Knobbed acrosome defect is associated with a region containing the genes STK17b and HECW2 on porcine chromosome 15

RESEARCH ARTICLE Open Access Knobbed ac osome de ec is associa ed wi h a egion con aining he genes STK17b and HECW2 on po cine ch omosome 15 Anu Si onen 1* , Pekka Uima i 1 , Szabolcs Nagy 2 , Sándo Paku 3 , Magnus Ande sson 4 , Johanna Vilkki 1 Abs ac Backg ound: Male in e ili y is an inc easing p oblem in all domes ic species including man. Localiza ion and iden i ica ion o genes in ol ed in de ec s causing male in e ili y p o ide aluable in o ma ion o speci ic e en s in spe m de elopmen . Co ec condensa ion o he spe m head and de elopmen o he ac osome a e equi ed o e ile spe m. In he Finnish Yo kshi e pig popula ion a knobbed ac osome de ec (KAD) has been epo ed which appea s o be o gene ic o igin. In p e ious s udies we ha e shown ha a la ge numbe o a ec ed spe ma ozoa ha e a cys ic swelling an e io o he apical pa o he ac osome. Resul s: Cha ac e iza ion o he knobbed ac osome a ec ed spe m e ealed ha bo h he ac osomal g anules and ch oma in a e a ec ed. This ype o KAD appea s o be a p e iously unknown and se ious o m o he de ec . A genome wide scan wi h Po cineSNP60 Geno yping BeadChip de ined he KAD associa ed egion wi hin 0.7 Mbp on po cine ch omosome 15. Two genes, STK17b and HECW2, loca ed wi hin his egion we e sequenced. The exp ession o hese genes appea ed compa able in KA-a ec ed and con ol boa s. The known unc ion o HECW2 in ac osome de elopmen highligh ed his gene as a good candida e esponsible o he KAD. One nonsynonymous SNP was iden i ied wi hin he HECW2 gene. Howe e , as his mu a ion was ound in homozygous s a e in indi iduals wi h no mal spe m, his is no likely o be he causal mu a ion. Conclusions: In his s udy we iden i ied wo candida e genes o a se e e de ec a ec ing bo h he spe m ac osome and ch oma in ha causes in e ili y. One o hese genes, HECW2, plays an impo an ole in ubiqui ina ion, a p e equisi e o ch oma in emodelling and ac osome o ma ion, highligh ing he in ol emen o his gene in he knobbed ac osome de ec and male in e ili y. Backg ound Male in e ili y is becoming inc easingly p e alen pa ly due o en i onmen al ac o s, bu many de ec s in spe m de elopmen a ise om a gene ic cause. P oblems in he p oduc ion and ma u a ion o spe m a e he mos common causes o male in e ili y esul ing in low spe m numbe s, mo phologically abno mal spe m o low spe m mo ili y [1-3]. Despi e e o s o e eal he genes and hei unc ions in spe ma ogenesis, li le is known abou he unde lying causes o male in e ili y. The e o e, he localiza ion and iden i ica ion o mu a- ions speci ically a ec ing spe ma ogenesis p o ide in aluable in o ma ion o in es iga ing he causes o male in e ili y. Mammalian spe ma ogenesis is a complex p ocess, whe e diploid spe ma ogonia de elop in o haploid, highly specialized spe ma ozoa. Spe ma ogenesis includes many es is-speci ic p ocesses ha a e con olled by complex egula o y mechanisms [4,5]. Du ing spe miogenesis, hap- loid ound spe ma ids unde go d ama ic biochemical and mo phological changes ha a e go e ned by specialized gene exp ession and in e ac ions be ween a ious genes and hei p o ein p oduc s [6]. Iden i ica ion o genes in ol ed in spe m de elopmen is a p e equisi e o unde - s anding he molecula mechanisms o spe ma ogenesis. Spe m de elopmen is known o be dis up ed du ing spe miogenesis in se e al ac osomal de ec s; e.g. globo- zoospe mia in humans, whe e spe ma ozoa lack an * Co espondence: [email p o ec ed] 1 Ag i ood Resea ch Finland, MTT, Bio echnology and Food Resea ch, Genomics, FI-36100 Jokioinen, Finland Full lis o au ho in o ma ion is a ailable a he end o he a icle Si onen e al.BMC Genomics 2010, 11:699 h p://www.biomedcen al.com/1471-2164/11/699 © 2010 Si onen e al; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. ac osome [7-9] and he knobbed ac osome de ec (KAD) in bulls, boa s, s allions, ams, and dogs [10-15]. The ac osome is an o ganelle ha de elops o e he an e io hal o he head in he spe ma ozoa. I is a cap-like s uc u e de i ed om he Golgi appa a us. The ac o- some con ains diges i e enzymes, which b eak down he zona pellucida o he o um, allowing he spe m o deli- e i s haploid nucleus in o he o a. Dis u bances o ac osomal de elopmen and unc ion signi ican ly impai he e ilizing capaci y o spe ma ozoa [16]. Knobbed ac osome de ec has been ecen ly desc ibed in he Finnish Yo kshi e pig popula ion [15]. Tes icula weigh s o boa s wi h KAD did no di e om con ol boa s. Howe e , a ec ed boa s had a smalle semini e - ous ubulediame e andlowe numbe o Se olicells ela i e o con ol boa s [15]. In es iga ion o he pedi- g ees o KA-a ec ed boa s sugges ed an au osomal ecessi e inhe i ance o he de ec . Gene ally wo com- mon boa s we e iden i ied in he pedig ee o he boa s wi h he KAD. Fe ili y o KA-a ec ed boa s is se e ely comp omised. Depending on he amoun o knobbed spe ma ozoa (25-81%) a ec ed boa s had poo non- e u n a e om no p egnancies o 47%, hus KA- a ec ed boa s p oduced no o sp ing o on a e age 2.5 ewe pigle s pe li e han con ol boa s. He e we ha e cha ac e ized u he he se e i y o he spe m head abno mali ies in KA-a ec ed boa s. A whole genome scan wi h mic osa elli e ma ke s showed inc eased homozygosi y in KA-a ec ed boa s in ch omosomes 3, 8, 14 and 15 [15]. Howe e , no s a is i- cally signi ican associa ion was de ec ed wi h a ailable mic osa elli e ma ke s. In his s udy we ha e used he Po cineSNP60 Geno yping BeadChip (Illumina) in o de o inc ease ma ke densi y and accu a ely map he KAD associa ed egion in pigs. All a ec ed boa s we e homo- zygous o SNPs co e ing 432 kb on po cine ch omo- some 15. The coding egion o wo genes was loca ed wi hin his homozygous egion and sequenced om bo h a KA-a ec ed and con ol boa . Resul s Mic oscopical analysis o he KAD In he con ocal lase scanning mic oscopy h ee- dimensional econs uc ions o he spe ma ozoa wi h ac osomal g anules indica ed ha he g anules p o- uded on bo h sides and con ained a acuolum (Figu e 1A). TEM analyses con i med he h ee-dimen- sional p o usion o he g anules and he occu ence o acuoles wi hin he g anules. The nucleus was also shown o be a ec ed as e iden om he Y-shaped o m a he apical end (Figu e 1B) sugges ing ha he de ec a ec s bo h he ch oma in and ac osome. These indings highligh ha his pa icula and p e iously unknown KA-de ec appea s o be a se ious o m o he ac osomal g anule de ec . SNP quali y measu es Based on all a ailable SNPs and he me hod o es ima e IBDs in he Plink so wa e package (pi_ha ) he a e age ela