Polymorphism in the gene encoding toll-like receptor 10 may be associated with asthma after bronchiolitis

1 Scien i ic RepoR s | 7: 2956 | DOI:10.1038/s41598-017-03429-x www.na u e.com/scien i ic epo s Polymo phism in he gene encoding oll-like ecep o 10 may be associa ed wi h as hma a e b onchioli is Sa i Tö mänen1, Ma i Ko ppi2, Johanna Te äsjä i3, Juho Vuonon i a3, Pe i Koponen2, Me ja Helminen1, Qiushui He3,4 & Ki si Nuoli i a 5 Toll-like ecep o s (TLRs) ecognise mic obes ha con ibu e o he se e i y o b onchioli is and he subsequen isk o as hma. We e alua ed whe he pos -b onchioli is as hma was associa ed wi h polymo phisms in he TLR3 s3775291, TLR4 s4986790, TLR7 s179008, TLR8 s2407992, TLR9 s187084, and TLR10 s4129009 genes. The gene polymo phisms we e s udied a he age o 6.4 yea s (mean) in 135 child en hospi alised o b onchioli is in in ancy. The ou come measu e was cu en o p e ious as hma. Cu en as hma was mo e common (30%) in child en wi h he a ian AG o GG geno ype in he TLR10 s4129009 gene e sus hose who we e homozygous o he majo allele A (11%) (p = 0.03). The adjus ed odds a io (aOR) was 4.30 (95% CI 1.30–14.29). As hma e e was mo e common (34.6%) in gi ls wi h he TLR7 a ian AT o TT geno ype e sus hose who we e homozygous o he majo allele A (12.5%) (p = 0.03). The adjus ed OR was 3.93 (95% CI 1.06–14.58). Co esponding associa ions we e no seen in boys. The e we e no signi ican associa ions be ween TLR3, TLR4, TLR8, o TLR9 polymo phisms and pos -b onchioli is as hma. Polymo phism in he TLR10 gene inc eases and in he TLR7 gene may inc ease he isk o as hma in p eschool-aged child en a e in an b onchioli is. B onchioli is in in ancy inc eases he isk o subsequen wheezing and childhood as hma1. Al hough many as hma isk ac o s, such as as hma in pa en s, a opy o eosinophilia in child en, and hino i us ae iology o b onchioli is2, a e well documen ed, p edic ing he ou come o an indi idual pa ien is no possible. Inna e immu- ni y, which is highly egula ed by genes, plays a c ucial ole in bo h in ec ion and in lamma ion3. The de elop- men o as hma is a complica ed and mul i ac o ial p ocess in which genes in e ac wi h he en i onmen 4. In ea ly li e, he Th2-domina ed immune esponses shi owa ds Th1-domina ed esponses5, bu among gene ically suscep ible indi iduals, en i onmen al ac o s like i uses may lead o he pe sis ence o Th2-domina ed immu- ni y and o subsequen a opy and as hma6. Toll-like ecep o s (TLRs) a e pa e n- ecognising p o eins ha , a e ecognising o eign ma e ial like mic obes, a e able o igge he p oduc ion o media o s o inna e immuni y and, subsequen ly, a e complex signalling p ocesses, he de elopmen o adap i e immune esponses7, 8. TLRs 1, 2, 4, 5, 6, and 10 a e loca ed on he cell su ace, whe eas TLRs 3, 7, 8, and 9 a e loca ed inside he cells9, ecognising mic obial componen s a e endocy osis. TLR1, TLR2, TLR6, and TLR10 comp ise he TLR2 sub amily, and TLR1, TLR2, TLR6, and TLR10 gene polymo phisms seem o play a ole in suscep ibili y o as hma, a opic eczema, and alle gic hini is10–12. TLR3 ecognises double-s anded i al ibonucleic acid (RNA), and, in mice, TLR3 ac i a ion by i uses combined wi h alle gen inhala ion esul ed in alle gic ai way disease13. TLR4 ecognises bac e ial lipopolysaccha ides and he F glycop o ein o he espi a o y syncy ial i us (RSV)14. TLR7 and TLR8, which a e egula ed by genes loca ed in he X ch omosome, ecognise single-s anded i al RNA15. An Ame ican s udy ound ha TLR7 con ibu ed o human ai way elaxa ion ia he p oduc ion o ni ic oxide16. The e is e idence ha polymo phisms in he TLR7 1Tampe e Uni e si y Hospi al, Tampe e, Finland. 