CLP. Linkage studies in human families are very difficult since 70% of CLP cases do not occur as part of an inherited syndrome. Thus, mouse models have been utilized as a valuable source of elucidating craniofacial development since early facial development in the mouse shows striking similarities to the human. We currently have a unique mouse model called CL/Fr that demonstrates CLP at a much higher incidence (35%) than that observed in the background "A" strain mouse (4%) that are used to study CLP, and we are the sole laboratory worldwide with an established, working colony. Using classical mouse breeding strategies, it has been shown that at least two disease loci are involved in the defect while gene targeting analyses strongly imply a role of wnt9b in the disease process. Wnt9b is typically present in the facial prominence fusion area (nasal fin), and its absence in the wnt9b knockout mouse leads to a CLP phenotype. The specific aims of this project are to: 1) to perform a comparative genomic hybridization (CGH) analysis of the CL/Fr genome to ascertain whether additional disease loci might be present. 2) to sequence the IAP transposon, which has been identified 6kB downstream of the Wnt9b gene, to evaluate if sequence differences between the "A" strain and CL/Fr exist that may account for the different incidence of cleft lip and palate 3) ascertain whether there are modifications in the structure of the IAP transposon and determine whether the methylation pattern of the transposon affect CLP formation. Although CL/Fr showed numerous differences compared to "A" strain, no candidate loci were revealed with CGH. Sequencing of the Wnt9b gene and the IAP transposon revealed no sequence differences between substrains of the "A" strain and CL/Fr exist that may account for the different incidence of cleft lip and palate. Thus, Heritable cleft lip and Palate (CLP) is more likely associated with a modifier gene and/or altered methylation of the the IAP transposon that reduces expression of the transcription factor wnt-9b in the CL/Fr mouse model. We have found CpG Islands, genomic with higher chances of methylation, with loose parameters on 5' Wnt9b (AL596108); 3' Wnt 9b and transposon sequence. Loose parameters found 2 CpG islands within the Wnt9b gene; 4 CpG islands within transposon, 1 CpG island between transposon and gene. The the CpG Islands were PCR amplified with all sequences verified, pending one. We are currently looking to compare the methylation patterns of Cl/Fr and A strain using the EZ DNA methylation kit, Zymo Research. The results of this proposed study will provide important information that will provide a better understanding of this very common disease in human populations, an action critical for developing diagnostic, therapeutic and preventive strategies.

Supported, in part, by 1R01-DK-064752 (SL) and an Alpha Omega Alpha Carolyn L. Kuckein Student Research Fellowship.

Updated on January 6, 2011.

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