Haplotype of the Interleukin 17A gene is associated with osteitis after Bacillus Calmette-Guerin vaccination

1 SCIENTIFIC REPORTs | 7: 11691 | DOI:10.1038/s41598-017-12113-z www.na u e.com/scien i ic epo s Haplo ype o he In e leukin 17A gene is associa ed wi h os ei is a e Bacillus Calme e-Gue in accina ion Ma i Ko ppi1, Johanna Te äsjä i2, Milla Liehu-Ma iskainen1, Ee o Lauhkonen1, Juho Vuonon i a2, Ki si Nuoli i a 3, Liisa K öge 4, Lau a Pöyhönen5, Minna K. Ka jalainen6,7 & Qiushui He2,8 Bacillus Calme e-Gue in (BCG) os ei is was mo e common in Finland han elsewhe e a he ime when uni e sal BCG accina ions we e gi en o Finnish newbo ns. The e is e idence ha IL-17 plays a ole in he de ense agains ube culosis. The aim o his s udy was o e alua e he associa ions o IL17A s4711998, IL17A s8193036 and IL17A s2275913 single-nucleo ide polymo phisms (SNPs) wi h he isk o BCG os ei is a e newbo n accina ion. IL17A s4711998, s8193036 and s2275913 SNPs we e de e mined in 131 adul s had p esen ed wi h BCG os ei is a e newbo n BCG accina ion. We analyzed, using he HaploView and PLINK p og ams, whe he allele o haplo ype equencies o hese SNPs di e be ween he o me BCG os ei is pa ien s and Finnish popula ion con ols. O he h ee IL17A SNPs s udied, s4711998 associa ed nominally wi h BCG os ei is; mino allele equency was 0.215 in 130 BCG os ei is cases and 0.298 in 99 con ols (p = 0.034). F equency o he second common haplo ype (GTA) di e ed signi ican ly be ween BCG os ei is cases and con ols (0.296 s. 0.184, p = 0.040 a e mul i- es ing co ec ion). The GTA haplo ype o he IL17A SNPs s4711998, s8193036 and s2275913 was associa ed wi h os ei is a e BCG accina ion. In e leukin-17A (IL-17A) belongs o p o-in lamma o y cy okines p oduced by T helpe 17 (Th17) cells1,2. Th17 cells and Th17-de i ed cy okines pa icipa e in he de ense agains di e en pa hogens, like Mycobac e ium ube - culosis1. In addi ion, Th17 cells and Th17-de i ed cy okines a e in ol ed in he pa hogenesis o diseases p esen - ing wi h ch onic in lamma ion, such as as hma and au oimmune diseases2. Six s udies we e included in a sys ema ic e iew on he ole o IL-17A in ac i e and la en ube culosis (TB), and h ee s udies on he ole o IL-17A in Bacillus Calme e-Gue in (BCG) accina ion3. The au ho s concluded ha IL-17A ac s as an e ec o p o ec i e molecule in pa ien s wi h TB and in child en a e BCG accina ion3. In a ecen me a-analysis, he IL17A s3748067 single-nucleo ide polymo phism (SNP) was associa ed wi h he suscep ibili y o TB in Asian popula ions4. The e was no signi ican associa ion be ween IL17A s2275913 polymo phism and TB isk, bu a signi ican associa ion was documen ed in wo la e s udies in Chinese5 and B azilian6 popula ions. The me a-analysis did no include he IL17A s8193036 and IL17A s4711998 SNPs4, which we selec ed in addi ion o IL17A s2275913 o he p esen s udy. BCG os ei is was mo e common in Finland han elsewhe e a he ime when BCG accina ions we e gi en o all Finnish newbo ns. Du ing he yea s 1960–1988, al oge he 222 child en su e ed om BCG os ei is in in ancy7,8, and in 2008–2009, we collec ed ques ionnai e da a om 160 o hem and blood samples om 132 o hem o immunological and gene ic s udies9,10. 1Cen e o Child Heal h Resea ch, Uni e si y o Tampe e and Uni e si y Hospi al, Tampe e, Finland. 