Genome-Wide Association Study of Polymorphisms Predisposing to Bronchiolitis

1 Scien i ic RepoR s | 7:41653 | DOI: 10.1038/s ep41653 www.na u e.com/scien i ic epo s Genome-Wide Associa ion S udy o Polymo phisms P edisposing o B onchioli is Anu Pasanen1, Minna K. Ka jalainen1, Louis Bon 2, Eija Piippo-Sa olainen3, Ma ja Ruo salainen3, Emma Goksö 4, Kuldeep Kumawa 5, Hennie Hodemaeke s6, Ki si Nuoli i a7, Tuomas Ja i8, Gö an Wenne g en4, Mikko Hallman1, Mika Räme 1,9,* & Ma i Ko ppi10,* B onchioli is is a majo cause o hospi aliza ion among in an s. Se e e b onchioli is is associa ed wi h la e as hma, sugges ing a common gene ic p edisposi ion. Gene ic backg ound o b onchioli is is no well cha ac e ized. To iden i y polymo phisms associa ed wi h b onchioli is, we conduc ed a genome- wide associa ion s udy (GWAS) in which 5,300,000 single nucleo ide polymo phisms (SNPs) we e es ed o associa ion in a Finnish–Swedish popula ion o 217 child en hospi alized o b onchioli is and 778 con ols. The mos p omising SNPs (n = 77) we e geno yped in a Du ch eplica ion popula ion o 416 cases and 432 con ols. Finally, we used a se o 202 Finnish b onchioli is cases o u he in es iga e candida e SNPs. We did no de ec genome-wide signi ican associa ions, bu se e al sugges i e associa ion signals (p < 10−5) we e obse ed in he GWAS. In he eplica ion popula ion, h ee SNPs we e nominally associa ed (p < 0.05). O hem, s269094 was an exp ession quan i a i e ai locus (eQTL) o KCND3, p e iously shown o be associa ed wi h occupa ional as hma. In he addi ional se o Finnish cases, he associa ion o ano he SNP ( s9591920) wi hin a noncoding RNA locus was u he s eng hened. Ou esul s p o ide a i s genome-wide examina ion o he gene ics unde lying b onchioli is. These p elimina y indings equi e u he alida ion in a la ge sample size. B onchioli is is a common lowe espi a o y in ec ion (LRI) ha p ima ily a ec s child en unde wo yea s o age1,2. Respi a o y syncy ial i us (RSV) is ecognized as he mos impo an causa i e agen o b onchioli is wo ldwide3. Typically, RSV epidemics occu in annual cycles and peak du ing he cold season in empe a e cli- ma es and du ing he ainy season in wa m clima es4. Du ing RSV epidemics, mos in an s become in ec ed and de elop mild disease. Howe e , a ac ion o chil- d en, be ween 2% and 3% o an age coho , de elop se e e b onchioli is ha equi es hospi aliza ion5. RSV b on- chioli is is he mos common in ec ion- ela ed cause o hospi aliza ion among in an s and young child en in de eloped coun ies6. The only means o p e en ion is injec ing high- isk child en mon hly wi h an i-RSV mon- oclonal an ibody7. The e is no accine o e ec i e an i i al he apy a ailable o RSV in ec ion. Thus, ea men o b onchioli is is mainly suppo i e and includes supplying oxygen and main aining hyd a ion8. The e a e se e al congeni al and en i onmen al isk ac o s, such as p ema u i y, immune de iciency, congeni- al hea disease, and obacco smoke exposu e, ha p edispose child en o se e e b onchioli is4. Howe e , he isk ac o s do no comple ely explain he a ia ion in disease se e i y, and hos gene ic ac o s likely con ibu e o he 1PEDEGO Resea ch Uni , Medical Resea ch Cen e Oulu, Uni e si y o Oulu, and Depa men o Child en and Adolescen s, Oulu Uni e si y Hospi al, Oulu, Finland. 2Wilhelmina Child en’s Hospi al, Uni e si y Medical Cen e U ech , U ech , The Ne he lands. 3Kuopio Uni e si y Hospi al, Pedia ics, Uni e si y o Eas e n Finland, Kuopio, Finland. 4Depa men o Pedia ics, Uni e si y o Go henbu g, Queen Sil ia Child en’s Hospi al, Go henbu g, Sweden. 5Depa men o Immunology, Labo a o y o T ansla ional Immunology, Uni e si y Medical Cen e U ech , U ech , The Ne he lands. 6RIVM, Na ional Ins i u e o Public Heal h and he En i onmen , GZB, Cen e o Heal h P o ec ion, Bil ho en, The Ne he lands. 7Depa men o Pedia ics, Seinäjoki Cen al Hospi al, Seinäjoki, Finland. 8Depa men o Pedia ics, Uni e si y o Tu ku and Tu ku Uni e si y Hospi al, Tu ku, Finland. 9BioMediTech, Uni e si y o Tampe e, Tampe e, Finland. 10Cen e o Child Heal h Resea ch, Tampe e Uni e si y and Tampe e Uni e si y Hospi al, Tampe e, Finland. *These au ho s con ibu ed equally o his wo k. Co espondence and eques s o ma e ials should be add essed o A.P. (email: [email p o ec ed]) Recei ed: 04 No embe 2016 Accep ed: 20 Decembe 2016 Published: 31 Janua y 2017 OPEN www.na u e.com/scien i ic epo s/ 2 Scien i ic RepoR s | 7:41653 | DOI: 10.1038/s ep41653 se e i y o i al b onchioli is. Some e idence o gene ic suscep ibili y was p o ided by a win s udy ha showed a highe conco dance in hospi aliza ion a es be ween iden ical wins han in a e nal wins9. Tha s udy es ima ed he he i abili y o b onchioli is o be 22%. Gene ic s udies o suscep ibili y o b onchioli is ha e mos ly ocused on a ian s in selec ed candida e genes, o en ela ed o immuni y. A ew s udies ha e e alua ed he ele ance o la ge g oups o genes and ha e linked a ia ions in he se e i y o RSV disease wi h inna e immuni y– ela ed genes and he IL13/IL4 locus in he 5q31 cy okine clus e 10,11. Mo eo e , gene ic s udies ha e demons a ed RSV b onchioli is–associa ed loci in genes encoding p o eins such as oll-like ecep o s (TLRs), su ac an p o ein D (SFTPD), Vi amin D ecep o (VDR), and a ious cy okines, o which some ha e been sugges ed in mo e han one s udy3,10,12,13. Howe e , mos gene ic associa ions ha e no