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Scien i ic RepoR s | 6:31165 | DOI: 10.1038/s ep31165
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Pos -b onchioli is wheezing is
associa ed wi h oll-like ecep o 9
s187084 gene polymo phism
Ki si Nuoli i a1,*, Sa i Tö mänen2,*, Johanna Te äsjä i3, Juho Vuonon i a3, Pe i Koponen2,
Ma i Ko ppi2, Me ja Helminen2, Ville Pel ola4 & Qiushui He3,5,*
Inna e immuni y ecep o s play a c i ical ole in hos de ence, as well as in alle gy and as hma. The aim
o his explo a o y s udy was o e alua e whe he he e a e associa ions be ween TLR7 s179008, TLR8
s2407992, TLR9 s187084 o TLR10 s4129009 polymo phisms and i al indings, clinical cha ac e is ics
o subsequen wheezing in in an s wi h b onchioli is. In all, 135 ull- e m in an s we e hospi alized
o b onchioli is a age less han 6 mon hs: 129 o hem we e ollowed-up un il he age o 1.5 yea s.
The ou come measu es we e epea ed wheezing, use o inhaled co icos e oids, a opic de ma i is
du ing he i s 1.5 yea s o li e and o al se um immunoglobulin E (IgE). The e we e no signi ican
associa ions be ween he geno ypes o allele equencies o TLR7 s179008, TLR8 s2407992, TLR9
s187084 o TLR10 s4129009 polymo phisms and clinical cha ac e is ics o he se e i y o b onchioli is
du ing hospi aliza ion. Du ing ollow-up, epea ed wheezing was mo e common in child en wi h TLR9
s187084 a ian geno ype CC (30.5%) han in child en wi h TLR9 wild- ype geno ype TT (12.2%)
(p = 0.02, aOR 2.73, 95% CI 1.02–7.29). The TLR10 s4129009 mino allele G was associa ed wi h
ele a ed o al se um IgE. TLR9 s187084 gene polymo phism may be associa ed wi h pos -b onchioli is
wheezing, and TLR10 s4129009 gene polymo phism may be associa ed wi h a opy.
B onchioli is is he mos common lowe espi a o y ac in ec ion (LRTI) in young child en1. Among he a ious
espi a o y i uses causing b onchioli is, espi a o y syncy ial i us (RSV) is he single mos impo an one2. The
clinical cou se o p ima y RSV in ec ion is highly a iable, and gene ic a ia ions in genes egula ing he immune
esponse e iden ly a e impo an in de e mine whe he a child su e s om a mild uppe espi a o y in ec ion o
a mo e se e e LRTI- like b onchioli is a e exposu e o RSV3.
B onchioli is in in ancy is associa ed wi h subsequen wheezing in ea ly childhood4. The mechanisms o
i us-induced wheezing and he ole o ai way hype - esponsi eness, howe e , a e poo ly unde s ood. Signalling
ia oll-like ecep o s (TLRs) seems o play an impo an ole in igge ing ai way in lamma ion5,6.
TLRs a e key molecules in inna e immuni y ha de ec conse ed s uc u es, which a e p esen in a b oad
ange o pa hogens, and ei he p omo e o inhibi in lamma o y and immune esponses7. TLR3, TLR7, TLR8 and
TLR9 ecognize i al p oduc s esponding o i al double-s anded (ds) RNA, i al single-s anded (ss) RNA o
cy o oxic g anule p o eins con aining DNA om ce ain bac e ia and some i uses, espec i ely8,9. TLR10 is, in
addi ion o TLR1, TLR2 and TLR6, a membe o he TLR2 sub amily. Al hough TLR10 is a pa e n- ecogni ion
p o ein wi hou known ligand speci ici y, he e is e idence ha i is a modula o y ecep o wi h mainly inhibi o y
p ope ies10.
I has been epo ed ha gene ic ac o s migh in luence suscep ibili y o RSV in ec ion in ea ly li e. TLR4 gene
polymo phisms ha e been associa ed wi h se e e RSV in ec ion11,12. In addi ion, TLR9 and TLR10 gene polymo -
phisms13 and TLR 3 gene polymo phisms14 we e associa ed wi h RSV b onchioli is.