2Depa men o Medical Mic obiology and Immunology, Uni e si y o Tu ku, Tu ku, Finland. 3Depa men o Pedia ics, Seinäjoki Cen al Hospi al, Seinäjoki, Finland. 4Depa men o Pedia ics, Kuopio Uni e si y Hospi al, Kuopio, Finland. 5S . Giles Labo a o y o Human Gene ics o In ec ious Diseases, Rocke elle B anch, The Rocke elle Uni e si y, New Yo k, NY, USA. 6PEDEGO Resea ch Uni and Medical Resea ch Cen e Oulu, Uni e si y o Oulu, Oulu, Finland. 7Depa men o Child en and Adolescen s, Oulu Uni e si y Hospi al, Oulu, Finland. 8Depa men o Medical Mic obiology, Capi al Medical Uni e si y, Beijing, China. Ma i Ko ppi, Johanna Te äsjä i, Minna K. Ka jalainen and Qiushui He con ibu ed equally o his wo k. Co espondence and eques s o ma e ials should be add essed o M.K. (email: ma i.ko ppi@s a .u a. i) Recei ed: 26 May 2017 Accep ed: 4 Sep embe 2017 Published: xx xx xxxx OPEN www.na u e.com/scien i ic epo s/ 2 SCIENTIFIC REPORTs | 7: 11691 | DOI:10.1038/s41598-017-12113-z The aim o he p esen s udy was o e alua e he associa ion be ween he IL17A s4711998 (−877A > G), IL17A s8193036 (−737C > T) and IL17A s2275913 (−197G > A) SNPs and he isk o BCG os ei is a e new- bo n accina ion. We compa ed he allele and haplo ype equencies o he h ee SNPs be ween 131 o me BCG os ei is pa ien s and 99 Finnish popula ion con ols ob ained om he 1000 Genomes P ojec 11. Resul s O he h ee IL17A SNPs, s4711998 associa ed nominally wi h BCG os ei is (Table1). The mino allele was p o ec i e (mino allele equency, MAF, cases/con ols 0.215/0.298, OR = 0.61, p = 0.034). Howe e , he s a- is ical signi icance was los a e mul i- es ing co ec ion. We u he analyzed he h ee-SNP haplo ypes o associa ion wi h BCG os ei is, and de ec ed ha he second common haplo ype, GTA consis ing o majo alleles o s4711998 (G) and s8193036 (T), and he mino allele A o s2275913, was o e ep esen ed in BCG os ei- is cases compa ed o con ols (0.291 s. 0.184, p = 0.008) (Table2). This associa ion emained signi ican a e mul i- es ing co ec ion. The esul s sugges ha he mino allele A o he SNP IL17A s4711998 may be p o ec i e o complica ions like os ei is a e BCG accina ion, and ha he associa ed GTA haplo ype may be p edisposing. Discussion The main esul o he p esen s udy was ha he haplo ype GTA o IL17A s4711998, s8193036 and s2275913 polymo phisms was mo e common in o me BCG os ei is pa ien s han in he Finnish popula ion con ols. The di e ence be ween cases and con ols emained signi ican a e mul i- es ing co ec ion. The e was also a nominally signi ican di e ence be ween cases and con ols in he allele equency o IL17A s4711998. The majo allele G was he p edisposing and he mino allele A he p o ec i e allele. As published ecen ly om his coho , he mino allele A and he a ian geno ypes GA and AA o he IL17A s2275913 we e mo e common in BCG os ei is pa ien s han in 405 heal hy in an s om Sou h-Wes Finland12. The esul is in ag eemen wi h ou p esen esul , hough now he mos signi ican polymo phism was IL17A s4711998. Six s udies we e included in a ecen sys ema ic e iew on he ole o IL-17A in ac i e and la en TB disease3, and he conclusion was ha IL-17A was low in ac i e TB bu inc eased du ing he con e sion om ac i e o la en TB. Th ee s udies included in he me a-analysis e alua ed he ole o IL-17A in BCG accina ion3,13–15, and BCG accina ion induced high se um le els