been eplica ed in subsequen s udies. Se e e i al b onchioli is du ing childhood, especially a e RSV b onchioli is o hino i us b onchioli is, is associa ed wi h he de elopmen o as hma la e in li e5,14. I is unknown whe he he isk o as hma inc eases because o he b onchioli is o whe he a common gene ic backg ound unde lies bo h diseases15. Polymo phisms in genes including IL-10, IL-13, TLR4, VDR, CCR5, and ADAM33 ha e been associa ed wi h bo h RSV b onchi- oli is and a isk o as hma16,17. This may suppo a gene ic p edisposi ion o bo h in an i al b onchioli is and subsequen as hma. An inc eased isk o childhood-onse as hma a e hino i us in ec ion has been associa ed wi h 17q21 locus18. Rega ding b onchioli is, gene ic associa ions sugges ed in di e en s udies need o be alida ed in u he s udies. A mo e comp ehensi e cha ac e iza ion o gene ic de e minan s will lead o imp o ed ecogni ion o isk g oups and will likely con ibu e o he de elopmen o new and mo e-speci ic ea men s o espi a o y in ec- ions. The e o e, he aim o he p esen s udy was o conduc a hypo hesis- ee genome-wide associa ion s udy (GWAS) o iden i y gene ic polymo phisms ha p edispose in an s o i al b onchioli is. Resul s Sugges i e associa ions in GWAS o b onchioli is and RSV b onchioli is. We de ec ed se e al sug- ges i e associa ion signals (p < 1 × 10−5) in he GWAS pe o med on 217 in an s hospi alized o i al b onchi- oli is and 778 con ols. (Supplemen a yFigu eS1). The s ingen genome-wide signi icance le el (p < 5 × 10−8) was no eached. Table1 con ains he esul s o he SNPs in he s onges sugges i e GWA loci (p < 5 × 10−6). Cases we e s a i ied acco ding o p esence o RSV, and RSV-posi i e b onchioli is cases (n = 121) we e es ed agains he same GWAS con ols (“RSV GWAS”). Again, he signals did no each he le el o genome-wide sig- ni icance (p < 5 × 10−8) (Supplemen a yFigu eS2). Table2 lis s he s onges sugges i e GWA loci (p < 5 × 10−6) in he RSV GWAS. Th ee o he bes loci we e sha ed be ween he b onchioli is GWAS and he RSV GWAS. The sha ed loci we e si ua ed in he egions nea he genes VSTM4, C10o 71, and DRGX in ch omosome 10 and in he egion nea LOC105375265 and LOC105375266 in ch omosome 7. Based on he esul s ob ained om he b onchioli is GWAS and he RSV GWAS, we selec ed one ep esen a i e SNP o he eplica ion phase om each o he bes sugges i e GWA egion. All SNPs selec ed o eplica ion geno yping and he espec i e esul s a e p esen ed in Supplemen a yTableS1. SNPs associa ed wi h RSV b onchioli is in p e ious s udies. We sc eened o SNPs ha we e epo ed o be associa ed wi h RSV b onchioli is in p e ious s udies and SNPs in he icini y (wi hin 20 kb) o such a ian s in ou GWAS da a. The exac SNPs epo ed in p e ious s udies did no show associa ions o we e no p esen in ou GWAS da a. The esul s o he a ian s nea he epo ed b onchioli is-associa ed SNPs a e p esen ed in Supplemen a yTableS2 ( a ian s wi h p < 0.01 in he b onchioli is GWAS o RSV GWAS a e shown). The bes SNP was s56039226 in he CX3CR1 in on (p = 5.7 × 10−5). This SNP is loca ed 11 kb ups eam o he nonsynonymous CX3CR1 a ian , s3732378 (p e iously T280M). The A-allele–con aining geno ypes o s3732378 we e associa ed wi h RSV b onchioli is se e i y in a p e ious s udy by Amana idou e al.19. The exac SNP was p esen in ou GWAS da a, bu i did no show an associa ion (p = 0.095). Howe e , he A-allele was also sligh ly mo e equen in he GWAS cases compa ed o he con ols (allele equencies o 0.16 and 0.14 in cases and con ols, espec i ely). SNPs associa ed wi h as hma in p e ious s udies. When we sc eened SNPs ha ha e been epo ed o be associa ed wi h as hma in he NHGRI-EBI GWAS Ca alog (wi h p ≤ 10−6), no associa ions we e seen in ou da a. The bes SNP was s12436663 (p = 5.8 × 10−4, OR = 1.96 o T allele in RSV GWAS) wi hin MRPP3 in on. The egions (20 kb) a ound he as hma-associa ed SNPs showed mode a e signals in ou da a. The esul s a e shown o a ian s wi h p ≤ 0.0001 in GWAS, RSV GWAS o GWAS wi h cases < 1yea (Supplemen a yTableS3). Many o he a ian s a e known eQTLs. The a ian s showing s onges sugges i e signals we e in he a ea o VCAN, HLA-DQA1, and NIN. Those genes ha e a ious oles including cell adhesion, cy okine p oduc ion, an i- gen binding and GTP binding20. Associa ions o s269094, s9591920, and s1537091 wi h b onchioli is in he eplica ion popula ions. In he eplica ion phase, we analyzed 77 o he mos p omising SNPs based on he esul s o he GWA analysis in an independen popula ion collec ed in he Ne he lands (416 cases and 432 con ols). The sugges i e associa ions o h ee o he SNPs ( s269094, s9591920, and s1537091) wi h b onchioli is we e nom- inally eplica ed (p < 0.05), and me a-analysis o hese SNPs yielded lowe p alues compa ed o he o iginal GWAS (Table3). The esul s we e simila o he RSV GWAS and GWAS o in an s aged < 1 yea . The esul s o he SNPs wi h a p alue < 0.1 in he Du ch popula ion and ORs in he same di ec ion in he GWAS subse s, and me a-analyses, a e shown in Supplemen a yTableS4. To iden i y genes o which exp ession is po en ially a ec ed by ou candida e SNPs (p alue < 0.05 in he Du ch eplica e), we que ied geno ype- issue exp ession (GTEx) da a21. Rs269094 was p e iously iden i ied as www.na u e.com/scien i ic epo s/ 3 Scien i ic RepoR s | 7:41653 | DOI: 10.1038/s ep41653 an eQTL o KCND3 in spleen (p = 7.1 × 10−10); he C-allele is associa ed wi h highe exp