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A Large Deletion in RPGR Causes XLPRA in Weimaraner Dogs

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A Large Deletion in RPGR Causes XLPRA in Weimaraner Dogs

Regina Kropatsch et al. Canine Genet Epidemiol.

Abstract

Background: Progressive retinal atrophy (PRA) belongs to a group of inherited retinal disorders associated with gradual vision impairment due to degeneration of retinal photoreceptors in various dog breeds. PRA is highly heterogeneous, with autosomal dominant, recessive or X-linked modes of inheritance. In this study we used exome sequencing to investigate the molecular genetic basis of a new type of PRA, which occurred spontaneously in a litter of German short-hair Weimaraner dogs.

Results: Whole exome sequencing in two PRA-affected Weimaraner dogs identified a large deletion comprising the first four exons of the X-linked retinitis pigmentosa GTPase regulator (RPGR) gene known to be involved in human retinitis pigmentosa and canine PRA. Screening of 16 individuals in the corresponding pedigree of short-hair Weimaraners by qPCR, verified the deletion in hemizygous or heterozygous state in one male and six female dogs, respectively. The mutation was absent in 88 additional unrelated Weimaraners. The deletion was not detectable in the parents of one older female which transmitted the mutation to her offspring, indicating that the RPGR deletion represents a de novo mutation concerning only recent generations of the Weimaraner breed in Germany.

Conclusion: Our results demonstrate the value of an existing DNA biobank combined with exome sequencing to identify the underlying genetic cause of a spontaneously occurring inherited disease. Identification of the genetic cause has allowed the development of a diagnostic test, which should help to eradicate the PRA causing mutation from the respective canine line. Thus, planning of future pairings is facilitated and manifestation of this type of PRA can be prevented.

Keywords: Exome sequencing; Progressive retinal atrophy; RPGR (retinitis pigmentosa GTPase regulator) gene; Weimaraner.

Figures

Fig. 1
Fig. 1
Ophthalmoscopic appearance of the central fundus. a Healthy unrelated female Weimaraner dog (~3 years) with a normally appearing fundus (right eye). b PRA-affected male Weimaraner dog (~2.5 years) showing the typical landmarks of PRA with vessel attenuation primarily of the arterioles in the papillary area and a hyperreflective tapetum lucidum (left eye)
Fig. 2
Fig. 2
The deletion in the X chromosomal RPGR gene as identified in a PRA pedigree of Weimaraner dogs via whole exome sequencing; the breakpoint (BP) region is indicated. a Pedigree structure and RPGR deletion genotypes of 18 investigated individuals of the XLPRA Weimaraner family. PRA segregates in two generations of the family. Squares represent males, circles indicate females, crossed-out symbols represent deceased dogs. Filled squares show ophthalmologically diagnosed PRA-affected male dogs. Half-filled circles indicate females with ophthalmologically confirmed mild PRA symptoms. Open symbols represent male and female dogs with normal sight as revealed by general veterinarian examinations, respectively. An asterisk below solid square symbols indicates PRA-diagnosed dogs, which were used for whole exome sequencing. Genotypes of RPGR deletion screening are shown below the symbols. XM (colored in red) refers to an allele with RPGR deletion, X and Y symbols illustrate normal X- (with wildtype RPGR alleles) and Y-chromosomes, respectively. b Integrated Genomics Viewer (IGV) display of the canine RPGR deletion and surrounding regions (CFAX: 3310100–33106500, CanFam3.1 UCSC genome browser) as well as graphical illustration of exon-intron boundaries from the 5′UTR to exon 5. As viewed in IGV, the control and male PRA-affected dog are represented by two separate panels. The upper panel is a histogram where the height of each mountain-like grey area is representative of the read depth at that location. The lower panel is a graphical view of some of the reads that align to that location. Lack of reads (horizontal bars in lower panel) is characteristic for complete loss of exonic sequences. The deletion comprising exons 1–4 (~5 kb) is obvious in the male PRA-affected dog in hemizygous state in contrast to the PRA-unaffected dog. Thus the gap region includes exon 1 in the canine genomic RPGR sequence explaining only non-specific read alignments in the lower panel for the control. c QPCR-based copy number analysis of the deleted exons 3–4 and the non-deleted exon 5 of RPGR gene in four individuals of the pedigree of Weimaraners in comparison to a healthy control. Error bars indicate the standard deviation of three replicates. d Chromatogram and graphical representation of the BP region in the RPGR gene in a male PRA-affected Weimaraner. The graphical illustration indicates part of intron 4 sequence and of 5′UTR of RPGR. Deleted sequences of intron 4 and 5′UTR are coloured in light grey, non-deleted sequences are indicated by coloured letters. The BP region comprises three nucleotides (TTC) from either end which are underlined. The chromatogram also shows the BP (marked with arrows) as well as flanking sequences of intron 4 and 5′UTR of RPGR as identified in a male PRA-affected Weimaraner

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