A Coding Variant in the Gene Bardet-Biedl Syndrome 4 (BBS4) Is Associated with a Novel Form of Canine Progressive Retinal Atrophy

Abstract

Progressive retinal atrophy is a common cause of blindness in the dog and affects >100 breeds. It is characterized by gradual vision loss that occurs due to the degeneration of photoreceptor cells in the retina. Similar to the human counterpart retinitis pigmentosa, the canine disorder is clinically and genetically heterogeneous and the underlying cause remains unknown for many cases. We use a positional candidate gene approach to identify putative variants in the Hungarian Puli breed using genotyping data of 14 family-based samples (CanineHD BeadChip array, Illumina) and whole-genome sequencing data of two proband and two parental samples (Illumina HiSeq 2000). A single nonsense SNP in exon 2 of BBS4 (c.58A > T, p.Lys20*) was identified following filtering of high quality variants. This allele is highly associated (P_CHISQ = 3.425e^-14, n = 103) and segregates perfectly with progressive retinal atrophy in the Hungarian Puli. In humans, BBS4 is known to cause Bardet-Biedl syndrome which includes a retinitis pigmentosa phenotype. From the observed coding change we expect that no functional BBS4 can be produced in the affected dogs. We identified canine phenotypes comparable with Bbs4-null mice including obesity and spermatozoa flagella defects. Knockout mice fail to form spermatozoa flagella. In the affected Hungarian Puli spermatozoa flagella are present, however a large proportion of sperm are morphologically abnormal and <5% are motile. This suggests that BBS4 contributes to flagella motility but not formation in the dog. Our results suggest a promising opportunity for studying Bardet-Biedl syndrome in a large animal model.

Keywords: Hungarian Puli; blindness; infertility; obesity; whole-genome sequencing.

Figure 1

Positions of SNP array markers that segregate with the PRA phenotype and candidate genes are identified. Concordant markers are indicated in blue. Color opacity describes the density of concordant markers with darker hues corresponding with higher concordant marker density. Candidate genes are depicted in red. The locus with the highest frequency and density of markers is chr30: 25,254,123–39,976,525, with 103 markers and 12 candidate genes residing on the region. Following this is chr4: 556,510–10,473,708 with 61 markers and three candidate genes and chr20: 9,562,689 – 20,226,838 with 60 markers and three candidate genes.

Figure 2

BBS4 protein sequence alignment of affected dogs containing the c.58A > T SNP and of the wild-type protein. The SNP in affected dogs results in a premature stop codon (p.Lys20*). Hyphens (-) refer to missing amino acids in the affected dogs relative to the wild-type protein.

Figure 3

Segregation of the BBS4 SNP (c.58A > T, p.Lys20*) in the Hungarian Puli family. DNA samples were available for all individuals with an identifier (n = 17). PRA is consistent with an autosomal recessive form in this family. Genotypes confirmed through Sanger sequencing represented by unfilled (homozygous wild type A/A), filled (homozygous mutant T/T), or half filled (heterozygous A/T) circles (females) or squares (males) support this mode of inheritance.

Figure 4

Sanger sequencing of a PCR fragment containing the c.58A > T SNP at position chr30: 36,063,748 on CanFam 3.1 in exon 2 of BBS4. The numbers above the amino acid code (S = serine, Q = glycine, K = lysine, P = proline, R = arginine) denote their position in the protein sequence. Nucleotides (W = A or T) are arranged in the 5′ to 3′ direction. (A) All affected Hungarian Puli cases (USCF516, USCF519, and USCF1311) have the T/T genotype which results in a nonsense mutation (p.Lys20*). (B) Carrier individuals including all parents (USCF347, USCF524, and USCF525) have the A/T genotype. (C) Unaffected individuals have the A/A genotype. This is identical to the canine reference sequence (CanFam 3.1 build).