Characterization of Phenotype and Col11a1 Expression in Cleft Palate Mice

Objectives: Cleft palate (CP) is a common birth defect occurring in 1 in 2000 live births.  CP can be isolated or associated with a recognized syndrome. This study investigated a transgene (Tg) insertion mouse line (OVE1226b) which demonstrates CP, limb dysplasia, and craniofacial dysmorphology as an autosomal recessive trait.  Localization of the Tg complex and characterization of the phenotype of CP embryos were goals of this study.

Methods: A BAC OVE1226b Tg/+ genomic library was constructed and screened by PCR using primers specific for the Tg element (TYBS minigene).  Skeletal staining, microCT, craniofacial measurements, and RT-PCR were performed on late gestation (17.5 & 18.5dpc) embryos (wildtype, Tg/+, and Tg/Tg).

Results: A BAC clone was identified and end-sequencing performed.  One end contained sequence from the Tg complex and the other an intergenic region approximately 180kb from the 3’ end of Col11a1. CP embryos exhibit micrognathia, tongue protrusion, U-shaped cleft, and rhizomelic shortening of the limbs.  Comparing 17.5dpc CP and wildtype embryos no significant differences were observed for tip of nose to eye, tip of nose to pinna, overall head or crown to rump lengths. For 18.5dpc CP and wildtype embryos significant differences were observed for tip of nose to eye (p<0.001), tip of nose to pinna (p=0.001). No significant differences were observed for overall head or crown to rump lengths. Skeletal clearing and microCT demonstrate reduced skeletal mineralization.  Humeri BMD were less in CP embryos compared to wildtype at 17.5dpc p <0.001 and 18.5dpc p=0.014. RT-PCR of Col11a1 gene expression showed 62-fold downregulation comparing CP to wildtype (p=0.007).

Conclusions: The OVE1226b line recapitulates phenotypic and molecular features paralleling those seen in cho/cho (chrondrodysplasia) mice carrying a mutation in Col11a1. COL11A1 mutations are associated with fibrochondrogenesis, Stickler type II, Marshall syndrome, and some cases of Robin sequence. (NIH/NIDCR DE015180 and institutional funds)