ession. Ano he co e- la ed SNP in he same egion ( s269101, 2 = 0.42 in 1000 genomes phase3 Finnish indi iduals) showed a end owa d associa ion in ou GWAS (p = 4.2 × 10−5) and in he eplica e (p = 0.054), u he suppo ing he esul . By explo ing he NHGRI-EBI GWAS Ca alog, we also disco e ed ha a ian s in o nea KCND3 ha e been asso- cia ed wi h diisocyani e-induced as hma and he cy okine esponse o smallpox22. The wo o he candida e SNPs we e no iden i ied as known eQTLs. We u he sc eened he h ee SNPs (wi h p < 0.05 in he Du ch popula ion) in he Finnish case da a collec ed in Tu ku, using he No dicDB popula ion con ols as a e e ence (Supplemen a yTableS5). Rs9591920 showed an associa ion (p = 0.042, OR = 1.5). The allele equencies o he wo o he SNPs, s269094 and s1537091, did no di e be ween cases and con ols in his analysis. Sugges i e GWA locus ╤Selec ed SNP in o ma ion Ch Range Ж Span (kb) SNPs Lowes pSNP name Loca ionЖGenes** A1 MAF OR‡ (95% CI) pCases Con ols 1 246851155..246968053 116 119 3.2 × 10−6 s61202512 246860213 SCCPDH, CNST A 0.464 0.327 1.67 (1.32–2.11) 1.6 × 10−5 2 235168096..235202022 33 29 2.8 × 10−6 s11886348 235193436 LOC105373933 G 0.079 0.027 3.35 (2.02–5.55) 2.8 × 10−6 3 5377711..5570764 193 24 4.7 × 10−6 s79062720 5442550 MIR4790, EDEM1 C 0.064 0.021 3.79 (2.14–6.70) 4.7 × 10−6 3 39318192..39593234 275 38 3.2 × 10−6 s56039226 39318192 CX3CR1 A 0.041 0.011 4.43 (2.15–9.14) 5.7 × 10−5 3 107206802..107233889 27 6 3.0 × 10−6 s6437736 107208688 BBX, CCDC54 A 0.366 0.491 0.58 (0.47–0.73) 3.0 × 10−6 3 153780474..153973898 193 9 5.0 × 10−6 s36086140 153973898 ARHGEF26 T 0.093 0.037 2.86 (1.82–4.48) 5.0 × 10−6 4 24735463..24736258 0.8 2 2.6 × 10−6 s4697072 24735463 SOD3, CCDC149 G 0.410 0.532 1.77 (1.40–2.25) 2.6 × 10−6 5 167829645..168048306 218 6 6.0 × 10−7 s4976604 167849667 WWC1 C 0.064 0.021 3.80 (2.15–6.71) 4.3 × 10−6 7 46319511..46524796†205 9 1.5 × 10−7 s62452699 46492326 LOC105375265, LOC105375266 T 0.143 0.060 2.83 (1.92–4.17) 1.5 × 10−7 10 50452677..50460878†8 10 4.9 × 10−7 s17009617 50453933 VSTM4, C10o 71 A 0.090 0.032 3.39 (2.11–5.46) 4.9 × 10−7 10 50454331..50553085†98 50 3.9 × 10−7 s76728164 50465201 C10o 71, DRGX T 0.088 0.032 2.93 (1.86–4.62) 3.7 × 10−6 10 50454331..50553085†98 50 3.9 × 10−7 s1001338 50498449 C10o 71, DRGX G 0.086 0.031 3.10 (1.95–4–93) 1.7 × 10−6 10 128584158..128805211 221 12 2.1 × 10−6 s111255454 128777619 DOCK1 G 0.065 0.021 3.90 (2.22–6.84) 2.1 × 10−6 12 17376616..17514609 137 37 3.8 × 10−6 s2137526 17377995 LOC105369677, LOC105369676 T 0.233 0.148 1.98 (1.48–2.65) 3.8 × 10−6 13 59304857..59481192 176 40 2.0 × 10−6 s9591920 59315119 LOC105370220 T 0.190 0.098 2.16 (1.57–2.97) 2.0 × 10−6 13 67847391..67877199 29 4 4.6 × 10−7 s142670120 67850108 PCDH9, LOC105370246 DEL 0.099 0.037 3.23 (2.05–5.10) 4.6 × 10−7 16 73130946..73140092 9 5 2.4 × 10−6 s76079505 73136490 ZFHX3, C16o 47 T 0.203 0.100 2.22 (1.59–3.09) 2.4 × 10−6 17 72973962..73220824 246 12 2.1 × 10−6 s113409681 72973962 HID1, CDR2L A 0.076 0.025 3.58 (2.11–6.05) 2.1 × 10−6 20 56486748..56527179 40 12 1.3 × 10−6 s379083* 56527179 MIR4532, LOC100129869 T 0.112 0.043 2.82 (1.86–4.29) 1.3 × 10−6 21 42284256..42343366 59 52 2.6 × 10−6 s117674297 42343366 DSCAM, BACE2 A 0.092 0.038 2.96 (1.88–4.65) 2.6 × 10−6 Table 1. S onges sugges i e GWA loci (p < 5 × 10−6) iden i ied by b onchioli is GWAS and SNPs selec ed o eplica ion geno yping om each locus. ╤Each sugges i e GWA locus comp ises se e al SNPs in LD ( 2 > 0.5, p < 0.01). Loci we e o med wi h de aul op ions o LD-based esul clumping (PLINK 1.9). We selec ed one a ian o eplica ion geno yping om each locus. Signals in spo adic impu ed a ian s (no shown) we e conside ed un eliable and no chosen o eplica ion geno yping. Va ian s wi h a mino allele equency o < 0.02 wi hin ei he cases o con ols we e no chosen o eplica ion geno yping. ЖRange and loca ion e e o human genome build 37 (GRCh37/hg19) coo dina es. **Co esponding locus shown o a ian s wi hin genes; wo nea es loci shown o in e genic SNPs. ‡OR o A1 allele unde addi i e model in logis ic eg ession, wi h sex and h ee p incipal componen s as co a ia es. †Sugges i e GWA loci sha ed be ween GWAS and RSV GWAS (o loci wi h p < 5 × 10−6). GWA, genome-wide associa ion; SNP, single nucleo ide polymo phism; Ch , ch omosome; MAF, mino allele equency; OR, odds a io; CI, con idence in e al; LD, linkage disequilib ium. www.na u e.com/scien i ic epo s/ 4 Scien i ic RepoR s | 7:41653 | DOI: 10.1038/s ep41653 Discussion In he p esen s udy, we iden i ied se e al sugges i e GWA loci associa ed wi h i al b onchioli is. Th ee o he SNPs selec ed o eplica ion geno yping showed nominal associa ions (p < 0.05) also in an ex e nal eplica ion popula ion. O hose a ian s, s269094 is a known eQTL o KCND3, which has been linked o di e se unc ions including egula ion o neu o ansmi e elease and hea a e20. Mo e ecen ly, he gene has also been associ- a ed wi h diisocyani e-induced occupa ional as hma and cy okine esponse in smallpox accine ecipien s23,24. A he p o ein le el, KCND3 in e ac s wi h ano he memb ane p o ein, DPP10, o which gene ic a ian s ha e been associa ed wi h condi ions including as hma20,25. The associa ion o ano he SNP wi h p < 0.05 in he Du ch popula ion, s9591920, was nominally eplica ed in he addi ional Finnish case se . The a ian esides wi hin an uncha ac e ized noncoding