Sironen et al. BMC Genomics 2010, 11:699 http://www.biomedcentral.com/1471-2164/11/699 Page 4 of 8 The exons (based on human AB037722), position of the fragment within the HECW2 [GenBank HM562353] and STK17b [GenBank HM594868] mRNA and the length of the PCR amplicons are shown. Figure 3 Haplotype counts in KAD cases and controls on chromosome 15 (91861321-95156189 bp, Pig genome build 9). SNPs that are shared among KAD cases are marked in grey background and the SNPs with the smallest P-value from the recessive model are in bold face. the phenotype of KA-affected boars does not implicate a defect in apoptosis the expression and sequence of STK17b mRNA was determined. Analysis of the porcine HECW2 gene The expression pattern of different HECW2 fragments (Table 2) appeared to be comparable in the KA-affected and control boar. The full-length mRNA of porcine HECW2 [GenBank HM562353] was sequenced from the testis of one KA-affected and one control boar. The total length of the sequenced HECW2 transcript was 4802 bp with a high homology with other mammalian species. When compared to the human HECW2 gene, the porcine sequence started at position 177 bp in the exon 2. In the pig, exon 1 did not appear to be expressed in the testis. However, based on the genome sequence, exon 1 was highly conserved compared with the human suggesting that it may have an important role in HECW2 expression, at least in some tissues. In man, the protein coding region starts at mRNA position 184 bp (exon 2). The human HECW2 protein consists of 1572 aa. Current data suggests that the corresponding pig protein sequence is 1574 aa with a 96% homology to the human sequence. Similarly, the full-length HECW2 protein in the mouse includes 1578 aa and has 95% homology to HECW2 in the pig. Sequencing of the porcine HECW2 mRNA and the exon 1 from genomic DNA of a KA-affected and control boar showed two SNPs at mRNA positions 1563 (SNP1) and 2233 bp (SNP2). SNP1 causes a change in the pro- tein sequence at position 519 aa from isoleucine to threonine (Figure 4). This SNP was further genotyped for all 14 KA-affected and 10 control boars. All KA- affected boars were homozygous for this SNP, but also four control boars had the same homozygous allele, dis- counting this as the causal mutation of the KAD. In Table 2 Primers used for sequencing of the candidate genes HECW2 and STK17b. Position, bp Length, bp Forward primer Gene HECW2 HECW2 HECW2 HECW2 HECW2 HECW2 HECW2 HECW2 HECW2 HECW2 STK17b STK17b Exons 1 2-4 4-7 5-2-10 10-12/13 12-15 14-20/21 20-22/23 21/22-30 28- 1-8 3-9 - 1-351 332-744 483-1534 1403-2609 2543-2896 2783-3560 3461-3800 3654-4675 4567-4815 1-927 413-1100 404 421 413 1052 1207 354 778 340 1022 249 928 707 CTGGGACGTGTTTCAAGGTT AGACGGGATGGCTAGCTCA AAAAACAGGGGTGTGACTGG TGAAGAACCCTGCTGTGATG GAGGAAGACCACGAGTTCCA CCGCAGGTGCTGCAGAGGTC TTCCTCATCAGCCCAGAGTT ATGTCATACGTGCCTCCACA AAGGCCCAGGGAAATTAAAG AGGGAGTAATGGCCCAAGAA GTAAGCTCCGGTCTCCGTCT TTTGCTGTGGTTAGGCAATG Reverse primer GATCTCTGACGCTTGCCTTC GATTTTTATCTCCGGCTCCA CGGTGCCAGATTGGATTAGT GTCTTCCGGCTTTGTCTGAG TACTCTGGTATCGCCGGTTC GGTGTCCCGCCGGACTTTGG GCTGAGTACCTGGCGAGTTC CACTGTAATCCAGCCCTTCC GGATGGGTATGGAGGGAGAT CTAGAGGGCAGCTTCTGGA TGTTGCTGTGGTAATAGGATCATA AAAAGCCTCTGGATGAAGTCTGT