2Cen e o Child Heal h Resea ch, Tampe e Uni e si y and Uni e si y Hospi al, Tampe e, Finland. 3Depa men o Medical Mic obiology and Immunology, Tu ku Uni e si y, Tu ku, Finland. 4Depa men o Medical Mic obiology, Capi al Medical Uni e si y, Beijing, China. 5Depa men o Paedia ics, Seinäjoki Cen al Hospi al, Seinäjoki, Finland. Sa i Tö mänen, Ma i Ko ppi, Qiushui He and Ki si Nuoli i a con ibu ed equally o his wo k. Co espondence and eques s o ma e ials should be add essed o K.N. (email: [email p o ec ed]) Recei ed: 30 Sep embe 2016 Accep ed: 28 Ap il 2017 Published: xx xx xxxx OPEN www.na u e.com/scien i ic epo s/ 2 Scien i ic RepoR s | 7: 2956 | DOI:10.1038/s41598-017-03429-x and TLR8 genes a e associa ed wi h suscep ibili y o as hma and ela ed a opic diso de s15 and o suscep ibili y o espi a o y i al in ec ions17. Signalling ia TLR7 and TLR9 a ec s he unc ion o eosinophils, engende ing a link be ween i al in ec ion and alle gic exace ba ions18. Al hough TLR10 is a pa e n- ecogni ion ecep o wi hou known ligand speci ici y, i has shown o be a modula o y ecep o wi h mainly inhibi o y p ope ies19. We ha e p ospec i ely ollowed 166 child en who we e hospi alised o b onchioli is a less han 6 mon hs o age2. A 5 o 7 yea s o age, 127 o he child en a ended a clinical con ol isi , and ques ionnai e da a we e a ailable o ano he 39 child en2. We ha e p e iously s udied he TLR1 s5743618, TLR2 s5743708, and TLR6 s5743810 polymo phisms and epo ed hei associa ions wi h pos -b onchioli is as hma a p eschool age10. The p esen s udy was ca ied ou o complemen his explo a o y s udy se ies by e alua ing whe he he TLR3 s3775291, TLR4 s4986790, TLR7 s179008, TLR8 s2407992, TLR9 s187084, and TLR10 s4129009 polymo - phisms a e associa ed wi h pos -b onchioli is as hma. The aim o his s udy was o compa e hese polymo phisms be ween child en wi h and wi hou cu en as hma, cu en a opic de ma i is, o cu en alle gic hini is a p e- school age, o wi h and wi hou as hma e e combining cu en and p e ious as hma in child en hospi alised o b onchioli is in in ancy. Resul s The mean age o he 135 pa ien s was 6.4 yea s a he con ol isi , and 51% we e males. As hma e e was p esen in 37 pa ien s (27.4%), cu en as hma in 18 (13.3%), a opic de ma i is in 46 (34.1%), and alle gic hini is in 39 (28.9%). The geno ypes and mino allele equencies (MAF) and popula ion da a on he MAFs a e lis ed in Table1. The MAFs o he cases and he Finnish popula ion MAF da a20 did no di e subs an ially in e ms o TLR3 s3775291, TLR4 s4986790, TLR7 s179008, TLR8 s2407992, TLR9 s187084, o TLR10 s4219009 genes. The TLR3 geno ype was wild (CC) in 45.9% and a ian (TC o TT) in 54.1% o he cases. The TLR4 geno ype was wild (AA) in 83.7% and a ian (AG) in 16.3% o he cases. The TLR9 geno ype was wild (TT) in 32.1% and a ian (TC o CC) in 67.9% o he cases. The e we e no signi ican associa ions be ween he TLR3, TLR4, o TLR9 geno ypes and as hma e e , cu en as hma, cu en a opic de ma i is, o cu en alle gic hini is (Table2). In emales, he TLR7 geno ype was wild (AA) in 60.6% and a ian (AT o TT) in 39.4% o he cases. In males, allele A was p esen in 79.4% and allele T in 20.6%. As hma e e was p esen in 34.6% o he gi ls who had he a ian AT o TT geno ype compa ed o 12.5% o hose who we e homozygous o he majo allele A (p = 0.03) (Table2). The odds a io (OR) adjus ed o age was 3.71 (95% con idence in e als [CI] 1.08–12.77). This associa ion was signi ican in logis ic eg ession adjus ed i s o ea ly-li e isk ac o s, and hen sepa a ely o cu en con ounde s (da a no shown). The associa ion emained signi ican in logis ic eg ession adjus ed o age, ea ly-li e isk ac o s, and cu en con ounde s in he same model (OR 3.93, 95% CI 1.06–14.58). The co esponding igu es in boys we e 33.3% (allele A p esen ) and 37.5% (allele T p esen ) (p = 1.00). The e we e no signi ican associa ions be ween he TLR7 geno ypes and cu en as hma, cu en a opic de ma i is, o cu en alle gic hini is in ei he gi ls o boys (Table3). In emales, he TLR8 geno ype was wild (GG) in 34.8% and a ian (GC o CC) in 65.2% o he cases. In males, allele G was p esen in 50.7% and allele C in 49.3%. The e we e no signi ican associa ions be ween he TLR8 geno ypes and as hma e e , cu en as hma, cu en a opic de ma i is, o cu en alle gic hini is in ei he gi ls o boys (Table3). The TLR10 geno ype was wild (AA) in 84.3% and a ian (AG o GG) in 16.7% o he cases. Cu en as hma was p esen in 30.0% o he child en who had he a ian AG o GG geno ype compa ed o 10.6% o hose who we e homozygous o he majo allele A (p = 0.03) (Table2). The OR adjus ed o age and gende was 3.74 (95% CI 1.19–11.78). This associa ion was signi ican in logis ic eg ession adjus ed i s o ea ly-li e isk ac o s, and hen sepa a ely o cu en con ounde s (da a no shown). The associa ion emained signi ican in logis ic eg es- sion adjus ed o age, gende , ea ly-li e isk ac o s, and cu en con ounde s in he same model (OR 4.30, 95% CI 1.30–14.29). The e we e no s a is ically signi ican associa ions be ween TLR10 gene polymo phisms and as hma e e , cu en a opic de ma i is, o cu en alle gic hini is (Table2). Discussion The e we e h ee main esul s in ou s udy on he associa ion o TLRs wi h as hma a 5 o 7 yea s o age a e hos- pi alisa ion o b onchioli is a less han 6 mon hs o age. Fi s ly, cu en as hma was mo e common in child en SNP (Majo > Mino ) Majo /Majo Majo /Mino Mino /Mino MAF FIN TLR3 s3775291 (C > T) 0.46 0.41 0.13 0.33 0.33 TLR4 s4986790 (A > G) 0.84 0.16 0 0.08 0.12 TLR7 s179008 (A > T) 0.61 (gi ls) 0.35 (gi ls) 0.04 (gi ls) 0.22 (gi ls and boys) 0.31 (gi ls and boys) 0.79 (boys) 00.21 (boys) TLR8 s2407992 (G > C) 0.35 (gi ls) 0.48 (gi ls) 0.17 (gi ls) 0.45 (gi ls and boys) 0.36 (gi ls and boys) 0.79 (boys) 00.49 (boys) TLR9 s187084 (T > C) 0.32 0.43 0.25 0.46 0.45 TLR10 s4129009 (A > G) 0.84 0.15 0.01 0.08 0.08 Table 1. Geno ypes and mino allele equencies o genes encoding oll-like ecep o s 3, 4, 7, 8, 9, and 10 in 135 child en hospi alised o b onchioli is and in he Finnish popula ion. MAF = mino allele equency, FIN = Finnish MAFs as in e . 20. N = 135 o TLR3, TLR4, TLR7, and TLR8; N = 134 o TLR9 and TLR10. www.na u e.com/scien i ic epo s/ 3 Scien i ic RepoR s | 7: 2956 | DOI:10.1038/s41598-017-03429-x who had he a ian TLR10 s4129009 geno ype. Secondly, as hma e e was mo e common in gi ls who had he a ian TLR7 s179008 geno ype. Bo h indings we e obus o adjus men s wi h known ea ly-li e isk ac o s o as hma as well as wi h cu en con ounde s a he age o 5 o 7 yea s. Howe e , TLR10 and TLR7 gene polymo - phisms had no signi ican associa ions wi h cu en alle gy. And hi dly, he e we e no signi ican associa ions be ween TLR3 s3775291, TLR4 s4986790, TLR8 s2407992, o TLR9 s187084 polymo phisms and ea lie o cu en as hma o alle gy. TLRs play a pi o al ole in p omo ing and con olling inna e immune esponses. Func ional gene