edness among cases and con ols was 0.24 and 0.26, espec i ely. These le els o ela edness a e ypical in he s udied Finnish Yo kshi e pig popula ion. The a e - age sample call a e was 95%. The e we e 2815 SNPs ha did no wo k o any o he samples analysed. Excluding hese SNPs, he a e age SNP call a e was 0.9982 (s.d. = 0.007) and he a e age mino allele e- quency was 0.25 (s.d. = 0.14). O e all, he da ase con- ained 9216 monomo phic SNPs. Obse ed dis ibu ion o P- alues in he Ha dy-Weinbe g equilib ium es s a- is ics did no di e om expec a ions. In o al, 183 SNPs (excluding SNPs on he X-ch omosome) had a P- alue <1.0E-06 being lowe han expec ed. Genome wide associa ion analysis The associa ion es was pe o med o 47055 SNPs. The Manha an plo o he log10 based P- alues is p esen ed in Figu e 2. The ecessi e model iden i ied a KAD asso- cia ed egion co e ing app oxima ely 3 Mbp be ween 93 and 96 Mbp (pig genome build 9) on ch omosome 15. A B Figu e 1 Mic oscopical examina ion o he KA-de ec .A. Con ocal lase scanning mic oscopy images o spe ma ozoa wi h ac osomal g anule om wo KA-a ec ed boa s. Yellow lines indica e he pe pendicula planes o he 3d- econs uc ions. A ows indica e a acuolum inside he g anule. B. TEM analysis o an ac osomal g anule. Two acuoles a e p esen and he apical pa o he nucleus shows a Y-shape. Si onen e al.BMC Genomics 2010, 11:699 h p://www.biomedcen al.com/1471-2164/11/699 Page 2 o 8 A e pe mu a ion, i e SNPs we e s a is ically signi ican (P- alue = 0.0002, Table 1). Fou o hese SNPs (ALGA0086494, DRGA0015302, MARC0011300, and CASI0005693) a e loca ed wi hin a 1.4 Mbp egion and we e in comple e linkage disequilib ium (D’=1.0, 2 = 1.0). 12 ou o 14 KAD cases had inhe i ed wo iden ical copies o he haplo ype co e ing hese and o he SNPs be ween hem, indica ing an ex ended homozygos- i y in his egion, and hus a common ances al o igin (Figu e 3). All KA-a ec ed boa s sha ed a 0.7 Mbp homozygous egion be ween SNPs DIAS0000367 and ALGA0086503 (Figu e 3, addi ional ile 1). The CASI0005693 SNP was in s onge linkage disequilib ium wi h ALGA0086494 and o he signi ican SNPs com- pa ed wi h neighbou ing SNPs, highligh ing ha he posi ion o CASI0005693 may change ollowing a mo e e ined genome build in his egion (see addi ional ile 1). The i h signi ican SNP (MARC0020403) was loca ed 4 Mbp om he o he ou SNPs, and was shown o be in linkage disequilib ium (D’= 1.0, 2 = 0.13). Candida e genes HECW2 and STK17b The mos p omising candida e gene Ubiqui in-p o ein ligase E3 (HECW2) was loca ed wi hin he haplo ype o wo SNPs wi h he highes P- alues; ALGA0086494 and DRGA001532 (Table 1, addi ional ile 1). All KA- a ec ed animals we e homozygous o hese SNPs and only wo con ol animals had he same homozygo e alleles as KA-a ec ed boa s (Figu e 3). Fu he mo e, one o hese wo animals appea ed o ha e a SME- de ec and ano he was emo ed om b eeding a young age due o weak leg con o ma ion and he e o e no esh spe m samples we e a ailable o analysis. The SME-de ec is a cys mal o ma ion in he spe m head, wi h indica ions ha his is o ac osomal o igin [17,18]. HECW2 is exp essed in he es is [19] and unc ions in ubiqui in media ed p o eolysis [20]. Ubiqui in signals ha e been de ec ed du ing ac osome de elopmen [21] and deubiqui ina ing enzyme mUBPy is up egula