RNA (ncRNA) locus (LOC105370220)20. Thus, i is di icul o assign po en ial unc- ions o his egion. In gene al, in e genic o noncoding SNPs ha e been associa ed wi h many complex pheno- ypes in p e ious GWASs. The SNPs wi hin ncRNA loci could a ec ei he he exp ession o unc ion o he RNA molecule, which may u he egula e a ge gene exp ession26. The hi d a ian showing nominal associa ion in he Du ch coho , s1537091, is loca ed in an in e genic egion, nea es loci being long in e genic non-p o ein coding RNAs wi hou known unc ion. Fu he s udy is equi ed o alida e he associa ions o ou candida e a ian s and s udy hei po en ial mechanism in b onchioli is suscep ibili y. We de ec ed only modes signals o he egions a ound SNPs epo ed by p e ious s udies o be associa ed wi h b onchioli is. This migh be due o allele equency o LD s uc u e di e ences among s udy popula ions, alse disco e ies by chance due o limi ed popula ion size o gene-en i onmen in e ac ions. The con ounding e ec o popula ion s a i ica ion is di icul o ake in o accoun in candida e gene s udies; in he p esen s udy, we could accoun o he sligh s uc u e caused by di e ing gene ic o igins by exploi ing he genome-wide da a and p incipal componen s and hus educe he isk o alse-posi i e indings. Fu he , we could exclude popula- ion ou lie s and close ela i es based on in o ma ion p o ided by genome-wide sampling. In ou da a, he bes a ian nea he SNP ha was p e iously epo ed o be associa ed wi h b onchioli is was in CX3CR1 in on (p = 5.7 × 10−5). Sugges i e RSV GWA locus╤Selec ed SNP in o ma ion Ch RangeЖ Span (kb) SNPs Lowes pSNP name Loca ionЖGenes** A1 MAF OR‡ (95% CI) pCases Con ols 1 153512691..153996110 483 6 5.2 × 10−7 s75940909 153932075 SLC39A1 A 0.063 0.011 5.58 (2.69– 11.93) 9.2 × 10−6 1 154011426..154260767 249 4 1.1 × 10−6 s186841738 154187935 C1o 43 A 0.062 0.009 6.73 (3.02– 15.01) 3.1 × 10−6 2 36592600..36617435 24 2 2.4 × 10−6 s112913823 36617435 CRIM1 G 0.086 0.025 4.38 (2.37–8.09) 2.4 × 10−6 3 185675973..185677613 1 9 4.9 × 10−6 s6444089 185676350 TRA2B, ETV5 G 0.277 0.410 0.48 (0.34–0.66) 8.7 × 10−6 3 185675973..185677613 1 9 4.9 × 10−6 s55675198 185677613 TRA2B, ETV5 G 0.252 0.391 0.46 (0.33–0.64) 6.2 × 10−6 4 32653789..32869901 216 52 4.7 × 10−6 s4547837 32688632 LOC107986223, LOC101927363 T 0.054 0.010 6.55 (2.93– 14.65) 4.7 × 10−6 7 46319511..46524796†205 9 6.9 × 10−7 s62452699 46492326 LOC105375265, LOC105375266 T 0.149 0.061 3.37 (2.09–5.45) 6.9 × 10−7 7 148765688..148959272 193 21 5.5 × 10−7 s139743624 148870713 ZNF398 A 0.051 0.010 6.51 (2.86– 14.86) 8.5 × 10−6 10 50452677..50460878†8 10 3.1 × 10−7 s17009617 50453933 VSTM4, C10o 71 A 0.111 0.032 4.16 (2.41–7.19) 3.1 × 10−7 10 50454331..50553085†98 50 1.2 × 10−7 s76728164 50465201 C10o 71, DRGX T 0.106 0.033 3.46 (2.05–5.85) 3.6 × 10−6 10 50454331..50553085†98 50 1.2 × 10−7 s1001338 50498449 C10o 71, DRGX G 0.112 0.031 3.95 (2.33–6.69) 3.6 × 10−7 15 39354517..39515134 160 46 6.5 × 10−7 s75360543 39505815 LOC105370777 G 0.104 0.034 4.10 (2.35–7.14) 6.5 × 10−7 Table 2. S onges sugges i e GWA loci (p < 5 × 10−6) om RSV b onchioli is GWAS and SNPs selec ed o eplica ion geno yping om each locus. ╤Each sugges i e GWA locus comp ises se e al SNPs in LD ( 2 > 0.5, p < 0.01). Loci we e o med wi h de aul op ions o LD-based esul clumping (PLINK 1.9). We selec ed one a ian o eplica ion geno yping om each locus. Signals in spo adic impu ed a ian s (no shown) we e conside ed un eliable and no chosen o eplica ion geno yping. ЖRange and loca ion e e o human genome build 37 (GRCh37/hg19) base pai posi ions. **Co esponding locus shown o a ian s wi hin genes; wo nea es genes shown o in e genic SNPs. ‡OR o A1 allele unde addi i e model in logis ic eg ession, wi h sex and h ee p incipal componen s as co a ia es. †Sugges i e GWA loci sha ed be ween GWAS and RSV GWAS (o loci wi h p < 5 × 10−6). GWA, genome-wide associa ion; RSV, espi a o y syncy ial i us; SNP, single nucleo ide polymo phism; Ch , ch omosome; MAF, mino allele equency; OR, odds a io; CI, con idence in e al; LD, linkage disequilib ium. www.na u e.com/scien i ic epo s/ 5 Scien i ic RepoR s | 7:41653 | DOI: 10.1038/s ep41653 Due o sugges ed sha ed gene ic con ibu ion among b onchioli is and as hma, a ian s ha ha e been associa ed wi h as hma in p e ious s udies, and egions (20 kb) a ound he a ian s, we e sc eened in ou da a. Mode a e signals (p < 0.0001) we e de ec ed in a ian s wi hin genes including VCAN and HLA-DQA1. HLA-DQ egion has been associa ed wi h di e en ypes o as hma in se e al s udies27, and an associa ion o VCAN wi h as hma was disco e ed mo e ecen ly28. As hma clus e s in amilies and likely has a s ong gene ic componen , wi h he i abili y es ima es as high as 60%29. Hund eds o gene ic polymo phisms ha e been associa ed wi h di - e en as hma pheno ypes in pas gene ic s udies. Howe e , as hma is a complex and he e ogeneous disease which is likely con ibu ed by a combina ion o many genes in in e ac ion wi h en i onmen al ac o s30. Thus, he po en- ial oles o he sugges ed genes in LRI suscep ibili y, and an o e lap wi h biological pa hways ela ed o as hma, wa an u he s udy. The p esen s udy had se e al limi a ions. The sample size was ela i ely small, and hus especially he s a i- ied GWAS subse analyses migh ha e been unde powe ed. In he disco e y GWAS, he bes sugges i e associa- ion signals, ha