poly- mo phism al e s he amino acid s uc u e o he ecep o , as has been shown in he cases o TLR3 s3775291 (Leu > Phe), TLR4 s4986790 (Asp > Gly), TLR7 s179008 (Glu > Leu), TLR9 s187084 (A g > T p), and TLR10 s4219009 (Ile > Leu) gene polymo phisms21, 22. The consequence o he mu a ion is dependen on i s loca ion. Mu a ion in he ex acellula domain o he ecep o may u he lead o an al e ed binding a ini y and subse- quen immune esponse23, whe eas mu a ion in he cy oplasmic TIR ( oll/in e leukine-1 ecep o ) domain, as in he case o TLR10 s4219009, may esul in an al e ed downs eam signalling, despi e no mal binding12, 24. Al hough polymo phism in he TLR8 s2407992 (2040 C/G) does no change he amino acid (651Leu > Leu), i can po en ially a ec TLR8 splicing15. TLR10 is a modula o y pa e n- ecogni ion ecep o wi h mainly inhibi o y p ope ies, and i is able o educe TLR2 esponses by inc easing he p oduc ion o an i-in lamma o y IL-1Ra19. Fu he , a ecen me a-analysis e ealed ha polymo phisms o he IL-1Ra encoding genes we e associa ed wi h as hma, especially in Caucasian popula ions25. Ou inding ha he TLR10 gene polymo phism was associa ed wi h cu en as hma is in acco d- ance wi h hese obse a ions. The gene ic a ia ion in TLR10 s4129009, which was also de e mined in he p esen s udy, was associa ed wi h as hma isk in wo independen samples om he USA26. In addi ion, in a Canadian– Aus alian s udy, a weak associa ion was obse ed be ween ano he TLR10 polymo phism ( s11096957) and a opic as hma27. In a la ge Ge man s udy, a p o ec i e e ec o gene ic a ian s on a opic as hma was iden i ied in he TLR2-associa ed he e odime ne wo k consis ing o TLR1, TLR6, and TLR1012. Co esponding indings in he genes encoding TLR1, TLR2, and TLR6 we e also seen in he p esen pos -b onchioli is coho , bu he di ec ion o he e ec was opposi e10. The a ian geno ype in he TLR1 gene was associa ed wi h as hma du ing he i s 6 yea s o li e, and as hma was p esen in only wo child en wi h he wild geno ype in all h ee polymo phisms10. In he mos ecen s udy om his coho 28, polymo phism o TLR6 was associa ed wi h b onchial hype - eac i i y, and i all o he ou genes including TLR10 p esen ed wi h he wild geno ype, exe cise-induced esponses in esis ance a 5HZ by impulse oscillome y we e signi ican ly smalle han in hose wi h one o mo e a ian geno ypes. These indings a e in acco dance wi h ou cu en obse a ions s essing he ole o he a ian TLR10 geno ypes in he eme gence o pos -b onchioli is as hma. The di e ences be ween he Ge man12 and Finnish coho s may be due o di e en as hma pheno ypes, alle gic as hma in he Ge man s udy, and pos -b onchioli is as hma in he cu en s udy. The Ge man s udy epo ed ha p ima y cells de i ed om ca ie s o p o ec i e TLR1, TLR6, and TLR10 a ian s showed augmen ed in lamma o y esponses, inc eased Th1 cy okine exp ession, and educed Th2-associa ed IL-4 p oduc ion a e speci ic s imula ion12. The supp essed sec e ion o alle gy- ela ed cy okines, like IL-4, IL-15, and IL-13, seems o be associa ed wi h as hma pheno ypes no ela ed o alle gy29. We ound p elimina y e idence ha he ole o he TLR7 gene in as hma may di e be ween gi ls and boys, which may be explained by he si ua ion o he TLR7 s179008 in he X ch omosome30. A ecen expe imen al s udy epo ed ha a TLR7 gene de ec and ea ly pneumo i us in ec ion in mice in e ac ed wi h each o he , i s leading o a se e e b onchioli is-like disease, hen o Th2-domina ed immuni y, and inally o an as hma-like pa hology31. A Danish s udy e