Table 1. Genotyping results for RNF34 and KDM2B mutations. 65 3 A/A 65 RNF34 genotype (c.704–716del) del/del del/wt wt/wt KDM2B genotype (c.2503G.A) 56 172 AG 56 150 1b 291 288 GG 1b 316 288 Cases Parentsa Romagnola controls (n = 466) Other breed controls (n = 288) Cases Parentsa Romagnola controls (n = 466) Other breed controls (n = 288) (n = 65) (n = 57) (n = 65) (n = 57) aCattle recorded as parent of affected calves. bFor this cow only one single suspicious offspring was recorded by the breeding organization and the diagnosis of this calf had not been confirmed by necropsy. Genotyping of additional flanking markers revealed that this animal did not carry the disease-associated haplotype. Therefore, the reported calf most likely represented a phenocopy and the parent is indeed free of the deleterious mutation. doi:10.1371/journal.pone.0045634.t001 transcription regulatory system that affects mammalian develop- ment and cell differentiation [11]. During the embryonic and fetal differentiation, chromatin modifications are important to drive the correct temporal expression of specific genes. Transcription factors act in concert with histone acetylatases and methylases to influence gene transcription by modifying the architecture of chromatin at specific loci [12]. Histone methylation had been thought of as an irreversible epigenetic mark until the first lysine specific histone demethylase LSD1 (also known as KDM1) was discovered [13]. Subsequent to the discovery of LSD1, another larger family of more than 30 histone demethylases sharing a motif designated the Jumonji C (JmjC) domain, structurally different from LSD1 but including KDM2B, was described [10]. This enzyme catalyzes the removal of methyl groups from a specific lysine residue on histone H3 and is capable of suppressing cellular senescence [9]. Recently, it was shown that KDM2B acts in concert with other key reprogramming factors in early gene activation and promotes induced pluripotent stem cell generation from somatic cells [14]. To our knowledge, no human patients with KDM2B mutations have so far been identified. Several other JmjC-domain-containing histone demethylases have been implicated in disease including prostate cancer [15] and X-linked intellectual disability [16+17]. Kdm2b-deficient mice exhibit failure of neural tube closure, resulting in exencephaly and death shortly after birth [18]. These recent findings in mice suggest that the KDM2B gene is involved in neural development. Our report indicates that KDM2B function is also important for the development of endo- and mesodermal derived organs like bone and liver. In conclusion, our results suggest that the KDM2B c.2503G.A variant represents the causative mutation for paunch calf syndrome in Romagnola cattle. Especially, the genetic makeup of three inbred cattle provides extremely strong support for the causality of this mutation. Selection against this mutation can now be used to eliminate paunch calf syndrome from Romagnola cattle in production systems. Furthermore, we discovered a hint for a possible essential role of this histone demethylase during the KDM2B Mutation in Cattle development of endo- and mesodermal organs. This study highlights the potential of naturally-occurring mutants in domestic animals to gain further insights into gene functions. Materials and Methods Ethics Statement All animal work has been conducted according to the national and international guidelines for animal welfare. There is no permit number as this study is not based on an invasive animal experiment. All cattle owners agreed that the samples can be used for our study. The data were obtained during diagnostic procedures that would have been carried out anyway. This is a very special situation in veterinary medicine. As the data are from client-owned cattle that underwent veterinary exams, there was no ‘‘animal experiment’’ according to the legal definitions in Italy. Animals We collected samples from 65 paunch calf syndrome affected calves (35 male, 30 female) from different farms. For most of the cases detailed pedigree records were available and their relation- ship is illustrated in Figure S1. The phenotype classification was primarily based on the owners’ reports. Most of the affected animals were seen by a veterinarian and for some cases necropsy results were available. Additionally, we collected 57 samples recorded as parents (14 sires, 43 dams) of affected offspring and 466 healthy Romagnola cattle (352 male, 114 female) resulting in a total of 588 samples from this breed. For the association study we also used 288 normal cattle from 25 genetically diverse Bos taurus breeds. Genome Wide Association Mapping Genotyping was performed using the BovineHD BeadChip (Illumina), including 777,962 evenly distributed SNPs on the 29 bovine autosomes and X chromosome (median distance between markers: 2.68 kb), and standard protocols recommended by the manufacturer. A total of 241,791 SNPs were excluded based on genotyping rate (call frequency ,80%; 5,949 SNPs) and minor allele frequency (MAF ,10%; 237,307 SNPs). For single-marker association studies the genotype frequencies at 536,171 SNPs were compared between cases and controls using a standard allelic association test implemented with PLINK [19]. Genome-wide corrected empirical p-values were determined applying the max(T) permutation procedure implemented in PLINK with 500,000 permutations. To identify extended homozygous regions with allele sharing across all affected animals the options –homozyg- group and –homozyg-match were applied. All given positions correspond to the UMD3.1 cattle genome assembly [8]. The corresponding human chromosome segments (build 37.2) were identified by BLASTN searches of bovine SNP flanking sequences to the human genome sequence. Sequence Capture Enrichment DNA fragmentation was conducted using a microTube in a Covaris S2 instrument, resulting in 100 to 150 bp average fragment length at the sample peak, as monitored using an Agilent Bioanalyzer. Ends were repaired using a DNA end polishing enzyme mixture (Agilent). Double stranded custom adaptors containing barcode sequences (sequences available upon request; Agilent) were ligated to the blunt ended genomic DNA fragments using T4 DNA ligase (Agilent). Resulting fragments were purified by elution from E-gel (Invitrogen) to enrich approximately 200 bp length fragments. These fragments were nick translated and amplified by PCR using primers specific for PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45634