o IL-17A. Thus, IL-17A seems o ac as an e ec o p o ec i e molecule like in e e on gamma (IFN-γ) in pa ien s wi h TB and in child en a e BCG accina ion3. No p e ious s udies a e a ailable on he associa ion o IL17A gene polymo phisms wi h esponses o BCG accina ion o wi h complica ions a e BCG accina ion. Ins ead, some da a a e a ailable on di e en IL17 polymo phisms and TB isk, as summa ized in a ecen me a-analysis4. IL17A s3748067 and IL17F s763780 SNPs we e associa ed wi h he suscep ibili y o TB in Asian popula ions, bu no in Eu opean popula ions. IL17A s2275913 polymo phism, which was de e mined in he p esen s udy, had no such associa ion, and he IL17A s4711998 and s8193036 SNPs, which also we e de e mined in he p esen s udy, we e no e iewed. Recen ly, IL17A s2275913 polymo phism was associa ed wi h he isk o TB in he Chinese5 and B azilian6 popula ions. IL17A s8193036 polymo phism was de e mined in he B azilian s udy, bu he da a did no ul ill he HWE c i- e ia and we e no analyzed6. IL17A haplo ypes we e no e alua ed in any o he p e ious s udies. SNP Mino allele Case/con ol MAF OR (95% CI) p alue s4711998 A 0.215/0.298 0.61 (0.39–0.96) 0.034 s8193036 C 0.350/0.394 0.85 (0.59–1.21) 0.364 s2275913 A 0.500/0.429 1.33 (0.91–1.93) 0.138 Table 1. Associa ion o IL17A single-nucleo ide polymo phisms (SNPs) wi h BCG os ei is SNP, single- nucleo ide polymo phism; MAF, mino allele equency; OR, odds a io, CI, con idence in e al. OR o he mino allele in logis ic eg ession unde he addi i e model. P alue 0.034 no signi ican a e mul iple- es ing co ec ion. Haplo ype Haplo ype equency Case/con ol haplo ype equency p alue GTG 0.275 0.254/0.302 0.255 GTA 0.245 0.291/ 0.184 0.008 GCA 0.115 0.111/0.120 0.748 GCG 0.114 0.128/0.095 0.278 ACA 0.088 0.074/0.107 0.210 ATG 0.087 0.068/0.112 0.101 ACG 0.054 0.044/0.066 0.307 ATA 0.022 0.029/0.013 0.235 Table 2. Associa ion o IL17A h ee-SNP haplo ypes wi h BCG os ei is. Haplo ypes o med by alleles o IL17A SNPs s4711998, s8193036 and s2275913. P alue 0.008 signi ican a e mul iple- es ing co ec ion wi h 100,000 pe mu a ions (p = 0.040). www.na u e.com/scien i ic epo s/ 3 SCIENTIFIC REPORTs | 7: 11691 | DOI:10.1038/s41598-017-12113-z The s eng h o he p esen s udy is he unique ma e ial, 130 pa ien s who p esen ed wi h i mly diagnosed BCG os ei is a e BCG accina ion as newbo ns. This ma e ial is he la ges one e e published on BCG in ec- ions. All BCG os ei is cases we e e hnic Finns, and his homogenei y is a bene i in gene ic s udies. O iginally, he o al numbe o BCG os ei is pa ien s was 222, and as published ecen ly, none o hem had died o ube cu- losis, BCG in ec ion o o he se e e in ec ion10. Thus, he p esen sample o 130 o me BCG pa ien s p obably ep esen s well he o iginal BCG os ei is pa ien s a he ime when BCG accina ion was o e ed o all Finnish newbo ns. Cu en ly, only isk g oups a e accina ed in Finland. The main sho coming o he s udy is he small numbe o pa ien s and con ols o gene ic analyses. Thus, he e is a need o eplica ion o ou indings in a la ge coho and in o he popula ions. Howe e , a collec ion o a BCG os ei is coho ha is la ge han he p esen is no ealis ic. The e o e, an associa ion be ween he GTA haplo ype o he h ee IL17A SNPs and o he Th17-associa ed pheno ypes, i p esen , would o e indi ec e idence o ou p esen obse a