Sironen et al. BMC Genomics 2010, 11:699 http://www.biomedcentral.com/1471-2164/11/699 Conclusions In this study we demonstrate the exact KAD phenotype in mature sperm. In addition to the acrosome, the sper- matid chromatin is also affected. We have located the homozygous region for KAD within 0.5 Mbp on porcine chromosome 15 containing two genes STK17b and HECW2. The role of ubiquitination in chromatin remo- delling and acrosome formation is consistent with HECW2 being involved in this defect. While a causal mutation for KAD was unable to be identified, our results indicate that the observed phenotype may be caused by a malfunction in the ubiquitination system. Identification of the causal variation for the KAD requires further ana- lysis of the genomic region containing the HECW2 gene. Methods Animal material Experimental material included 14 Finnish Yorkshire boars affected with KAD and 21 control boars. All affected boars were clinically examined and shown to display symptoms typical of the syndrome, but no other abnorm- alities. Sperm from affected and control boars was collected and the DNA obtained following phenol/chloro- form extraction. Samples were diluted to 100 ng/μl in TE- buffer and used as templates for PorcineSNP60 Genotyp- ing BeadChip (Illumina). Genomic DNA was also used for sequencing of the HECW2 exon 1 and SNP1. For microscopical examination representative semen samples from KA-affected and control boars were fixed in formaldehyde for confocal laser scanning and trans- mission electron microscopy analyses. Confocal laser scanning microscopy Spermatozoa were labeled with LIVE/DEAD Reduced Bio- hazard Viability kit (red, L23102, Invitrogen). The labelling protocol was in accordance with the recommendations of the manufacturer. In brief, 50 μl DMSO was added to one vial of fluorescent dye and thoroughly mixed to make a stock solution. Spermatozoa were suspended in PBS at approximately 1 × 106/ml. One μl of fluorescent dye was added to the suspension. After 30 min incubation at room temperature spermatozoa were washed and resuspended in 1 ml PBS twice. One drop of suspension was put on a Superfrost slide and coverslipped and subsequently ana- lyzed on a BioRad MRC 1024 confocal laser scanning microscope. Three-dimensional reconstructions were pre- formed using Volocity LE free software (http://www. improvision.com). Transmission electron microscopy (TEM) Cells were fixed in 2.5% glutaraldehyde in PBS (pH 7.2) for 2 hours at 4°C. After washing, samples were post- fixed in 1% OsO4 and 0.5% K-ferrocyanide in PBS for Page 6 of 8 2 hours, dehydrated with a graded series of acetone, and embedded in Spurr’s mixture. Semithin sections were stained by 0.5% toluidine blue (pH 8.5) Areas of interest were trimmed out by comparing the cut surface of the blocks with the semithin sections. Ultrathin sections were cut by an RMC MT-7 ultramicrotome, stained with 2% uranylacetate and lead citrate and analyzed on Philips CM10 electron microscope. Genotyping For high throughput genotyping DNA samples were analyzed by PorcineSNP60 Genotyping BeadChip (Illu- mina Ltd, San Diego, USA) in the Institute for Molecu- lar Medicine Finland (FIMM, Helsinki, Finland). The PorcineSNP60 BeadChip has recently been developed as an outcome of the porcine whole genome sequencing project [39]). Expression profiling and sequencing of STK17b and HECW2 For sequencing the full-length mRNA of the candidate genes STK17b and HECW2, samples of testicular tissue from a KA-affected and a control boar were collected and stored in RNAlater buffer (Qiagen). Total RNA pur- ification was performed with RNeasy Protect Mini kit (Qiagen). Extracted RNA was reverse transcribed (RT- PCR) using oligo T primers and an ImProm-II Reverse Transcription System (Promega) according to the manu- facturer’s instructions and amplified using gene specific primers (Table 2). Expression of gene fragments was assessed by gel electrophoresis. For sequencing the PCR amplicons were purified using ExoSAP-IT™ (Amersham Biosciences), while PCR fragments were sequenced in both directions with the same primers used in the amplification procedures. Sequencing was performed on MegaBace 500 capillary DNA sequencer (Amersham Biosciences) using DYEnamic ET Terminator Kits with Thermo Sequenase™ II DNA Polymerase (Amersham Biosciences). Statistical analysis A recessive mode of inheritance was tested for each SNP separately. The recessive model was selected because the pedigree of KA-affected boars suggested a recessive mode of inheritance and the low frequency of the defect in the Finnish Yorkshire pig population. In the recessive model, for each SNP the frequency of homozygote ani- mals for the minor allele (or for the major allele) was compared to frequency of heterozygote and other homo- zygote animals between cases and controls. In order to correct for multiple testing a permutation procedure was adapted to create empirical genome-wide P-values. Asso- ciation tests and permutation were carried out using the