Confirmation of a non-synonymous SNP in PNPLA8 as a candidate causal mutation for Weaver syndrome in Brown Swiss cattle

Abstract

Background: Bovine progressive degenerative myeloencephalopathy (Weaver syndrome) is a neurodegenerative disorder in Brown Swiss cattle that is characterized by progressive hind leg weakness and ataxia, while sensorium and spinal reflexes remain unaffected. Although the causal mutation has not been identified yet, an indirect genetic test based on six microsatellite markers and consequent exclusion of Weaver carriers from breeding have led to the complete absence of new cases for over two decades. Evaluation of disease status by imputation of 41 diagnostic single nucleotide polymorphisms (SNPs) and a common haplotype published in 2013 identified several suspected carriers in the current breeding population, which suggests a higher frequency of the Weaver allele than anticipated. In order to prevent the reemergence of the disease, this study aimed at mapping the gene that underlies Weaver syndrome and thus at providing the basis for direct genetic testing and monitoring of today's Braunvieh/Brown Swiss herds.

Results: Combined linkage/linkage disequilibrium mapping on Bos taurus chromosome (BTA) 4 based on Illumina Bovine SNP50 genotypes of 43 Weaver-affected, 31 Weaver carrier and 86 Weaver-free animals resulted in a maximum likelihood ratio test statistic value at position 49,812,384 bp. The confidence interval (0.853 Mb) determined by the 2-LOD drop-off method was contained within a 1.72-Mb segment of extended homozygosity. Exploitation of whole-genome sequence data from two official Weaver carriers and 1145 other bulls that were sequenced in Run4 of the 1000 bull genomes project showed that only a non-synonymous SNP (rs800397662) within the PNPLA8 gene at position 49,878,773 bp was concordant with the Weaver carrier status. Targeted SNP genotyping confirmed this SNP as a candidate causal mutation for Weaver syndrome. Genotyping for the candidate causal mutation in a random sample of 2334 current Braunvieh animals suggested a frequency of the Weaver allele of 0.26 %.

Conclusions: Through combined use of exhaustive sequencing data and SNP genotyping results, we were able to provide evidence that supports the non-synonymous mutation at position 49,878,773 bp as the most likely causal mutation for Weaver syndrome. Further studies are needed to uncover the exact mechanisms that underlie this syndrome.

Fig. 1

Identification of a candidate causal mutation at position 49,878,773 bp on BTA4. a Results of the combined linkage/linkage disequilibrium mapping approach. Colored dots indicate the positions of the six microsatellite markers (RM188, MAF50, RM067, TGLA116, BM1224, BM6458) of the indirect genetic test for Weaver syndrome. A maximum peak value (LRT = 73.9) was detected between microsatellite markers RM188 and MAF50 at position 49,812,384 bp. The 2-LOD drop-off method used to determine the corresponding confidence interval sets its boundaries at positions 49,514,652 and 50,367,484 bp. b Detailed overview of the confidence interval between 49,514,652 and 50,367,484 bp. Six genes (NRCAM, PNPLA8, NME8, SFRP4, EPDR1, STARD3NL), two novel microRNA genes (ENSBTAG00000045095, ENSBTAG00000044255) and two uncharacterized protein coding genes (ENSBTAG00000014795 and ENSBTAG00000035945) are located within this confidence interval. The vertical blue line indicates the position of the maximum peak value (LRT = 73.9, 49,812,384 bp). The vertical red line indicates the position of the candidate causal mutation at position 49,878,773 bp (rs800397662), which was identified by analyzing whole-genome sequence data. c Detailed structure of the PNPLA8 gene that carries the candidate causal mutation. Bold sections represent exons, thin sections represent introns. The vertical red line indicates the position of the candidate causal mutation. d The 41-bp DNA sequence that harbors the candidate causal mutation. Red letters between brackets represent the SNP at position 49,878,773 bp (C: reference allele, T: alternative allele)

Fig. 2

Multi-species alignment of the PNPLA8 protein sequence. The red bar highlights the serine residue at position 568 of PNPLA8 which is conserved throughout eukaryotes. The grey bar highlights the Bos taurus reference sequence. (NCBI HomoloGene, http://www.ncbi.nlm.nih.gov/homologene, Accessed 25 Aug 2015)

Fig. 3

Familial relationships, genotypes and haplotype structure of animal W0277 and its progeny. Haplotypes in red represent the common Weaver haplotype identified by homozygosity mapping (48,688,283–50,412,884 bp), haplotypes in green represent the haplotype that was passed down from animal W0380 to W0277 and inherited by its progeny and haplotypes in grey represent all haplotypes that were inherited from non-carrier and non-affected ancestors. Letters in rectangles represent the animals’ genotypes for the SNP at position 49,878,773 bp (rs800397662) with C as the reference allele and T as the alternative allele