ions. In addi ion, we could no include any con ounding ac o s in he logis ic eg ession, because he con ols we e om he publicly a ailable 1000 Genomes Finnish popula ion11 and no clin- ical da a a e a ailable o hese con ols. As he e we e no genome-wide SNP da a a ailable, assessing popula ion s a i ica ion by gene ic me hods was no possible. Howe e , since bo h cases and con ols we e om he Finnish popula ion, i is unlikely ha popula ion s a i ica ion would ha e any subs an ial impac on he esul s. We included h ee IL17A polymo phisms in he s udy, bu he unc ions o hese SNPs a e poo ly known. The associa ion be ween IL17A s2275913 polymo phism and IL-17A p oduc ion has been documen ed in cell cul u es16 and in heal hy in an s17, bu he esul s we e inconclusi e. The IL17A s8193037 polymo phism was associa ed wi h plasma IL-17A le el in adul s wi h conges i e hea ailu e18. The IL17A s8193036 polymo phism in luenced IL-17A p oduc ion in blood cells ob ained om adul s wi h in lamma o y bowel disease19. No da a a e a ailable on he associa ion be ween he IL17A s4711998 polymo phism and IL-17A p oduc ion. Ins ead, nume ous s udies a e a ailable on he associa ion o hese SNPs wi h as hma and in lamma o y dis- eases. In h ee me a-analyses, he highe as hma isk was associa ed wi h IL17A s819303620, s227591621 and s471199822 polymo phisms. In wo me a-analyses, he IL17A s2275913 SNP inc eased he isk o heuma ic diseases23, and IL17A polymo phisms, when analyzed as combined, we e associa ed wi h he isk o ulce a i e coli is24. Mos s udies we e done in Asian popula ions, and he e o e, he esul s canno be di ec ly applied o o he such as Eu opean popula ions. In conclusion, he GTA haplo ype o IL17A s4711998, IL17A s8193036 and IL17A s2275913 was signi i- can ly associa ed wi h os ei is a e BCG accina ion as newbo n. Ma e ial and Me hods As published p e iously, we collec ed whole blood samples om 132 Finnish o me BCG os ei is pa ien s, and sen he samples o he labo a o y o he Na ional Ins i u e o Heal h and Wel a e, Tu ku, Finland9,10. DNA was isola ed om 200 μL o whole blood, and DNA samples we e ozen a −70 °C. Fo he p esen s udy, he sam- ples we e ans e ed o he labo a o y o Medical Mic obiology and Immunology, Uni e si y o Tu ku, Tu ku, Finland, whe e IL17A s4711998, s8193036 and s2275913 SNPs we e de e mined. The eason, why we included jus hese h ee SNPs in his s udy, is he a ailable in o ma ion on hei associa ion wi h many diseases. As sum- ma ized in me a-analyses20–24, IL17A s2275913 and s8193036 SNPs ha e been associa ed wi h in ec ious, alle - gic, heuma ic and e en malignan diseases. IL17A s4711998 has been o en included in hese s udies, hough such associa ions ha e been a e. BCG os ei is in he s udy subjec s was diagnosed by BCG cul u e and/o ypical his ology du ing yea s 1960– 1988 when he s udy subjec s we e in an s7,8. This is he la ges BCG os ei is ma e ial e e published. A ha ime, mo e han 95% o all newbo ns ecei ed he BCG accina ion in Finland, and he diagnos ics o se e e complica- ions like BCG os ei is was cen alized in he Public Heal h Ins i u e, Helsinki, Finland. Con ols. Con ols o he IL17A s4711998, s8193036 and s2275913 polymo phisms we e ob ained om he Finnish popula ion da a (N = 99) o he 1000 Genomes P ojec 11. Geno yping. All 132 BCG os ei is pa ien s we