ed in he es is o wobble mouse, which is in e ile due o he lack o a unc ional ac osome [22]. Ano he gene wi hin he KAD homozygous egion was se ine/ h eonine kinase 17b (STK17b, DRAK2, addi ional ile 1). STK17b is a se ine/ h eonine kinase, which has a ole in he egula ion o apop osis [23-25]. STK17b is highly exp essed in he es es whe e he apop osis plays an impo an ole du ing spe ma ogenesis. E en hough KAD associa ed egion Figu e 2 Manha an plo o he log10 based P- alues ac oss all ch omosomes o he KAD in Finnish Yo kshi e pig popula ion. The KA- a ec ed haplo ypes o ma ked KAD associa ed egion suppo ed he expec ed ecessi e mode o inhe i ance and had he lowes P- alues. Table 1 Geno ype coun s o cases and con ols, nominal, and pe mu a ed P- alues om he ecessi e model o he bes KAD associa ed SNPs. SNP Ch Posi ion, bp Geno ypes cases Geno ypes con ols Recessi e, P- alue Pe mu a ed P- alue ALGA0086494 15 93805621 14/0/0 2/8/11 1.41E-07 0.0002 DRGA0015302 15 93835894 14/0/0 2/8/11 1.41E-07 0.0002 MARC0011300 15 94070930 14/0/0 2/8/11 1.41E-07 0.0002 CASI0005693 15 95156189 14/0/0 2/8/11 1.41E-07 0.0002 MARC0020403 15 90155667 12/2/0 0/7/14 1.66E-07 0.0002 Si onen e al.BMC Genomics 2010, 11:699 h p://www.biomedcen al.com/1471-2164/11/699 Page 3 o 8 he pheno ype o KA-a ec ed boa s does no implica e a de ec in apop osis he exp ession and sequence o STK17b mRNA was de e mined. Analysis o he po cine HECW2 gene The exp ession pa e n o di e en HECW2 agmen s (Table 2) appea ed o be compa able in he KA-a ec ed and con ol boa . The ull-leng h mRNA o po cine HECW2 [GenBank HM562353] was sequenced om he es is o one KA-a ec ed and one con ol boa . The o al leng h o he sequenced HECW2 ansc ip was 4802 bp wi h a high homology wi h o he mammalian species. When compa ed o he human HECW2 gene, he po cine sequence s a ed a posi ion 177 bp in he exon 2. In he pig, exon 1 did no appea o be exp essed in he es is. Howe e , based on he genome sequence, exon 1 was highly conse ed compa ed wi h he human sugges ing ha i may ha e an impo an ole in HECW2 exp ession, a leas in some issues. In man, he p o ein coding egion s a s a mRNA posi ion 184 bp (exon 2). The human HECW2 p o ein consis s o 1572 aa. Cu en da a sugges s ha he co esponding pig p o ein sequence is 1574 aa wi h a 96% homology o he human sequence. Simila ly, he ull-leng h HECW2 p o ein in he mouse includes 1578 aa and has 95% homology o HECW2 in he pig. Sequencing o he po cine HECW2 mRNA and he exon 1 om genomic DNA o a KA-a ec ed and con ol boa showed wo SNPs a mRNA posi ions 1563 (SNP1) and 2233 bp (SNP2). SNP1 causes a change in he p o- ein sequence a posi ion 519 aa om isoleucine o h eonine (Figu e 4). This SNP was u he geno yped o all 14 KA-a ec ed and 10 con ol boa s. All KA- a ec ed boa s we e homozygous o his SNP, bu also ou con ol boa s had he same homozygous allele, dis- coun ing his as he causal mu a ion o he KAD. In Figu e 3 Haplo ype coun s in KAD cases and con ols on ch omosome 15 (91861321-95156189 bp, Pig genome build 9). SNPs ha a e sha ed among KAD cases a e ma ked in g ey backg ound and he SNPs wi h he smalles P- alue om he ecessi e model a e in bold ace. Table 2 P ime s used o sequencing o he candida e genes HECW2 and STK17b. Gene Exons Posi ion, bp Leng h, bp Fo wa d p