we e almos genome-wide signi ican , we e seen in he a ea o LOC105375265 on ch omosome 7 and in he a ea o VSTM4 and DRGX on ch omosome 10. The signals in hese egions we e iden i ied bo h in he GWAS and he RSV GWAS bu no in he Du ch eplica ion popula ion. Despi e he lack o eplica ion, hese signals migh well e lec ue associa ions in he Finnish popula ion. The e is no wo ldwide consensus on ea men and diagnosis o b onchioli is. The Ame ican Academy o Pedia ics de ines 24 mon hs as an uppe age limi o b onchioli is2,31. In Finland and Sweden, clinicians ha e begun o conside b onchioli is o be mainly a disease o in an s unde 12 mon hs o age32. In ou GWAS, mos o he cases we e < 12 mon hs o age a he ime o he diagnosis. Mo eo e , o he mos pa he esul s o he GWAS < 1 yea we e simila o hose o he main GWAS. Howe e , he di e ing clinical de ini ions o included b onchioli is cases we e one limi a ion o he cu en s udy. Fu he limi a ion was he di e ing p opo ion o RSV posi i e samples be ween he GWAS and he eplica ion popula ions. In s udies o come, selec ing b onchi- oli is s udy cases acco ding o mo e speci ied c i e ia will likely help de ec ing g ea e e ec s and suscep ibili y genes associa ed wi h speci ic pheno ypes. The p esen GWAS iden i ied se e al a ian s ha we e sugges i ely associa ed wi h b onchioli is. Al hough no signi ican associa ions we e de ec ed, ou s udy p o ides he i s genome-wide examina ion o b onchioli is and a la ge body o da a ha may bene i u u e s udies on complex gene ic de e minan s and biological pa hways ha cause a ia ion in b onchioli is suscep ibili y. Fu he eplica ion and unc ional alida ion is needed o e - i y and unco e he po en ial oles o ou candida e SNPs. The ela ionship be ween in an i al b onchioli is and as hma, and a plausible gene ic o e lap o he wo diseases, emain an impo an a ea o s udy. Ma e ials and Me hods S udy design and popula ions. The s udy popula ions comp ised a o al o 2045 s udy subjec s consis ing o 835 b onchioli is cases and 1210 con ols (974 emales and 1071 males). The s udy was app o ed by he e hics commi ees o Tampe e Uni e si y Hospi al and Kuopio Uni e si y Hospi al, Finland, and he egional e hics commi ee o Go henbu g, Sweden. The s udy was ca ied ou in acco dance wi h he app o ed guidelines o he WMA Decla a ion o Helsinki. In o med consen s we e ob ained om he s udy subjec s o p o ided by hei pa en s. The main elemen s o he s udy we e: (1) a GWAS in a Finnish–Swedish disco e y popula ion o 217 cases and 778 con ols, (2) an analysis o he mos p omising a ian s in a Du ch eplica ion popula ion o 416 cases and 432 con ols, and (3) a u he examina ion o he mos p omising a ian s in an addi ional Finnish se o 202 cases. Mo eo e , GWAS cases we e s a i ied acco ding o age o p esence o RSV and he subg oups o RSV posi i e cases (“RSV GWAS”) and hose o < 12 mon hs o age we e s udied sepa a ely (Fig.1). The inclusion c i e ia o all he a ec ed child en we e (1) clinically diagnosed b onchioli is, (2) an age < 24 mon hs a he ime o diagnosis and (3) na i e Finnish, Swedish o Du ch e hnici y. B onchioli is was de ined as he p esence o LRI and espi a o y symp oms ypical o b onchioli is, including wheezing and labo ed b ea h- ing2. RSV in ec ion was es ed by an igen immuno luo escen o adioimmuno assays, i al isola ion, PCR o nasopha yngeal cells o by se um an ibody assays. O e all, RSV was de ec ed in 72% o he cases, and > 80% we e unde 12 mon hs o age. Cha ac e is ics o b onchioli is cases in GWAS disco e y coho s and alida ion coho s a e p esen ed in Tables4 and 5. The Va ian de ails GWAS Replica ion Me a-analysis§ SNP Ch Loca ionЖGenes** A1 OR‡ (95% CI) pOR╤ (95% CI) pOR (95% CI) p s269094 1 112639174 KCND3, LOC643355 C1.67 (1.30–2.15) 5.3 × 10−51.28 (1.03–1.60) 0.0292 1.44 (1.22–1.70) 1.5 × 10−5 s9591920 13 59315119 LOC105370220 T2.16 (1.57–2.97) 2.0 × 10−61.41 (1.05–1.88) 0.0227 1.71 (1.38–2.12) 9.8 × 10−7 s1537091 21 29004226 LOC105372763, LINC00113 A1.69 (1.34–2.14) 1.3 × 10−51.24 (1.01–1.52) 0.0408 1.42 (1.21–1.66) 1.1 × 10−5 Table 3. SNPs ha had a p alue o <0.05 in he Du ch eplica e, wi h ORs in he same di ec ion compa ed o GWAS. ЖLoca ion e e s o human genome build 37 (GRCh37/hg19) coo dina es. **Co esponding locus shown o a ian s wi hin genes; wo nea es genes shown o in e genic SNPs. ‡OR o A1 allele unde addi i e model in logis ic eg ession, wi h sex and h ee p incipal componen s as co a ia es. ╤OR o A1 allele unde addi i e model in logis ic eg ession, wi h sex as a co a ia e. §Me a-analysis pe o med unde ixed-e ec s model o logis ic eg ession esul iles o disco e y and eplica ion da a. SNP, single nucleo ide polymo phism; GWAS, genome-wide associa ion s udy; RSV, espi a o y syncy ial i us; OR, odds a io; CI, con idence in e al; Ch , ch omosome. www.na u e.com/scien i ic epo s/ 6 Scien i ic RepoR s | 7:41653 | DOI: 10.1038/s ep41653 popula ions a e u he desc ibed in he Supplemen a yMa e ial. B ie ly, he Finnish indi iduals (n = 936) in he GWAS disco e y popula ion o igina ed om Kuopio (64 cases, 64 con ols), Tampe e (124 cases), and Helsinki/ Kuopio (684 con ols) egions33–36. A subse o he GWAS popula ion was collec ed in Go henbu g, Sweden (29 cases, 30 con ols)37. Replica ion coho s o igina ed om The Ne he lands (n = 848) and Finland (n = 202)10,38,39. DNA sample p epa a ion and geno yping. Genomic DNA was ob ained om