e geno yped o IL17A s2275913 gene (−197G > A) by a newly de eloped high esolu ion mel ing analysis (HRMA) (Roche Diagnos ics Ligh Cycle 480, Basel, Swi ze land). The p ime s ( o wa d 5′-TCTGCCCTTCCCATTTTCCTTC-3′ and e e se 5′-GGTTAAAAT TTCCGCCCCCAATT-3′) we e designed wi h P ime -Blas design ool. The known IL17A s2275913 s anda ds (wild ype, he e ozygo e and homozygo e a ian s) we e used in each un. The me hod o IL17A s2275913 was published in de ail ecen ly25. Likewise, HRMA was used o geno yping o 130 BCG os ei is pa ien s o IL17A s4711998 (−877 A > G) and s8193036 (−737 C > T) SNPs. HRM analyses we e pe o med by Ligh Cycle 480 e sion 5.1 (Roche, Basal, Swi ze land) using SensiFAST HRMA mel ing mas e ki (Bioline, London, UK). In each un, he ol- ume was 20 µl consis ing o 4 µl genomic DNA (8.0 ng/µl) and 16 µl o mas e mix including 10 µl mel ing mas e mix and 0.2 µM o o wa d and e e se p ime s. P ime s o he IL17A s4711998 and s8193036 SNPs we e designed wi h P ime -Blas design ool (h p://www.ncbi.nlm.nih.go / ools/p ime -blas /) and we e o de ed om Sigma-Ald ich Company (Sain Louis, Missou i, USA). High Pe o mance Liquid Ch oma og aphy (HPLC) quali y p ime s used in he HRMA analysis we e as ollows: IL17A s4711998 o - wa d 5′-TCTTGTCCTAGTCCTCTGTATTC-3′ and e e se 5′-GTAAGATGAACTTGGACTCAGGTC-3′, and IL17A s8193036 o wa d 5′-CTCCTTTCTAGTTCTCATCACTCTC-3′ and e e se 5′-GGGGATAGA GACTGGACAAA-3′. HRMA polyme ase chain eac ion (PCR) p og am s a ed wi h an ini ial dena u a ion o 3 min a 95 °C ol- lowed by 39 cycles ampli ica ion o 5 s a 95 °C and annealing o 10s a 61 °C (IL17A s8193036) o a 60 °C (IL17A s4711998) and ex ension o 15 s a 72 °C. A e he PCR s ep, high esolu ion mel ing cycle condi ions we e as www.na u e.com/scien i ic epo s/ 4 SCIENTIFIC REPORTs | 7: 11691 | DOI:10.1038/s41598-017-12113-z ou lined by Roche: i s hea ed o 95 °C and held o 1 min, cooled o p e-hold empe a u e (40 °C) ollowed by mel ing in e al o collec ing luo escence om 60 °C o 95° a amp a e o 0.2 °C pe second. Based on he HRMA mel ing p o iles o he SNPs wo samples we e selec ed o ep esen each geno ype. The samples we e sequenced in he Finnish Ins i u e o Molecula Medicine (FIMM), Helsinki, Finland, o con i m he iden i y o he sequences. These samples we e used as posi i e con ols on each un. Func ion. The associa ion be ween IL17A s2275913 (−197G > A) polymo phism and IL-17A p oduc ion has been documen ed in heal hy adul s16 and in heal hy in an s17, hough he indings sugges ed di e en di ec- ions o he in luence. In i o s imula ed T cells om he adul s possessing he a ian A allele p oduced signi - ican ly mo e IL-17 han hose wi hou he A allele16, whe eas se um IL-17A concen a ions we e high in in an s wi h he wild GG, in e media e in in an s wi h he a ian GA, and low o unmeasu able in in an s wi h he a ian AA geno ype17. The p esence o he a ian GA o GG geno ype o he IL17A s8193037 (−737C > T) was associa ed wi h lowe plasma IL-17A le els compa ed o wild geno ypes in adul s wi h conges i e hea ailu e, bu he IL17A s2275913 had no such associa ion18. The IL17A s8193036 in luenced he DNA me hyla ion, and mRNA and p o ein exp ession o IL-17A in blood cells ob ained om adul s wi h in lamma o y bowel disease19. No da a a e a ailable on he associa ion be ween he IL17A s4711998 (−877A > G) SNP