ime Re e se p ime HECW2 1 - 404 CTGGGACGTGTTTCAAGGTT GATCTCTGACGCTTGCCTTC HECW2 2-4 1-351 421 AGACGGGATGGCTAGCTCA GATTTTTATCTCCGGCTCCA HECW2 4-7 332-744 413 AAAAACAGGGGTGTGACTGG CGGTGCCAGATTGGATTAGT HECW2 5-2-10 483-1534 1052 TGAAGAACCCTGCTGTGATG GTCTTCCGGCTTTGTCTGAG HECW2 10-12/13 1403-2609 1207 GAGGAAGACCACGAGTTCCA TACTCTGGTATCGCCGGTTC HECW2 12-15 2543-2896 354 CCGCAGGTGCTGCAGAGGTC GGTGTCCCGCCGGACTTTGG HECW2 14-20/21 2783-3560 778 TTCCTCATCAGCCCAGAGTT GCTGAGTACCTGGCGAGTTC HECW2 20-22/23 3461-3800 340 ATGTCATACGTGCCTCCACA CACTGTAATCCAGCCCTTCC HECW2 21/22-30 3654-4675 1022 AAGGCCCAGGGAAATTAAAG GGATGGGTATGGAGGGAGAT HECW2 28- 4567-4815 249 AGGGAGTAATGGCCCAAGAA CTAGAGGGCAGCTTCTGGA STK17b 1-8 1-927 928 GTAAGCTCCGGTCTCCGTCT TGTTGCTGTGGTAATAGGATCATA STK17b 3-9 413-1100 707 TTTGCTGTGGTTAGGCAATG AAAAGCCTCTGGATGAAGTCTGT The exons (based on human AB037722), posi ion o he agmen wi hin he HECW2 [GenBank HM562353] and STK17b [GenBank HM594868] mRNA and he leng h o he PCR amplicons a e shown. Si onen e al.BMC Genomics 2010, 11:699 h p://www.biomedcen al.com/1471-2164/11/699 Page 4 o 8 addi ion o hese wo SNPs, a dele ion o nine bp ( h ee aa) was de ec ed a e he nucleo ide a posi ion 3348 bp (1113 aa) when compa ed o he po cine e e ence sequence [Ensemble: ENSSSCG00000016068]. This dele- ion seems o be e y common in mammalian species (Figu e4).Thepo cine e e encesequence o HECW2 included exons 3-28 (based on human [GenBank AB037722]), howe e ou sequencing esul s indica e ha exons 2, 29 and 30 a e also exp essed in he pig es es. Analysis o STK17b mRNA The sequenced es icula mRNA o po cine STK17b [GenBank HM594868] con ained exons 1-9 (based on human exon numbe ing, [GenBank NM_004226]) and 1102 bp. T ansla ion s a codon was iden i ied a posi ion 282 wi hin exon 2. No change in he exp es- sion p o ile was iden i ied and he p o ein coding sequence was iden ical in he KA-a ec ed and con ol boa . Discussion While he esul s o homozygosi y mapping o he KAD in a p e ious s udy [15] we e no s a is ically signi ican , hey did indica e he mos p obable posi ions o he KAD-associa ed ch omosomal segmen s. In his s udy we con i med he associa ion be ween KAD and po cine ch omosome 15. The ini ial genome sc een wi h mic o- sa elli e ma ke s S0004 and SW2608 showed inc eased homozygosi y in KAD a ec ed boa s [15]. The genome scan wi h Po cineSNP60 Geno yping BeadChip (Illu- mina) localized he KAD associa ed egion be ween hese wo ma ke s on po cine ch omosome 15. The Po cineSNP60 BeadChip illus a ed a high call a e (<95%) in he Finnish Yo kshi e pig popula ion and only 15% o he SNPs we e monomo phic. In his s udy we de ec ed he KAD associa ed egion co e ing 2 Mbp indica ing ha he ma ke map in he ini ial sc een was no dense enough o de ec he signi ican inc ease in homozygosi y. Wi hin he associa ed egion we iden i ied and sequenced wo candida e genes Ubiqui in-p o ein ligase E3 (HECW2) and se ine/ h eonine kinase 17b (STK17b). The sequencing o hese wo genes e ealed wo SNPs wi hin HECW2 gene, bu no polymo phisms we e de ec ed in he p o ein coding sequence o STK17b. Al hough he iden i ied mu a ions appea ed no o be he causal cause o he KAD, HECW2 emains a good candida e gene o his de ec conside ing i s ole in ac osome de elopmen and ch oma in emodelling. P o