ealed ha TLR7 s179008 polymo phism was associa ed wi h as hma15. An Ame ican s udy ound ha TLR7 was exp essed in human ai way ne es and con ibu ed o elaxa ion o he ai ways ia he p oduc ion o ni ic oxide16. The e o e, no mal TLR7 unc ion may be p o ec i e agains ai way As hma e e Cu en as hma Cu en a opic de ma i is Cu en alle gic hini is TLR3 s3775291 N = 135 N = 37 N = 18 N = 46 N = 39 Wild CC N = 62 (%) 19 (30.6) 8 (12.9) 20 (32.3) 16 (25.8) Va ian CT, TT N = 73 (%) 18 (24.7) p = 0.45 10 (13.7) p = 0.55 26 (35.6) p = 0.72 23 (31.5) p = 0.57 TLR4 s4986790 N = 135 N = 37 N = 18 N = 46 N = 39 Wild AA N = 113 (%) 31 (27.4) 16 (14.2) 39 (34.5) 34 (30.1) Va ian AG, GG N = 22 (%) 6 (27.3) p = 1.00 2 (9.1) p = 0.74 7 (31.8) p = 1.00 5 (22.7) p = 0.61 TLR9 s187084 N = 134 N = 37 N = 18 N = 46 N = 39 Wild TT N = 43 (%) 11 (25.6) 4 (9.3) 10 (23.3) 13 (30.2) Va ian TC, CC N = 91 26 (28.0)p = 0.44 14 (15.4) p = 0.25 36 (40.0) p = 0.06 26 (28.6) p = 0.50 TLR10 s4129009 N = 134 N = 37 N = 18 N = 46 N = 39 Wild AA N = 113 (%) 28 (24.8) 12 (10.6) 39 (34.5) 31 (27.4) Va ian AG, GG N = 21 (%) 9 (42.9) p = 0.08 6 (28.6) p = 0.03 7 (33.3) p = 0.57 8 (38.1) p = 0.23 Table 2. Geno ypes o TLR3 s3775291, TLR4 s4986790, TLR9 s187084, and TLR10 s4129009 encoding genes in ela ion o as hma and alle gy a p eschool age in 135 o me b onchioli is pa ien s. www.na u e.com/scien i ic epo s/ 4 Scien i ic RepoR s | 7: 2956 | DOI:10.1038/s41598-017-03429-x hype - eac i i y and as hma, whe eas TLR7 polymo phism may p edispose o as hma, and ou obse a ions sugges ha he in luence is g ea e in gi ls han in boys. Polymo phism in he TLR8 s2407992 gene had no associa ion wi h cu en o ea lie as hma, a opic de - ma i is, o alle gic hini is. This inding con adic s he esul s o he Danish s udy15, in which he same TLR8 polymo phism was associa ed wi h as hma, a opic de ma i is, alle gic hini is, and ele a ed alle gen-speci ic immunoglobulin E. A Swedish case-con ol s udy ound an associa ion be ween TLR8 gene a ia ion and alle gic hini is in adul s9. Polymo phisms in he TLR3 s3775291, TLR4 s4986790, o TLR9 s187084 genes we e no associa ed wi h cu en o ea lie as hma o alle gies. A p e ious s udy om he p esen coho o e ed p elimina y e idence ha he wild TLR3 s3775291 geno ype inc eased he isk o epea ed pos -b onchioli is wheezing32. The nega i e esul o he p esen s udy a he p eschool age was due, a leas pa ly, o he mo e bene icial ou come o sub- jec s hospi alised o RSV b onchioli is compa ed o subjec s hospi alised o hino i us b onchioli is. He ein, subsequen as hma was mo e common a e hino i us b onchioli is han a e RSV b onchioli is2, 33. Recen me a-analyses ha e ailed o ind any associa ions be ween TLR4 s4986790 polymo phism and as hma34, and be ween TLR9 s187084 polymo phisms and as hma35. The e we e ce ain limi a ions in he p esen s udy. The numbe o pa ien s was ela i ely small o gene ic s udies. In addi ion, blood samples o gene ic s udies we e no a ailable om all cases wi h su icien ollow-up da a a ailable, al hough we conside he 81% p opo ion as accep able. The small numbe o pa ien s means a isk o ype-2 s a is ical e o . On he o he hand, we ca ied ou mul iple analyses o polymo phisms o six di e en TLR-encoding genes, which means a isk o ype-1 s a is ical e o . We conside ed ou s udy as an explo a o y s udy aimed a inding p elimina y e idence o associa ions, i p esen , which needs o be con i med o ejec ed in u u e con i ma o y s udies. The e o e, and because only wo polymo