whole blood samples, buccal swabs, o bu y coa s. DNA om blood samples collec ed om he GWAS subjec s was ex ac ed wi h he Ul aClean® Blood DNA Isola ion Ki (MO BIO Labo a o ies, Inc., Ca lsbad, CA, USA). DNA om in an s in he Du ch eplica ion coho was isola ed om blood samples o buccal swabs wi h he QIAamp DNA Blood Ki (Qiagen, Hilden, Ge many). DNA ex ac ion om bu y coa s collec ed om he Du ch popula ion con ols was done by diges ion wi h p o einase K ollowed by sal ing ou wi h po assium ace a e and chlo o o m–isoamyl alcohol ex ac ion. Genome-wide geno yping was pe o med wi h he Human Geno yping A ay Ki s (Illumina, San Diego, CA, USA. (Supplemen a yTableS6). The Technology Cen e a he Ins i u e o Molecula Medicine Finland (FIMM), Uni e si y o Helsinki, did he geno yping. FIMM also pe o med he a ge ed SNP geno yping in he eplica ion phase wi h he Sequenom iPLEX Gold assay (San Diego, CA, USA). No dicDB con ols a e desc ibed in de ail in Bilgu a e al.40. Quali y con ol and impu a ion o he genome-wide da a. A e quali y con ol (QC), 995 s udy subjec s we e a ailable o he analyses: 464 male subjec s and 531 emale subjec s/217 cases and 778 con ols. The e we e 79,563 au osomal SNPs wi h a MAF o > 0.01. The da a was p ephased wi h SHAPEIT2, ollowed by geno ype impu a ion (i.e., s a is ical p edic ion o missing geno ypes) wi h IMPUTE241,42. Mo e de ailed desc ip- ion o he QC and impu a ion can be ound in he online Supplemen a yMa e ial. A e impu a ion, he amoun o SNPs (MAF > 0.01) was 5,304,323. Selec ion o SNPs o eplica ion analysis. Se en y-se en SNPs we e geno yped in he Du ch eplica ion popula ion. Because he s ingen genome-wide signi icance le el (p < 5 × 10−8) was no eached in he disco e y GWAS, less-s ic c i e ia we e applied o he selec ion o SNPs. p alues o < 5 × 10−4 and < 1 × 10−4 o gen- o yped and impu ed SNPs, espec i ely, we e used as h eshold alues o sugges i e associa ions. Fo impu ed SNPs, linkage disequilib ium (LD) was aken in o accoun by selec ing ela i ely independen ( 2 < 0.5) SNPs om each sugges i e GWA locus. Spo adic impu ed SNPs we e excluded. P o ided ha a sugges i e GWA locus had se e al a ian s wi h simila p alues, he bes SNP was chosen based on p edic ed o empi ical egula o y unc ions (including eQTLs, p omo e ac i i y, and p o ein binding si es) by explo ing anno a ion da abases, including HaploReg . 4.1, GTEx, and dbSNP21,43,44. We did no choose low- equency SNPs (MAF < 0.02 in cases o con ols) in he main GWAS popula ion o a oid geno yping monomo phic SNPs in he Du ch popula ion. Figu e 1. Flow cha o s udy design and popula ions. P ima y endpoin s udied in GWAS was i al b onchioli is. Cases we e u he s a i ied in o subse s acco ding o p esence o RSV and age. S a i ied pheno ypes s udied we e b onchioli is caused by espi a o y syncy ial i us (“RSV GWAS”) and b onchioli is in in an s < 12 mon hs o age. All Kuopio, FIN Tampe e, FIN Go henbu g, SWE n % n % n % n % All cases 217 100% 64 100% 124 100% 29 100% RSV posi i e 121 56% 21 33% 90 73% 10 36% RSV nega i e 94 43% 41 64% 34 27% 19 64% Age < 12 mon hs 173 80% 33 52% 124 100% 16 55% Age > 12 mon hs 42 19% 29 45% 0 0% 13 45% Males 114 53% 36 56% 62 50% 16 55% Hospi aliza ion a e 100% 100% 100% 100% Table 4. Cha ac e is ics o s udy cases in GWAS b onchioli is popula ions. GWAS, genome-wide associa ion s udy; FIN, Finland; SWE, Sweden. www.na u e.com/scien i ic epo s/ 7 Scien i ic RepoR s | 7:41653 | DOI: 10.1038/s ep41653 We es ed he homogenei y o odds a ios among he GWAS subpopula ions acco ding o ci y o o igin wi h he B eslow–Day es and excluded a ian s wi h p alues o < 0.01. S a is ical analysis. P incipal componen analysis (PCA). PCA wi h PLINK1.9 was pe o med on nonim- pu ed LD-p uned GWAS da a o delinea e componen s explaining possible emaining popula ion s uc u e. The adequa e numbe o componen s was e alua ed based on genome-wide in la ion ac o lambda (λ ), eigen alues, and inspec ion o co a ia e signi icances in logis ic eg ession. Th ee leading componen s we e inco po a ed in o subsequen associa ion analyses. SNP associa ion analysis. Allele equency di e ences we e assessed wi h logis ic eg ession unde an addi- i e model, wi h gende and h ee p incipal componen s as co a ia es. Analyses we e pe o med wi h PLINK 1.945. Lambda alues we e close o 1, indica ing an adequa ely con olled popula ion s uc u e (λ = 1.004 o he geno yped popula ion, λ = 1.06 o he impu ed popula ion). In he RSV GWAS, we s udied a subse o cases posi i e o RSV in ec ion (n = 121) agains he GWAS con ols (n = 778). Simila ly, cases ha we e < 12 mon hs o age (173 cases, 778 con ols) we e es ed as a g oup. Replica ion popula ions collec ed in he Ne he lands and Tu ku, Finland, we e s udied wi h logis ic eg ession analysis unde an addi i e model, wi h gende as a co a ia e. Measu es o basic QC, which en ailed emo ing a ian s wi h MAF < 0.01, geno yping a e < 0.9, o de ia ion om HWE (p < 0.001), we e pe o med. The eplica ion case se o Tu ku was s udied agains he No dicDB popula ion con ols (n = 684). The disco e y and he Du ch eplica ion popula ion we e s udied by me a-analysis unde a ixed-e ec s model o es ima e hei combined e ec . Con idence in e als o odds a ios o me a-analysis we e calcula ed wi h a me a package in R46,47. Finally, SNPs ha we e epo ed in he li e a u e o be associa ed wi h RSV b onchioli is o as hma, o a ian s wi hin 20 kb o such SNPs, we e sc eened in ou GWAS da a. Re e ences 1. Dawson-Caswell, M. & Muncie, H. L. J . Respi a o y syncy ial i us in ec ion in child en. Am. Fam. Physician 83, 141–146 (2011). 