ein ubiqui ina ion is one o he undamen al egula- o y pos - ansla ional modi ica ions con olling in acellu- la signalling e en s. Ubiqui in-p o eosome-dependen p o eolysis plays an impo an ole in selec i ely deg ading and ecycling p o eins in many basic cellula p ocesses including spe ma ogenesis [26]. Fo deg ada ion by he p o eosome, binding o ubiqui in wi h subs a e p o eins equi es he ac i i y o ubiqui in-ac i a ing enzyme E1, ubiqui in-conjuga ing enzyme E2, and subs a e-speci ic ubiqui in ligase E3 [27]. Ubiqui in ligase E3 in combina- ion wi h an E2 ubiqui in-conjuga ing enzyme causes he a achmen o ubiqui in o a lysine esidue on he a ge p o ein. In spe ma ogenesis ubiqui ina ion is equi ed o a - ious p ocesses; o example he eplacemen o he spe - ma ids nuclea his ones wi h p o amines du ing spe ma id elonga ion [26]. In spe ma ozoa, p o eosomes a e loca ed on he plasma memb ane o e lying he ac o- some, in he ac osomal and pos ac osomal egions, in he head- ail connec ing-piece, middle-piece o he ail, and esidual bodies [28-32]. P o eosome subuni Psmc3 and an ubiqui in p o ein ligase Rn 19a ha e been loca ed a he cy osolic side o ou e and inne memb anes o he ac osome [33]. The co-immunop ecipi a ion and localiza ion o Psmc3 and Rn 19a in spe miogenesis poin s o he pa icipa ion o he ubiqui in-p o eosome sys em in ac osome o ma ion, spe ma id head shaping, and de elopmen o he head- ail coupling appa a us and ail [33]. Mal unc ion o componen s in ubiqui ina ion sys em has been shown o be a cause o male in e ili y [27,34-36]. The e appea s o be a special equi emen o ce ain componen s o he ubiqui in sys em du ing spe - miogenesis, in pa icula [37], and i is p obable ha di - e en spe ma ogenic phases would equi e di e en specialized ac i i ies o he ubiqui in sys em. Mu a ions in ubiqui ina ion ela ed p o eins may also a ec speci i- cally spe ma ogenesis h ough hei es is speci ic in e - ac ing pa ne s [36]. A mal unc ion o ubiqui ina ion may cause di e se pheno ypes as exempli ied in he human and mouse by mu a ion o H 6b and Usp14 [35,38]. SNP DEL Figu e 4 Alignmen o HECW2 p o ein sequences a he de ec ed polymo phism posi ions in a ious species. The only di e ence be ween con ol (Yo kshi e) and KA-a ec ed (Ak ) animals was a change om isoleucine o h eonine (SNP). Howe e , his change was also p esen in he human p o ein sequence. Fu he mo e, a h ee aa dele ion was iden i ied when compa ed wi h he pig e e ence sequence. Si onen e al.BMC Genomics 2010, 11:699 h p://www.biomedcen al.com/1471-2164/11/699 Page 5 o 8 Conclusions In his s udy we demons a e he exac KAD pheno ype in ma u e spe m. In addi ion o he ac osome, he spe - ma id ch oma in is also a ec ed. We ha e loca ed he homozygous egion o KAD wi hin 0.5 Mbp on po cine ch omosome 15 con aining wo genes STK17b and HECW2. The ole o ubiqui ina ion in ch oma in emo- delling and ac osome o ma ion is consis en wi h HECW2 being in ol ed in his de ec . While a causal mu a ion o KAD was unable o be iden i ied, ou esul s indica e ha he obse ed pheno ype may be caused by a mal unc ion in he ubiqui ina ion sys em. Iden i ica ion o he causal a ia ion o he KAD equi es u he ana- lysis o he genomic egion con aining he HECW2 gene. Me hods Animal ma e ial Expe imen al ma e ial included 14 Finnish