phisms we e associa ed wi h as hma isk, we did no ega d any mul iplici y adjus men s as necessa y36, 37. Mul i a ia e logis ic eg ession was used, allowing adjus men s wi h ea ly-li e isk ac o s and cu en con ounding ac o s, bu he powe o he s udy was no su icien o inco po a ion o all six polymo phisms in he same model. The s eng hs o he p esen s udy a e he p ospec i e design, ela i ely long ollow-up pe iod o six yea s, ex ensi e i ological es panel a ailable du ing hospi alisa ion o b onchioli is, and ca e ul da a collec ion du - ing b onchioli is and subsequen con ol isi s. The homogenei y o s udy popula ions, as in he p esen s udy, is a clea bene i o gene ic s udies. We conside he e ealed signi ican associa ion be ween TLR10 polymo phism and cu en as hma a p eschool age a eliable inding, since he MAF igu es we e he same, 0.08, in bo h cases and popula ion-based con ols om he FIN da a20. Since TLR7 and TLR8 genes a e loca ed in he X ch omo- some, he analyses we e ca ied ou sepa a ely o bo h sexes, and indeed, he indings seemed o be di e en in gi ls and boys. RSV caused o e 70% o he b onchioli is cases, bu subsequen as hma was mo e common a e hino i us b onchioli is han a e RSV b onchioli is2. The e o e, he inal analyses on he ole o TLR10 s4129009 we e pe o med wi h mul i a ia e logis ic eg ession adjus ed o age, sex, ea ly-li e isk ac o s, and cu en isk ac o s o as hma, including he RSV ae iology o ea ly-li e b onchioli is, and he conclusions did no change. In conclusion, polymo phism in he TLR10 gene seems o inc ease he isk o pos -b onchioli is as hma in p eschool-aged child en, and polymo phism in he TLR7 gene seems o inc ease he isk o pos -b onchioli is as hma in p eschool-aged gi ls. This esul was a he s ong, since he associa ions we e obus o adjus men s o ea ly-li e and cu en isk ac o s o as hma. Me hods The s udy was conduc ed a he Depa men o Paedia ics, Tampe e Uni e si y Hospi al, Finland, and he design was p e iously desc ibed2. In b ie , 166 p e iously heal hy, ull- e m in an s hospi alised o b onchioli is a less han 6 mon hs o age in 2002–2004 a ended a ollow-up s udy in 2008–2010, when he child en we e 5 o 7 yea s As hma e e Cu en as hma Cu en a opic de ma i is Cu en alle gic hini is TLR7 s179008 Females N = 66 N = 14 N = 8 N = 23 N = 17 Wild AA N = 40 (%) 5 (12.5) 3 (7.5) 15 (37.5) 10 (25.0) Va ian AT, TT N = 26 (%) 9 (34.6) p = 0.03 5 (19.2) p = 0.25 8 (30.8) p = 0.61 7 (27.0) p = 1.00 TLR8 s2407992 Females N = 66 N = 14 N = 8 N = 23 N = 17 Wild GG N = 23 (%) 5 (21.7) 2 (8.7) 8 (34.8) 5 (21.7) Va ian GC, CC N = 43 (%) 9 (20.9) p = 1.00 6 (14.0) p = 0.70 15 (32.6) p = 1.00 12 (26.1) p = 0.77 TLR7 s179008 Males N = 68 N = 23 N = 10 N = 23 N = 22 Wild, allele A p esen N = 54 (%) 18 (33.3) 7 (13.0) 19 (35.2) 19 (35.2) Va ian , allele T p esen N = 14 (%) 5 (37.5) p = 1.00 3 (21.4) p = 0.68 3(21.4) p = 0.76 3 (21.4) p = 0.36 TLR8 s2407992 Males N = 69 N = 23 N = 10 N = 23 N = 22 Wild, allele G p esen N = 35 (%) 11 (31.4) 6 (17.1) 12 (34.3) 13 (37.1) Va ian , allele C p esen N = 34 (%) 12 (35.3) p = 0.80 4 (11.8) p = 0.73 11 (32.4) p = 1.00 9 (26.5) p = 0.44 Table 3. Geno ypes o TLR7 s179008 and TLR8 s2407992 encoding genes in ela ion o as hma and alle gy a p eschool age in 135 o me b onchioli is pa ien s. TLR7 and TLR8 a e p esen ed sepa a ely o emales and males. One es esul o a TLR7 male p esen ed AT he e ozygo e and was dele ed om he analyses. www.na u e.com/scien i ic epo s/ 5 Scien i ic RepoR s | 7: 2956 | DOI:10.1038/s41598-017-03429-x o age. In in ancy, b onchioli is was de ined as an acu e lowe espi a o y illness cha ac e ised by hino hea, cough, and di use wheezes o c ackles38. Ea ly-li e da a we e collec ed by in e iewing he pa en s du ing hos- pi alisa ion using s uc u ed ques ionnai es2. This showed ha 14.5% o he mo he s and 6% o he a he s had as hma, and 29.5% o he child en had a opic de ma i is o ood alle gy. Da a on he i al ae iology o b onchi- oli is we e s udied on admission by an igen de ec ion and polyme ase chain eac ion (PCR), and a i al in ec ion was iden i ied in mos cases: RSV in 70.5% and hino i us in 12.7%2. Du ing he ollow-up s udy, 127 child en a ended he clinical con ol isi , and an addi ional 39 child en e u ned he s udy ques ionnai e. The s uc u ed ques ionnai e comple ed by he pa en s o bo h g oups consis ed o sepa a e ques ions on wheezing episodes, as hma diagnoses, and use o b onchodila o s and inhaled co icos e oids (ICS) o he p eceding 12 mon hs. The p esence o a opic de ma i is and alle gic hini is, nigh cough in he absence o in ec ions, and p olonged cough o mo e han ou weeks we e also cha ed o he p eceding 12 mon hs. The subjec s o he p esen s udy consis ed o hose 135 child en o whom samples o gene ic s udies a e a ailable. De ini ion o as hma. Cu en as hma was de ined as he cu en use o con inuous main enance medica- ion wi h ICS o as hma, o su e ing om doc o -diagnosed episodes o wheezing, wi h a p olonged o nigh cough du ing he p eceding 12 mon hs and wi h a diagnos ic inding in he exe cise challenge es (ECT)2. The ECT consis ed o ee unning ou doo s o 8 minu es and measu emen s o p e- and pos -exe cise ai way esis - ance by impulse oscillome y (Jaege , Mas e Sc een IOS, Höchbe g, Ge many). An inc ease in esis ance o 35% o mo e a 5 Hz was conside ed o be pa hological2, 39. P e ious as hma be o e he con ol isi was de ined as he p e ious use o inhaled ICS as con inuous o in e mi en main enance medica ion o as hma2. I he child had ei he p e ious o cu en as hma, he e m as hma e e was used. Alle gic hini is was de ined as episodes o wa e y nasal discha ge no accompanied by e e o by o he symp oms o espi a o y ac in ec ion2. A opic de ma i is was de ined by doc o -diagnosed eczema and a opy2. Geno yping. Polymo phisms o TLR3 s3775291 (1234 C/T), TLR4 s4986790 (1194 A/G), TLR7 s179008 (171 A/T), TLR8 s2407992 (2040 C/G), TLR9 s187084 (1486 T/C), and TLR10 s4129009 (2322 A/G) we e selec ed due o hei e iden unc ional p ope ies. The geno yping o TLR3 s3775291 (1234 C/T) was pe o med by py osequencing (PSQ™96MA Py osequence , Bio age, Uppsala, Sweden) using a PSQ™96 Py o Gold Q96 ea- gen ki acco ding o he manu ac u e ’s p o ocol32. The geno yping o TLR4 s4986790 (299 A/G) was pe o med by he ABIPRISM 7000 Sequence De ec ion Sys em (Applied Biosys ems, CA)40 supplemen ed la e wi h py ose- quencing (PSQ™96MA Py osequence , Bio age, Uppsala, Sweden) using a PSQ™96 Py o Gold Q96 eagen ki 41, 42. The geno yping o TLR8 s2407992 (2040 C/G) was pe o med in he same manne as desc ibed o TLR3 s3775291 (1234 C/T). Fo TLR7 s179008 (171 A/T), he PCR p oduc s we e i s pu i ied using he QIA quick PCR Pu i ica ion Ki (Qiagen, Hilden, Ge many) acco ding o he manu ac u e ’s p o ocol. PCR p oduc s wi h low deoxy ibonucleic acid (DNA) con en we e elu ed o 30 µl o elu ion bu e . A e pu i ica ion, he PCR p od- uc s we e pipe ed o a 96-well pla e (5 µl) oge he wi h TLR7 s179008 (171 A/T) o wa d p ime (1.6 µl), and he 96-well pla e was sen o