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

Zena T Wolf,Claire M Wade,Nili Karmi,Zhonglin Jia,Kartika Jayashankar,Boaz Arzi,Saundra Sliskovic,Douglas J Rowland,Elizabeth J Leslie,Noa Safra,Danika L Bannasch,Jeffrey C Murray,Amy Young,Gregory S Barsh

doi:10.1371/journal.pgen.1004257

Abstract

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; praw = 4.64×10−15). 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.

Highlights

Cleft palate (CP) is one of the most commonly occurring craniofacial birth defects, affecting approximately 1 in 1500 live human births in the United States [1]
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, distal-less homeobox 5 (DLX5) and distal-less homeobox 6 (DLX6)

Summary

Introduction

Cleft palate (CP) is one of the most commonly occurring craniofacial birth defects, affecting approximately 1 in 1500 live human births in the United States [1]. Children born with CP may develop hearing loss, difficulties with speech and eating, and may be at an increased risk for psychiatric disorders and neurological deficits [2,3,4]. CP occurs when there is a failure in the formation of the secondary palate, which makes up all of the soft palate and majority of the hard palate. Secondary palate development is conserved across mammalian species and proceeds through highly regulated sequential steps: palatal shelf growth, elevation, fusion, and cell differentiation (reviewed in [5]). Disruptions in any of these pathways may cause a cleft palate and lead to the phenotypic spectrum of CP cases that is observed. CP may occur alone (nonsyndromic) or with other abnormalities (syndromic)

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Conclusion