As he inna e immune sys em is an impo an link be ween en i onmen al exposu e and he egula ion
o cy okine esponses, al e a ions in inna e immuni y may be undamen al o he balance be ween Th1- and
Th2-o ien ed esponses and o he subsequen de elopmen o as hma and alle gy. In a ecen e iew on he
1Depa men o Pedia ics, Seinäjoki Cen al Hospi al, Seinäjoki, 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,
Uni e si y o Tu ku, Tu ku, Finland. 4Depa men o Pedia ics and Adolescen Medicine, Tu ku Uni e si y Hospi al
and Child and You h Resea ch Ins i u e, Uni e si y o Tu ku, Tu ku, Finland. 5Depa men o Medical Mic obiology,
Capi al Medical Uni e si y, Beijing, China. *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 K.N. (email: [email p o ec ed])
Recei ed: 01 Feb ua y 2016
Accep ed: 15 July 2016
Published: 08 Augus 2016
OPEN
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ela ionship be ween genes encoding TLRs and as hma isk, an associa ion was ound be ween TLR9 gene poly-
mo phisms and as hma15. The e is mo e e idence ha polymo phisms in he TLR7 and TLR8 genes a e ela ed o
as hma and alle gy16–18, as well as o suscep ibili y o espi a o y i al in ec ions19,20.
We ha e p ospec i ely ollowed-up a g oup o child en hospi alized o b onchioli is a age less han 6 mon hs.
We ha e ea lie s udied he TLR1 s5743618, TLR2 s5743708, TLR3 s3775291, TLR4 s4986790 and TLR6
s5743810 polymo phisms, and epo ed hei associa ions wi h b onchioli is and pos -b onchioli is ou comes.
The aim o he p esen s udy was o complemen his explo a o y s udy se ies by e alua ing whe he he TLR7
s179008, TLR8 s2407992, TLR9 s187084 and TLR10 s4129009 polymo phisms a e associa ed wi h he p es-
ence, clinical cha ac e is ics and i al e iology o b onchioli is in ea ly in ancy. In addi ion, we s udied whe he
he e a e associa ions be ween hese polymo phisms and pos -b onchioli is ou comes including subsequen
in ec ions, pos -b onchioli is wheezing, need o co icos e oid ea men and a opic mani es a ions un il he age
o 1.5 yea s.
Resul s
Cases e sus con ols. The geno ypes and allele equencies o cases (child en hospi alized wi h b on-
chioli is) and con ols (heal hy child en om a bi h coho s udy) did no di e subs an ially wi h espec o
TLR7 s179008, TLR8 s2407992, TLR9 s187084 o TLR10 s4219009 polymo phisms (Table1). Because TLR7
s179008 and TLR8 s2407992 a e loca ed on he X ch omosome, he da a a e gi en sepa a ely o boys and gi ls.
The s udied alleles o TLR9 and TLR10 we e in HWE. Because TLR7 and TLR8 genes a e loca ed on he X ch o-
mosome, HWE was no s udied.
Hospi aliza ion in in ancy. The mean age o he 135 b onchioli is pa ien s was 10.0 weeks ( ange 1–25
weeks, SD 6.94) du ing hospi aliza ion. The s udy included 65 (48.1%) boys (Table2). The causa i e i us was
RSV in 101 (74.8%) cases and hino i us in 11 (8.1%) cases. In luenza A i us was ound in 7 (5.2%) and human
me apneumo i us in 2 (1.5%) cases, and pa ain luenza i us ype 3 (PIV3) was ound in 1 (0.8%) case. Two di e -
en i uses we e de ec ed in 8 (5.9%) samples: hino i us and adeno i us in wo, and hino i us and boca i us in
wo samples, and RSV and adeno i us, RSV and boca i us, In luenza A and boca i us, and PIV3 and adeno i us,
each in one sample. The e we e no di e ences in he clinical pic u e o in he ou comes be ween cases wi h mul i-
ple i uses s. a single i us de ec ed. The sample was nega i e o s udied i uses in 13 (9.6%) cases.