Yo kshi e boa s a ec ed wi h KAD and 21 con ol boa s. All a ec ed boa s we e clinically examined and shown o display symp oms ypical o he synd ome, bu no o he abno m- ali ies. Spe m om a ec ed and con ol boa s was collec ed and he DNA ob ained ollowing phenol/chlo o- o m ex ac ion. Samples we e dilu ed o 100 ng/μl in TE- bu e and used as empla es o Po cineSNP60 Geno yp- ing BeadChip (Illumina). Genomic DNA was also used o sequencing o he HECW2 exon 1 and SNP1. Fo mic oscopical examina ion ep esen a i e semen samples om KA-a ec ed and con ol boa s we e ixed in o maldehyde o con ocal lase scanning and ans- mission elec on mic oscopy analyses. Con ocal lase scanning mic oscopy Spe ma ozoa we e labeled wi h LIVE/DEAD Reduced Bio- haza d Viabili y ki ( ed, L23102, In i ogen). The labelling p o ocol was in acco dance wi h he ecommenda ions o he manu ac u e . In b ie , 50 μl DMSO was added o one ial o luo escen dye and ho oughly mixed o make a s ock solu ion. Spe ma ozoa we e suspended in PBS a app oxima ely 1 × 10 6 /ml. One μl o luo escen dye was added o he suspension. A e 30 min incuba ion a oom empe a u e spe ma ozoa we e washed and esuspended in 1 ml PBS wice. One d op o suspension was pu on a Supe os slide and co e slipped and subsequen ly ana- lyzed on a BioRad MRC 1024 con ocal lase scanning mic oscope. Th ee-dimensional econs uc ions we e p e- o med using Voloci y LE ee so wa e (h p://www. imp o ision.com). T ansmission elec on mic oscopy (TEM) Cells we e ixed in 2.5% glu a aldehyde in PBS (pH 7.2) o 2 hou s a 4°C. A e washing, samples we e pos - ixed in 1% OsO 4 and 0.5% K- e ocyanide in PBS o 2 hou s, dehyd a ed wi h a g aded se ies o ace one, and embedded in Spu ’s mix u e. Semi hin sec ions we e s ained by 0.5% oluidine blue (pH 8.5) A eas o in e es we e immed ou by compa ing he cu su ace o he blocks wi h he semi hin sec ions. Ul a hin sec ions we e cu by an RMC MT-7 ul amic o ome, s ained wi h 2% u anylace a e and lead ci a e and analyzed on Philips CM10 elec on mic oscope. Geno yping Fo high h oughpu geno yping DNA samples we e analyzed by Po cineSNP60 Geno yping BeadChip (Illu- mina L d, San Diego, USA) in he Ins i u e o Molecu- la Medicine Finland (FIMM, Helsinki, Finland). The Po cineSNP60 BeadChip has ecen ly been de eloped as an ou come o he po cine whole genome sequencing p ojec [39]). Exp ession p o iling and sequencing o STK17b and HECW2 Fo sequencing he ull-leng h mRNA o he candida e genes STK17b and HECW2, samples o es icula issue om a KA-a ec ed and a con ol boa we e collec ed and s o ed in RNAla e bu e (Qiagen). To al RNA pu - i ica ion was pe o med wi h RNeasy P o ec Mini ki (Qiagen). Ex ac ed RNA was e e se ansc ibed (RT- PCR) using oligo T p ime s and an ImP om-II Re e se T ansc ip ion Sys em (P omega) acco ding o he manu- ac u e ’s ins uc ions and ampli ied using gene speci ic p ime s (Table 2). Exp ession o gene agmen s was assessed by gel elec opho esis. Fo sequencing he PCR amplicons we e pu i ied using ExoSAP-IT™(Ame sham Biosciences), while PCR agmen s we e sequenced in bo h di ec ions wi h he same p ime s used in he ampli ica ion p ocedu es. Sequencing was pe o med on MegaBace 500 capilla y DNA sequence (Ame sham Biosciences) using DYEnamic ET Te mina o Ki s wi h The mo Sequenase™II DNA