he Ins i u e o Molecula Medicine labo a o y in Helsinki, Finland, o sequencing, as desc ibed ecen ly28. TLR9 s187084 (1486 T/C) geno yping was pe o med using BspTI es ic ion enzyme (The moFhishe Scien i ic, Wal ham, USA) o diges ion o he PCR p oduc 28. High- esolu ion mel ing analysis (HMR) (Roche Diagnos ics Ligh Cycle 480, Basel, Swi ze land) was used o geno yping o TLR10 s4129009 (2322 A/G), as desc ibed p e iously28. The e we e 135 samples a ailable o geno yping o he TLR3, TLR4, TLR7, and TLR8. Fo he geno yping o he TLR9 and TLR10, 134 samples we e a ailable. E hics. 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. The p o ocol o he s udy was app o ed by he E hics Commi ee o he Tampe e Uni e si y Hospi al Dis ic , Tampe e, Finland. Be o e we en olled he child en, we ob ained in o med pa en al consen , including he use o samples o gene ic s udies on b onchioli is and as hma isk, du ing bo h hospi alisa ion and he con ol isi . The pe sonal da a o he s udy subjec s we e no gi en o he wo labo a o ies ha pe o med he gene ic s udies, he Na ional Ins i u e o Heal h and Wel a e, Tu ku, Finland, o he Ins i u e o Molecula Medicine, Helsinki, Finland. S a is ics. S a is ical analyses we e ca ied ou wi h SPSS e sion 23.0 so wa e (IBM Co p, NY, USA). The chi-squa e es and Fishe ’s exac es we e used, when app op ia e, o analyse geno ype equencies be ween hose wi h and wi hou cu en as hma, as hma e e , cu en alle gic hini is, o cu en a opic de ma i is. Mul i a ia e analyses we e conduc ed i uni a ia e analyses e ealed s a is ically signi ican (p < 0.05) associa ions. Logis ic eg ession was i s adjus ed o gende and age, and hen o gende , age, ma e nal as hma, and RSV ae iology o b onchioli is (ea ly-li e isk ac o s), and u he o gende , age, and cu en a opic de ma i is (cu en con ound- e s). Finally, age, gende , ea ly-li e isk ac o s, and cu en con ounde s we e included in he same model. The esul s we e exp essed as OR and 95% CI. The FINETTI p og amme was used o e alua e he Ha dy–Weinbe g equilib ium (HWE) o he s udied TLR3, TLR4, TLR9, and TLR10 alleles, and hey we e in he HWE. Since TLR7 and TLR8 genes a e loca ed in he X ch omosome, he HWE was no s udied, and males and emales we e analysed sepa a ely. Re e ences 1. Piippo-Sa olainen, E. & Ko ppi, M. Wheezy babies—wheezy adul s? Re iew on long- e m ou come un il adul hood a e ea ly childhood wheezing. Ac a. Paedia . 97, 5–11 (2008). 2. 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). 3. 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Nasopha yngeal bac e ial coloniza ion and gene polymo phisms o mannose-binding lec in and oll-like ecep o s 2 and 4 in in an s. PloS ONE 6, e26198 (2011). Au ho Con ibu ions S.T. and P.K. had esponsibili y o pa ien sc eening, da a analysis, and w i ing he manusc ip . J.V., J.T., and Q.H. had esponsibili y o he gene ic analysis and pa icipa ed in w i ing he manusc ip . M.H. pa icipa ed in he p o ocol de elopmen and in w i ing he manusc ip . M.K. and K.N. we e esponsible o p o ocol de elopmen , planning, and in e p e a ion o he analyses, and hey pa icipa ed in w i ing he manusc ip . www.na u e.com/scien i ic epo s/ 7 Scien i ic RepoR s | 7: 2956 | DOI:10.1038/s41598-017-03429-x Addi ional In o ma ion Compe ing In e es s: G an s om Suomen Lääke ie een sää iö (Finnish Medical Founda ion) (Nuoli i a) and Tampe een Tube kuloosisää iÖ (Tampe e Tube culosis Founda ion) (Tö mänen and Nuoli i a). 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