A LINE-1 Insertion in DLX6 Is Responsible for Cleft Palate and Mandibular Abnormalities in a Canine Model of Pierre Robin Sequence

Cleft palate (CP) is one of the most commonly occurring craniofacial birth defects in humans. In order to study cleft palate in a naturally occurring model system, we utilized the Nova Scotia Duck Tolling Retriever (NSDTR) dog breed. Micro-computed tomography analysis of CP NSDTR craniofacial structures revealed that these dogs exhibit defects similar to those observed in a recognizable subgroup of humans with CP: Pierre Robin Sequence (PRS). We refer to this phenotype in NSDTRs as CP1. Individuals with PRS have a triad of birth defects: shortened mandible, posteriorly placed tongue, and cleft palate. A genome-wide association study in 14 CP NSDTRs and 72 unaffected NSDTRs identified a significantly associated region on canine chromosome 14 (24.2 Mb-29.3 Mb; p(raw)=4.64x10(-15)). Sequencing of two regional candidate homeobox genes in NSDTRs, distal-less homeobox 5 (DLX5) and distal-less homeobox 6 (DLX6), identified a 2.1 kb LINE-1 insertion within DLX6 in CP1 NSDTRs. The LINE-1 insertion is predicted to insert a premature stop codon within the homeodomain of DLX6. This prompted the sequencing of DLX5 and DLX6 in a human cohort with CP, where a missense mutation within the highly conserved DLX5 homeobox of a patient with PRS was identified. This suggests the involvement of DLX5 in the development of PRS. These results demonstrate the power of the canine animal model as a genetically tractable approach to understanding naturally occurring craniofacial birth defects in humans. Author Summary Cleft palate is one of the most commonly occurring birth defects in children, and yet its cause is not completely understood. In order to better understand cleft palate we have turned to man's best friend, the domestic dog. Common breeding practices have made the dog a unique animal model to help understand the genetic basis of naturally occurring birth defects. A genome-wide association study of Nova Scotia Duck Tolling Retrievers with naturally occurring cleft palate led to the investigation of two homeobox genes, DLX5 and DLX6. Dogs with this mutation also have a shortened lower jaw, which resembles those who have Pierre Robin Sequence (PRS). Investigation into people with PRS identifies a mutation within a highly conserved and functional region of DLX5 that may contribute to the development of PRS. This exemplifies how the dog will help us better understand common birth defects.