2. Rals on, S. L. e al. Clinical p ac ice guideline: he diagnosis, managemen , and p e en ion o b onchioli is. Pedia ics 134, e1474–502 (2014). 3. Amana idou, V., Apos olakis, S. & Spandidos, D. A. Gene ic di e si y o he hos and se e e espi a o y syncy ial i us-induced lowe espi a o y ac in ec ion. Pedia . In ec . Dis. J. 28, 135–140 (2009). 4. Rame , M., Ko ppi, M. & Hallman, M. Pa e n ecogni ion ecep o s and gene ic isk o s in ec ion: alue o clinical decision- making? Pedia . Pulmonol. 46, 101–110 (2011). 5. Meissne , H. C. Vi al B onchioli is in Child en. N. Engl. J. Med. 374, 62–72 (2016). 6. Tan, L. e al. Gene ic Va iabili y among Comple e Human Respi a o y Syncy ial Vi us Subg oup A Genomes: B idging Molecula E olu iona y Dynamics and Epidemiology. PLoS One 7, doi: 10.1371/jou nal.pone.0051439 (2012). 7. Andabaka, T. e al. Monoclonal an ibody o educing he isk o espi a o y syncy ial i us in ec ion in child en. Coch ane Da abase Sys . Re . (4), CD006602, doi, CD006602 (2013). 8. D ysdale, S. B., G een, C. A. & Sande, C. J. Bes p ac ice in he p e en ion and managemen o paedia ic espi a o y syncy ial i us in ec ion. The . Ad . In ec . Dis. 3, 63–71 (2016). 9. Thomsen, S. F. e al. Inc eased conco dance o se e e espi a o y syncy ial i us in ec ion in iden ical wins. Pedia ics 121, 493–496 (2008). 10. Janssen, R. e al. Gene ic suscep ibili y o espi a o y syncy ial i us b onchioli is is p edominan ly associa ed wi h inna e immune genes. J. In ec . Dis. 196, 826–834 (2007). 11. Fo on, J. T. e al. Gene ic associa ion s udy o RSV b onchioli is in in ancy a he 5q31 cy okine clus e . Tho ax 64, 345–352 (2009). 12. Choi, E. H., Lee, H. J. & Chanock, S. J. Human gene ics and espi a o y syncy ial i us disease: cu en indings and u u e app oaches. Cu . Top. Mic obiol. Immunol. 372, 121–137 (2013). 13. Lah i, M. e al. Su ac an p o ein D gene polymo phism associa ed wi h se e e espi a o y syncy ial i us in ec ion. Pedia . Res. 51, 696–699 (2002). 14. Backman, K., Piippo-Sa olainen, E., Ollikainen, H., Koskela, H. & Ko ppi, M. Adul s ace inc eased as hma isk a e in an RSV b onchioli is and educed espi a o y heal h- ela ed quali y o li e a e RSV pneumonia. Ac a Paedia . 103, 850–855 (2014). 15. Sly, P. D., Kusel, M. & Hol , P. G. Do ea ly-li e i al in ec ions cause as hma? J. Alle gy Clin. Immunol. 125, 1202–1205 (2010). 16. Wu, P. & Ha e , T. V. E idence o a causal ela ionship be ween espi a o y syncy ial i us in ec ion and as hma. Expe Re . An i In ec . The . 9, 731–745 (2011). 17. La kin, E. K. & Ha e , T. V. Genes associa ed wi h RSV lowe espi a o y ac in ec ion and as hma: he applica ion o gene ic epidemiological me hods o unde s and causali y. Fu u e Vi ol. 10, 883–897 (2015). 18. Caliskan, M. e al. Rhino i us wheezing illness and gene ic isk o childhood-onse as hma. N. Engl. J. Med. 368, 1398–1407 (2013). 19. Amana idou, V., Sou inos, G., Apos olakis, S., Tsilimigaki, A. & Spandidos, D. A. T280M a ia ion o he CX3C ecep o gene is associa ed wi h inc eased isk o se e e espi a o y syncy ial i us b onchioli is. Pedia . In ec . Dis. J. 25, 410–414 (2006). Ne he lands Tu ku, Finland n % n % All cases 416 100% 202 100% RSV posi i e* 416 100% 62 31% Males 237 60% 140 69% Age < 12 mon hs 95%** 50% Hospi aliza ion a e 100% 90% Table 5. Cha ac e is ics o b onchioli is cases in eplica ion popula ions. *Only RSV-posi i e b onchioli is cases o iginally selec ed10. **Cases we e andomly acqui ed om a popula ion o > 95% o he cases < 12 mon hs o age10. www.na u e.com/scien i ic epo s/ 8 Scien i ic RepoR s | 7:41653 | DOI: 10.1038/s ep41653 20. NCBI Resou ce Coo dina o s. Da abase esou ces o he Na ional Cen e o Bio echnology In o ma ion. Nucleic Acids Res. 44, D7–19 (2016). 21. GTEx Conso ium. The Geno ype-Tissue Exp ession (GTEx) p ojec . Na . Gene . 45, 580–585 (2013). 22. Bu de , T. e al. The NHGRI-EBI Ca alog o published genome-wide associa ion s udies. A ailable a : www.ebi.ac.uk/gwas. Accessed 23.6.2016. 23. Kennedy, R. B. e al. Genome-wide analysis o polymo phisms associa ed wi h cy okine esponses in smallpox accine ecipien s. Hum. Gene . 131, 1403–1421 (2012). 24. Yucesoy, B. e al. Genome-Wide Associa ion S udy Iden i ies No el Loci Associa ed Wi h Diisocyana e-Induced Occupa ional As hma. Toxicol. Sci. 146, 192–201 (2015). 25. B eue , K. e al. Inna eDB: sys ems biology o inna e immuni y and beyond– ecen upda es and con inuing cu a ion. Nucleic Acids Res. 41, D1228–33 (2013). 26. Edwa ds, S. L., Beesley, J., F ench, J. D. & Dunning, A. M. Beyond GWASs: illumina ing he da k oad om associa ion o unc ion. Am. J. Hum. Gene . 93, 779–797 (2013). 27. Kon akio i, E., Dom i, K., Papakos a, D. & Daniilidis, M. HLA and as hma pheno ypes/endo ypes: a e iew. Hum. Immunol. 75, 930–939 (2014). 28. Ree es, S. R. e al. Subepi helial Accumula ion o Ve sican in a Cock oach An igen-Induced Mu ine Model o Alle gic As hma. J. His ochem. Cy ochem. 64, 364–380 (2016). 29. Lee, J. U., Kim, J. D. & Pa k, C. S. Gene-En i onmen In e ac ions in As hma: Gene ic and Epigene ic E ec s. Yonsei Med. J. 56, 877–886 (2015). 30. Kim, H. Y., DeK uy , R. H. & Ume su, D. T. The many pa hs o as hma: pheno ype shaped by inna e and adap i e immuni y. Na . Immunol. 