A Contracted DNA Repeat in LHX3 Intron 5 Is Associated with Aberrant Splicing and Pituitary Dwarfism in German Shepherd Dogs

Annemarie M W Y Voorbij,Frank G Van Steenbeek,Bernard A Van Oost,Peter A Leegwater,Jeanette M Hanson-Nilsson,Ellen E C P Martens,Hans S Kooistra,Manon Vos-Loohuis

doi:10.1371/journal.pone.0027940

Annemarie M W Y Voorbij, Frank G Van Steenbeek + Show 6 more

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https://doi.org/10.1371/journal.pone.0027940

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Journal: PloS one	Publication Date: Nov 23, 2011
Citations: 65	License type: CC BY 4.0

Affiliation: Utrecht University, American University of the Caribbean

Abstract

Dwarfism in German shepherd dogs is due to combined pituitary hormone deficiency of unknown genetic cause. We localized the recessively inherited defect by a genome wide approach to a region on chromosome 9 with a lod score of 9.8. The region contains LHX3, which codes for a transcription factor essential for pituitary development. Dwarfs have a deletion of one of six 7 bp repeats in intron 5 of LHX3, reducing the intron size to 68 bp. One dwarf was compound heterozygous for the deletion and an insertion of an asparagine residue in the DNA-binding homeodomain of LHX3, suggesting involvement of the gene in the disorder. An exon trapping assay indicated that the shortened intron is not spliced efficiently, probably because it is too small. We applied bisulfite conversion of cytosine to uracil in RNA followed by RT-PCR to analyze the splicing products. The aberrantly spliced RNA molecules resulted from either skipping of exon 5 or retention of intron 5. The same splicing defects were observed in cDNA derived from the pituitary of dwarfs. A survey of similarly mutated introns suggests that there is a minimal distance requirement between the splice donor and branch site of 50 nucleotides. In conclusion, a contraction of a DNA repeat in intron 5 of canine LHX3 leads to deficient splicing and is associated with pituitary dwarfism.

Highlights

A defect in the differentiation of endocrine cells of the pituitary gland can lead to isolated or combined pituitary hormone deficiency (CPHD)
We found that pituitary dwarfism in German shepherd dogs (GSD) is associated with a deletion of a 7 bp repeat in intron 5 of LHX3, a gene encoding another important transcription factor involved in pituitary development
Pituitary dwarfism in German shepherd dogs has been seen for decades and dwarfs are born in purebred populations all over the world

Summary

Introduction

A defect in the differentiation of endocrine cells of the pituitary gland can lead to isolated or combined pituitary hormone deficiency (CPHD). The various cell types of the adenohypophysis arise in a distinct temporal order from progenitor cells. The corticotropic cells are the first to differentiate followed by gonadotropes, thyrotropes, lactotropes and somatotropes [1,2]. Several of the transcription factors involved in the differentiation cascade of the endocrine cells of the pituitary have been identified [3]. CPHD is mostly related to mutations in genes encoding the transcription factors POU1F1 (previously known as PIT1) and PROP1 [4,5,6,7,8,9]. Mutations in other genes that cause CPHD are rare

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