and IL-17A p oduc ion. S a is ical analyses. The IL17A s4711998, s8193036 and s2275913 SNPs we e analyzed o associa ion wi h BCG os ei is using logis ic eg ession unde he addi i e model wi h PLINK, e sion 1.0926. Es ima ion o haplo ype equencies and haplo ype associa ion analysis wi h he χ2 es we e pe o med wi h Haplo iew, e sion 4.3, 027 and mul iple- es ing co ec ion was done wi h 100,000 pe mu a ions. The IL17A s4711998, s8193036 and s2275913 SNPs did no de ia e om Ha dy-Weinbe g Equilib ium (HWE). Geno yping a es o he h ee SNPs we e 0.992 ( s4711998), 0.992 ( s8193036), and 0.985 ( s2275913) indica ing good geno yping quali y, and no indi iduals we e excluded. 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 s udy was accep ed by he E hics Commi ee o he Tampe e Uni e si y Hospi al Dis ic . W i en in o med consen was ob ained om he s udy subjec s, including pe mission o pe o m gene ic s udies con- ce ning suscep ibili y o mycobac e ial in ec ions. The samples we e s udied in he labo a o ies as anonymized and coded. Re e ences 1. To ado, E. & Coope , A. M. IL-17 and Th17 cells in ube culosis. Cy okine G ow h Fac o Re . 21, 455–462 (2010). 2. Isailo ic, N., Daigo, K., Man o ani, A. & Selmi, C. In e leukin-17 and inna e immuni y in in ec ions and ch onic in lamma ion. J Au oimmun. 60, 1–11 (2015). 3. Li, Q. e al. IL-17 and IFN-γ p oduc ion in pe iphe al blood ollowing BCG accina ion and Mycobac e ium ube culosis in ec ion in human. Eu Re Med Pha macol Sci. 16, 2029–2036 (2012). 4. Zhao, J., Wen, C. & Li, M. 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Second-gene a ion PLINK: ising o he challenge o la ge and iche da ase s. Gigascience. 4, 7 (2015). 27. Ba e , J. C., F y, B., Malle , J. & Daly, M. J. Haplo iew: analysis and isualiza ion o LD and haplo ype maps. Bioin o ma ics. 21, 263–265 (2005). Acknowledgemen s Tampe een Tube kuloosisää iö (Tampe e Tube culosis Founda ion suppo ed he s udy by a g an o Johanna Te äsjä i, Ki si Nuoli i a and Lau a Pöyhönen. Au ho Con ibu ions M.K. pa icipa ed in he planning o he s udy, in e p e a ion o he esul s and was esponsible in he w i ing o he manusc ip . M.L.-M. pa icipa ed in he in e p e a ion o he esul s and in he w i ing o he manusc ip . E.L. pa icipa ed in he in e p e a ion o he esul s and in he w i ing o he pape . J.T. pe o med he labo a o y wo k o IL17A gene ics. J.V. pe o med he labo a o y wo k o de e mina ion o IL-17A concen a ions. K.N. pa icipa ed in he planning o he s udy, in e p e a ion o he esul s and in he w i ing o he manusc ip . L.K. was esponsible o he ma e ial and clinical da a. L.P. pa icipa ed in he planning o he s udy, in e p e a ion o he esul s and in he w i ing o he manusc ip . M.K.K. was esponsible o he gene ic da a analyses wi h Haplo iew and PLINK and she pa icipa ed in w i ing he manusc ip . Q.H. was esponsible o he labo a o y wo k. All au ho s ha e e ised he manusc ip and accep ed i s con en . Addi ional In o ma ion Compe ing In e es s: The au ho s decla e ha hey ha e no compe ing in e es s. 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. Open Access This a icle is licensed unde a C ea i e Commons A ibu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C e- a i e Commons license, and indica e i changes we e made. 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