Sironen et al. BMC Genomics 2010, 11:699 http://www.biomedcentral.com/1471-2164/11/699 Page 5 of 8 SNP DEL Figure 4 Alignment of HECW2 protein sequences at the detected polymorphism positions in various species. The only difference between control (Yorkshire) and KA-affected (Akr) animals was a change from isoleucine to threonine (SNP). However, this change was also present in the human protein sequence. Furthermore, a three aa deletion was identified when compared with the pig reference sequence. addition to these two SNPs, a deletion of nine bp (three aa) was detected after the nucleotide at position 3348 bp (1113 aa) when compared to the porcine reference sequence [Ensemble: ENSSSCG00000016068]. This dele- tion seems to be very common in mammalian species (Figure 4). The porcine reference sequence for HECW2 included exons 3-28 (based on human [GenBank AB037722]), however our sequencing results indicate that exons 2, 29 and 30 are also expressed in the pig testes. Analysis of STK17b mRNA The sequenced testicular mRNA of porcine STK17b [GenBank HM594868] contained exons 1-9 (based on human exon numbering, [GenBank NM_004226]) and 1102 bp. Translation start codon was identified at position 282 within exon 2. No change in the expres- sion profile was identified and the protein coding sequence was identical in the KA-affected and control boar. Discussion While the results of homozygosity mapping of the KAD in a previous study [15] were not statistically significant, they did indicate the most probable positions of the KAD-associated chromosomal segments. In this study we confirmed the association between KAD and porcine chromosome 15. The initial genome screen with micro- satellite markers S0004 and SW2608 showed increased homozygosity in KAD affected boars [15]. The genome scan with PorcineSNP60 Genotyping BeadChip (Illu- mina) localized the KAD associated region between these two markers on porcine chromosome 15. The PorcineSNP60 BeadChip illustrated a high call rate (<95%) in the Finnish Yorkshire pig population and only 15% of the SNPs were monomorphic. In this study we detected the KAD associated region covering 2 Mbp indicating that the marker map in the initial screen was not dense enough to detect the significant increase in homozygosity. Within the associated region we identified and sequenced two candidate genes Ubiquitin-protein ligase E3 (HECW2) and serine/threonine kinase 17b (STK17b). The sequencing of these two genes revealed two SNPs within HECW2 gene, but no polymorphisms were detected in the protein coding sequence of STK17b. Although the identified mutations appeared not to be the causal cause for the KAD, HECW2 remains a good candidate gene for this defect considering its role in acrosome development and chromatin remodelling. Protein ubiquitination is one of the fundamental regula- tory post-translational modifications controlling intracellu- lar signalling events. Ubiquitin-proteosome-dependent proteolysis plays an important role in selectively degrading and recycling proteins in many basic cellular processes including spermatogenesis [26]. For degradation by the proteosome, binding of ubiquitin with substrate proteins requires the activity of ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and substrate-specific ubiquitin ligase E3 [27]. Ubiquitin ligase E3 in combina- tion with an E2 ubiquitin-conjugating enzyme causes the attachment of ubiquitin to a lysine residue on the target protein. In spermatogenesis ubiquitination is required for var- ious processes; for example the replacement of the sper- matids nuclear histones with protamines during spermatid elongation [26]. In spermatozoa, proteosomes are located on the plasma membrane overlying the acro- some, in the acrosomal and postacrosomal regions, in the head-tail connecting-piece, middle-piece of the tail, and residual bodies [28-32]. Proteosome subunit Psmc3 and an ubiquitin protein ligase Rnf19a have been located at the cytosolic side of outer and inner membranes of the acrosome [33]. The co-immunoprecipitation and localization of Psmc3 and Rnf19a in spermiogenesis points to the participation of the ubiquitin-proteosome system in acrosome formation, spermatid head shaping, and development of the head-tail coupling apparatus and tail [33]. Malfunction of components in ubiquitination system has been shown to be a cause of male infertility [27,34-36]. There appears to be a special requirement for certain components of the ubiquitin system during sper- miogenesis, in particular [37], and it is probable that dif- ferent spermatogenic phases would require different specialized activities of the ubiquitin system. Mutations in ubiquitination related proteins may also affect specifi- cally spermatogenesis through their testis specific inter- acting partners [36]. A malfunction of ubiquitination may cause diverse phenotypes as exemplified in the human and mouse by mutation of Hr6b and Usp14 [35,38].