Polyme ase (Ame sham Biosciences). S a is ical analysis A ecessi e mode o inhe i ance was es ed o each SNP sepa a ely. The ecessi e model was selec ed because he pedig ee o KA-a ec ed boa s sugges ed a ecessi e mode o inhe i ance and he low equency o he de ec in he Finnish Yo kshi e pig popula ion. In he ecessi e model, o each SNP he equency o homozygo e ani- mals o he mino allele (o o he majo allele) was compa ed o equency o he e ozygo e and o he homo- zygo e animals be ween cases and con ols. In o de o co ec o mul iple es ing a pe mu a ion p ocedu e was adap ed o c ea e empi ical genome-wide P- alues. Asso- cia ion es s and pe mu a ion we e ca ied ou using he Si onen e al.BMC Genomics 2010, 11:699 h p://www.biomedcen al.com/1471-2164/11/699 Page 6 o 8 so wa e package Plink [40]. Haplo ypes, he linkage dise- quilib ium plo and he Manha an plo we e p oduced wi h Haplo iew [41]. Addi ional ma e ial Addi ional ile 1: The KAD associa ed egion in he Finnish Yo kshi e pig localized on po cine ch omosome 15. The KAD associa ed homozygous egion in he Finnish Yo kshi e was iden i ied in ch omosome 15 be ween base pai s 93723216 and 94155055. A p omising candida e gene Ubiqui in-p o ein ligase E3 (HECW2) is loca ed wi hin his egion a he same posi ion as wo ma ke s wi h lowes P- alues (highligh ed by ci cles). Ano he gene STK17b was also loca ed wi hin he homozygous egion; howe e he known unc ion o his gene in apop osis would no in e a ole o STK17b in KAD. Acknowledgemen s Funding o his s udy was p o ided by he Finnish Minis y o Ag icul u e and Fo es y (Make a). The assis ance o Tiina Jaakkola and Ta ja Ho i uo i in DNA ex ac ion and Päi i Lahe mo (Ins i u e o Molecula Medicine Finland, FIMM) in geno yping wi h Po cineSNP60 Geno yping BeadChip (Illumina) is g ea ly app ecia ed. Au ho de ails 1 Ag i ood Resea ch Finland, MTT, Bio echnology and Food Resea ch, Genomics, FI-36100 Jokioinen, Finland. 2 Uni e si y o Pannonia, Ins i u e o En i onmen al Sciences, H-8200 Veszp em, Hunga y. 3 Fi s Depa men o Pa hology and Expe imen al Cance Resea ch, Semmelweis Uni e si y, H-1085 Budapes , Hunga y. 4 Uni e si y o Helsinki, Depa men o Clinical Ve e ina y Sciences, Helsinki, Finland. Au ho s’con ibu ions AS ca ied ou he molecula gene ics s udies, sequence alignmen s and d a ed he manusc ip . PU pe o med he s a is ical analysis and pa icipa ed in d a ing he manusc ip . SN pa icipa ed in he mic oscopical s udies and con ibu ed o d a ing o he manusc ip (mic oscopical s udies). SP ca ied ou he mic oscopical s udies. MA pa icipa ed in he design and coo dina ion o he s udy. JV pa icipa ed in he design and helped o d a he manusc ip . All au ho s ead and app o ed he inal manusc ip . Recei ed: 7 July 2010 Accep ed: 9 Decembe 2010 Published: 9 Decembe 2010 Re e ences 1. 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Submi you nex manusc ip o BioMed Cen al and ake ull ad an age o : • Con enien online submission • Tho ough pee e iew • No space cons ain s o colo igu e cha ges • Immedia e publica ion on accep ance • Inclusion in PubMed, CAS, Scopus and Google Schola • Resea ch which is eely a ailable o edis ibu ion Submi you manusc ip a www.biomedcen al.com/submi Si onen e al.BMC Genomics 2010, 11:699 h p://www.biomedcen al.com/1471-2164/11/699 Page 8 o 8