The quite uniform phenotype and a high degree of inbreeding suggested the existence of a single recessive founder mutation. Pedigree analysis indicated that all paunch calf syndrome cases with available pedigree records traced back to a once popular Romagnola sire born in 1969, between four to eight generations on both, the paternal and maternal side (Figure S1). Therefore, we applied an identical by descent mapping approach to fine-map the region containing the paunch calf syndrome mutation. Based on the pedigree records we hypothesized that the affected calves most likely were inbred to one single founder animal (Figure S1). Under this scenario the affected individuals were expected to be identical by descent (IBD) for the causative mutation and flanking chromosomal segments. To identify IBD regions between paunch calf syndrome cases we analyzed the cases for extended regions of homozygosity with simultaneous allele sharing. Only the disease- associated genome region on BTA 17 fulfilled our search criteria. All 16 cases were homozygous for the same disease-associated haplotype and shared identical alleles over 341 SNP markers corresponding to a 1.23 Mb interval (55,880,892–57,114,451 bp) on BTA 17 (Figure 2C). This cattle chromosome region contains 24 genes and loci according to the annotation of the corresponding human chromosome (HSA) 5 intervals (Figure 2D). A Missense Mutation in the KDM2B Gene is the Most Likely Causative Mutation To identify the mutation associated with paunch calf syndrome, we used a targeted sequence capture approach to re-sequence the critical 1.23 Mb interval in one affected calf. Sequencing coverage was limited to approximately 55% of the target region, as repetitive elements were masked during target enrichment probe design. Alignment of sequence reads showed that we obtained 98.6% of the single-copy sequences of the targeted BTA 17 interval, with 95.1% of the region covered by at least 50 high- quality reads. We mapped 17,986,641 sequences to the target region and a total of 8,030,795 sequence reads (44.6%) were reliably mapped to the target region with a mapping quality .20 resulting in a mean coverage of 406 for the probe design region. In total, 95% (87.4% with mapping quality .20) of the coding bases of the exome defined by UMD3.1 cattle genome annotation [8] were represented by at least 50 reads per base. Within the critical interval, we identified 400 homozygous sequence variants (382 SNPs and 18 indels) in comparison to the reference cattle genome sequence (Table S1). Visual inspection of the read-pairs revealed no indication for structural variants spanning coding regions. To distinguish potentially pathogenic mutations from other variants, we focused only on homozygous variants located in coding and splice site sequences, anticipating that variants in non-coding sequences were less likely to be pathogenic. Applying this filter, we identified two non-synonymous coding variants that were associ- ated with paunch calf syndrome. These were a 12 bp deletion (c.704–716del) in the ring finger protein 34, E3 ubiquitin protein ligase gene (RNF34) leading to a truncated stretch of aspartic acid residues, and a SNP (c.2503G.A) in the lysine (K)-specific demethylase 2B gene (KDM2B) causing an aspartic acid to asparagine substitution (p.D835N) (Figure S2). The c.2503G.A transition is located in KDM2B exon 18 and the encoded residue is located between the CXXC zinc finger and F-box domains of the KDM2B protein in a region of very high sequence conservation (Figure 3). Both polymorphisms were further investigated by genotyping additional cases, parents and unrelated controls using Sanger sequencing (Figure S1, Figure S2). We found perfect concordance between the presence of the KDM2B c.2503G.A variant and the paunch calf syndrome phenotype (Table 1). Only one reported KDM2B Mutation in Cattle paunch calf syndrome carrier yielded a discordant genotyping result and was homozygous for the wildtype allele, which is most likely due to a phenotyping error of the offspring (Table 1). The RNF34 deletion was not perfectly associated with paunch calf syndrome was thus excluded (Table 1). A total of 3 out of 255 Romagnola controls were genotyped as homozygous mutant for the RNF34 deletion and should by implication be affected if that were the causative mutation, but they were only heterozygous for the KDM2B SNP. Interestingly, these 3 animals were inbred to the assumed paunch calf syndrome founder (Figure 4). We performed SNP array genotyping of these individuals and detected that they were homozygous for all tested markers in the critical interval on BTA 17. In other words their SNP array genotypes in the critical region were identical to the SNP array genotypes of paunch calf syndrome cases. None of 523 healthy Romagnola cattle had the homozygous KDM2B A/A genotype (Table 1). The KDM2B variant was absent in all tested non-Romagnola cattle. It had an allelic frequency of 16% amongst 466 healthy Romagnola individuals that were not related to cases at the parent level. We carefully checked the available pedigree records of genotyped carriers and in all A/G animals we were able to identify at least one line of relationship to the assumed founder. The two oldest genotyped carriers were a grandson and a great-grandson of this sire, respectively (Figure S1). Discussion Using a genome-wide association study approach followed by targeted re-sequencing, we have identified the KDM2B c.2503G.A variant as most likely causative mutation for the lethal multi-organ developmental dysplasia in homozygous mutant animals. We exploited the special structure of cattle families to identify the causative mutation by a purely genetic approach. The rich pedigree records in cattle breeding allowed us to identify the possible founder animal of paunch calf syndrome, a Romagnola bull born in 1969. A few generations later several cattle received two copies of the same chromosome segment from this bull due to inbreeding. We hypothesize that this sire was a germline mosaic as he transmitted copies of the BTA 17 chromosome which acquired the causative mutation, while the other copies stayed in the ancestral state (Figure S1; Figure 4). Three inbred animals, which were IBD for all tested markers across the critical interval, were heterozygous at the KDM2B mutation. The recognition of these animals was a key element in our discovery and illustrates once again the potential of livestock specific population structures for genetic research [6]. In addition, we detected mutation carriers born two or three generation after the assumed founder, which provide additional support for the hypothesis of the origin of the causative mutation. The DNA-based test for the detection of paunch calf syndrome carriers test been already adopted by the Italian Romagnola Breeders’ Association and all the top sires indexed in the annual catalogues are identified as to whether they are carriers of the paunch calf syndrome causative mutation. Since summer 2011, there has been a requirement for young bulls short- listed for entrance into the performance testing program to be tested for the mutation; carriers are excluded from the programme. The 32% carrier frequency within the examined Romagnola population is very high compared to other recessive traits we studied [6+7]. This might explain the recently observed outbreak of the paunch calf syndrome disease due to intensive usage of a limited number of artificial insemination sires and subsequent increase of inbreeding level. PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45634