Figure 3. Multispecies alignment of the region around the KDM2B:p.D835N mutation. doi:10.1371/journal.pone.0045634.g003 Due to the recessive inheritance and the lethal effect of the mutation we hypothesized that most likely a loss of function mutation affecting the coding sequence of a gene would be responsible for paunch calf syndrome. Therefore, we subsequently KDM2B Mutation in Cattle concentrated on homozygous variants that were located within the coding sequences or within the splice sites of the annotated genes in the targeted region of the bovine genome. Our genetic data implicates KDM2B as a cause of paunch calf syndrome. Only the identified missense mutation in KDM2B co-segregates with the disease phenotype, and the re-sequencing of the entire paunch calf syndrome locus failed to identify other perfectly associated non- synonymous variants in transcribed sequences. The second non- synonymous variant in RNF34 is most likely functionally neutral as we detected the homozygous mutant RNF34 genotype in three normal control cattle. Unfortunately, there is no experimental crystal structure available to predict the structures of wildtype and mutant KDM2B proteins. The exchange of an acidic residue to an uncharged amino acid with the loss of a negative charge may disrupt the normal structure. Another possible structural effect could be that an aspartic acid residue accepts two hydrogen bonds instead of only one in case of an asparagine. In regard to the close vicinity of the mutant residue to the zinc finger domain of KDM2B the mutation could also affect its role as metal ligand. The KDM2B gene (also known as JHDM1B and FBXL10) encodes a histone H3 lysine 36 dimethyl (H3K36me2)-specific demethylase [9,10]. Histone methylation is one important Figure 4. Pedigree of selected inbred Romagnola cattle. The special structure of livestock families facilitates the mutation identification: The sire A, born 1969, is the assumed founder animal for paunch calf syndrome. The paunch calf syndrome mutation (indicated by an asterisk) most likely occurred in the germline of sire A. Beneath the animals the two copies of BTA 17 are indicated. Black symbols represent the ancestral chromosome, on which the mutation occurred. Gray symbols represent any other wild-type copy of BTA 17. Due to inbreeding loops three animals (cow 326, cow 19, bull 145) have inherited two copies of the same chromosome from their common ancestor (sire A). These three animals were genotyped as heterozygous carriers of the KDM2B mutation. For the cow 19 it is shown that her paternal copy of the critical 1.23 Mb interval is still in its ancestral wild-type state as her father (bull 198) was tested KDM2B clear, while her maternal copy carries the paunch calf syndrome mutation. The BTA 17 haplotypes were confirmed by SNP genotyping of available samples from cow 326, cow 19, bull 198 and bull 145. Based on pedigree and marker data these three animals were identical-by-descent for the critical segment on BTA 17, with the only exception of the causative paunch calf syndrome mutation. doi:10.1371/journal.pone.0045634.g004 PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45634