11, 577–584 (2010). 31. Ame ican Academy o Pedia ics Subcommi ee on Diagnosis and Managemen o B onchioli is. Diagnosis and managemen o b onchioli is. Pedia ics 118, 1774–1793 (2006). 32. Mecklin, M., Hesselma , B., Q is , E., Wenne g en, G. & Ko ppi, M. Diagnosis and ea men o b onchioli is in Finnish and Swedish child en’s hospi als. Ac a Paedia . 103, 946–950 (2014). 33. Leu, M. e al. No dicDB: a No dic pool and po al o genome-wide con ol da a. Eu . J. Hum. Gene . 18, 1322–1326 (2010). 34. Koponen, P., Helminen, M., Paassil a, M., Luukkaala, T. & Ko ppi, M. P eschool as hma a e b onchioli is in in ancy. Eu . Respi . J. 39, 76–80 (2012). 35. Ruo salainen, M., Piippo-Sa olainen, E., Hy a inen, M. K. & Ko ppi, M. Adul hood as hma a e wheezing in in ancy: a ques ionnai e s udy a 27 yea s o age. Alle gy 65, 503–509 (2010). 36. Ruo salainen, M., Hy a inen, M. K., Piippo-Sa olainen, E. & Ko ppi, M. Adolescen as hma a e hino i us and espi a o y syncy ial i us b onchioli is. Pedia . Pulmonol. 48, 633–639 (2013). 37. Gokso , E. e al. High isk o adul as hma ollowing se e e wheezing in ea ly li e. Pedia . Pulmonol. 50, 789–797 (2015). 38. Ja i, T. e al. Sho - and long- e m e icacy o p ednisolone o i s acu e hino i us-induced wheezing episode. J. Alle gy Clin. Immunol. 135, 691–8, e9 (2015). 39. Leh inen, P. e al. P ednisolone educes ecu en wheezing a e a i s wheezing episode associa ed wi h hino i us in ec ion o eczema. J. Alle gy Clin. Immunol. 119, 570–575 (2007). 40. Bilgu a , K. e al. Suscep ibili y loci o in ac anial aneu ysm in Eu opean and Japanese popula ions. Na . Gene . 40, 1472–1477 (2008). 41. O’Connell, J. e al. A Gene al App oach o Haplo ype Phasing ac oss he Full Spec um o Rela edness. PLoS Gene . 10, e1004234, doi: 10.1371/jou nal.pgen.1004234 (2014). 42. Howie, B. N., Donnelly, P. & Ma chini, J. A lexible and accu a e geno ype impu a ion me hod o he nex gene a ion o genome- wide associa ion s udies. PLoS Gene . 5, e1000529 (2009). 43. Boyle, A. P. e al. Anno a ion o unc ional a ia ion in pe sonal genomes using RegulomeDB. Genome Res. 22, 1790–1797 (2012). 44. She y, S. T. e al. dbSNP: he NCBI da abase o gene ic a ia ion. Nucleic Acids Res. 29, 308–311 (2001). 45. Chang, C. C. e al. Second-gene a ion PLINK: ising o he challenge o la ge and iche da ase s. Gigascience 4, 7-015-0047-8. eCollec ion 2015 (2015). 46. R De elopmen Co e Team. R: A language and en i onmen o s a is ical compu ing. R Founda ion o S a is ical Compu ing, Vienna, Aus ia. ISBN 3-900051-07-0, URL h p://www.R-p ojec .o g. (2008). 47. Schwa ze , G. me a: An R package o me a-analysis. h ps://c an. -p ojec .o g/doc/Rnews/Rnews_2007-3.pd . R News, 7(3), 40–45, 7, 40–41, 42, 43, 44, 45 (2007). Acknowledgemen s We hank Maa i Haa ala (Uni e si y o Oulu, Oulu, Finland) o echnical assis ance; Pe i Koponen, Piia Nu mi, Me ja Helminen (Cen e o Child Heal h Resea ch, Tampe e Uni e si y and Uni e si y Hospi al, Tampe e, Finland), He Quishui (Depa men o Medical Mic obiology and Immunology, Uni e si y o Tu ku, Tu ku, Finland), Riny Janssen (Cen e o Heal h P o ec ion, Na ional Ins i u e o Public Heal h and he En i onmen , Bil ho en, The Ne he lands) and Heidi Jokinen (Tu ku Uni e si y Hospi al, Tu ku, Finland) o assis ance in sample and pa ien da a collec ion; and Ki a Helle o language edi ing. CSC – IT Cen e o Science, Finland, is acknowledged o compu a ional esou ces. The geno yping o SNP ma ke s was pe o med by he Technology Cen e, Ins i u e o Molecula Medicine Finland (FIMM), Uni e si y o Helsinki. S udies wi h he anonymous Finnish popula ion con ols we e suppo ed by he No dic Cen e o Excellence in Disease Gene ics. The esea ch was inanced by he Alma and K.A. Snellman Founda ion (AP), he Päi ikki and Saka i Sohlbe g Founda ion (AP), he Sig id Jusélius Founda ion (MH), he Compe i i e S a e Resea ch Financing o he Expe Responsibili y A ea o Tampe e Uni e si y Hospi al (MiR), he Compe i i e S a e Resea ch Financing o he Expe Responsibili y A ea o Oulu Uni e si y Hospi al (MiR), EVO G an Tampe e Uni e si y Hospi al (MK), and Tampe e Tube culosis Founda ion (EP-S). Au ho Con ibu ions Mi.R., M.H., M.K.K., A.P. and M.K. designed he esea ch; A.P. and M.K.K. analyzed he da a; A.P. and M.K.K. w o e he manusc ip ; all au ho s e iewed/ e ised he manusc ip ; L.B., E.P.-S., M.R., E.G., K.K., H.H., K.N., T.J., G.W. and M.K. did/o ganized sample collec ion and con ibu ed ma e ials/ eagen s/clinical da a. Addi ional In o ma ion Supplemen a y in o ma ion accompanies his pape a h p://www.na u e.com/s ep Compe ing inancial in e es s: The au ho s decla e no compe ing inancial in e es s. www.na u e.com/scien i ic epo s/ 9 Scien i ic RepoR s | 7:41653 | DOI: 10.1038/s ep41653 How o ci e his a icle: Pasanen, A. e al. Genome-Wide Associa ion S udy o Polymo phisms P edisposing o B onchioli is. Sci. Rep. 7, 41653; doi: 10.1038/s ep41653 (2017). Publishe 's no e: Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a ilia ions. This wo k is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons license, unless indica ed o he wise in he c edi line; i he ma e ial is no included unde he C ea i e Commons license, use s will need o ob ain pe mission om he license holde o ep oduce he ma e ial. To iew a copy o his license, isi h p://c ea i ecommons.o g/licenses/by/4.0/ © The Au ho (s) 2017