Geno ypes and allele equencies Cases, No. (%) Con ols, No. (%)
TLR7 emales AA 43/70 (61.4) 77/145 (53.1)
TLR7 emales AT 23/70 (32.9) 53/145 (36.6)
TLR7 emales TT 4/70 (5.7) 15/145 (10.3)
TLR7 emales, majo allele A 109/140 (77.9) 207/290 (71.4)
TLR7 emales, mino allele T 31/140 (22.1) 83/290 (28.6)
TLR7 males, allele A p esen 54/65 (83.1) 116/168 (69.0)
TLR7 males, allele T p esen 11/65 (16.9) 52/168 (31.0)
TLR8 emales GG 19/70 (27.1) 43/144 (29.8)
TLR8 emales GC 41/70 (58.6) 70/144 (48.6)
TLR8 emales CC 10/70 (14.3) 31/144 (21.6)
TLR8 emales, majo allele G 79/140 (56.4) 156/288 (54.2)
TLR8 emales, mino allele C 61/140 (43.6) 132/288 (45.8)
TLR8 males, allele G p esen 32/65 (49.2) 89/168 (53.0)
TLR8 males, allele C p esen 33/65 (50.8) 79/168 (47.0)
TLR9 TT 42/133 (31.6) 87/270 (32.0)
TLR9 TC 55/133 (41.4) 130/270 (48.0)
TLR9 CC 36/133 (27.1) 53/270 (20.0)
TLR9 majo allele T 139/266 (52.3) 304/540 (56.3)
TLR9 mino allele C 127/266 (47.7) 236/540 (43.7)
TLR10 AA 113/135(83.7) 271/328 (82.6)
TLR10 AG 21/135 (15.6) 47/328 (14.4)
TLR10 GG 1/135 (0.7) 10/328 (3.0)
TLR10 majo allele A 247/270 (91.5) 589/656 (89.8)
TLR10 mino allele G23/270 (8.5) 67/656 (10.2)
Table 1. The geno ypes and allele equencies o TLR7 s179008 (171 A/T), TLR8 s2407992 (2040 C/G),
TLR9 s187084 (1486 T/C) and TLR10 s4219009 (2322 A/G) in he b onchioli is pa ien s (cases) and
popula ion-based con ols. The es showed he e ozygosis o he TLR7 gene in ou con ols and he e ozygosis
o he TLR8 gene in ou con ols, al hough he subjec s we e male. In one male con ol, he es esul s showed
he e ozygosis o bo h TLR7 and TLR8. These se en con ols we e dele ed om he analyses. The e we e no
s a is ically signi ican di e ences be ween cases and con ols.
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The e we e no signi ican associa ions be ween he TLR7 s179008 o TLR8 s4207992 geno ypes o allele
equencies and he clinical cha ac e is ics o b onchioli is in emales (Table3). Ins ead, he p esence o he mino
allele T in he TLR7 s179008 gene in males was associa ed wi h he RSV ae iology o b onchioli is (100% s.
70.4% in hose wi h he majo allele A) and wi h he need o eeding suppo (54.5% s. 24.1%, espec i ely)
(Table4). The i al ae iology o b onchioli is, and he need o eeding suppo o oxygen supplemen a ion du ing
b onchioli is hospi aliza ion did no di e be ween he child en wi h di e en geno ypes o allele equencies
o TLR9 s187084 o TLR10 s4219009 (Table5). The LOS in hospi al was oughly equal in all he g oups con-
s uc ed on he basis o TLR7 s179008, TLR8 s4207992, TLR9 s187084 o TLR10 s4219009 polymo phisms.
Follow-up o 1.5 yea s. In all, 129 oddle s a ended he ollow-up isi a he mean age o 18.1 mon hs
( ange 13–25 mon hs, SD 2.35). The e we e 24 (19.4%) child en who had p esen ed wi h epea ed wheezing
(≥ 2 episodes) du ing he ollow-up pe iod and 16 (12.4%) child en who had used inhaled co icos e oids (ICSs).
Pa en s epo ed ha 30 child en (23.3%) had doc o -diagnosed ood alle gy and 18 child en (14.0%) had
doc o -diagnosed a opic de ma i is du ing he i s 1.5 yea s o li e (Table2). In addi ion, 63 child en (48.8%) had
su e ed om ecu en (≥ 3) o i is media. To al se um IgE was measu ed om 125 child en: among hem, he
mean o al se um IgE was 43.4 IU/ml (SD 95.9, ange 1–621).
In males, he p esence o he TLR8 mino allele C was associa ed wi h ecu en o i is media, which was p es-
en in 22 (68.8%) o he 32 mino allele C ca ie s s. in 13 o he 33 (39.4%) majo allele G ca ie s (p = 0.04)
(Table6). Howe e , he signi icance o his associa ion was los when adjus ed o age (OR 2.75, 95% CI 0.96–
7.85). In emales, he esul was o he same di ec ion bu no s a is ically signi ican (p = 0.06) (Table7). The e
was a non-signi ican inding ha he TLR7 majo allele A, when p esen as homozygous, was connec ed o ecu -
en o i is media in emales (p = 0.07) (Table7), bu his was no seen in males (Table6).
In an s du ing b onchioli is N = 135
Age (mean, ange, SD) 10.0 weeks (1–25, SD 6.94)
Boys N(%) 65 (48.1)
RSV N (%) 101 (74.8)
Feeding suppo N (%) 45 (33.3)
Oxygen supplemen a ion N(%) 25 (18.5)
Leng h o hospi al s ay (mean, ange, SD) 4.74 days (0–22, Sd 4.74)
Child en a ollow-up isi N = 129
Age (mean, ange, SD) 18.1 mon hs (13–25, SD 2.35)
Boys 62 (48.1)
Pos -b onchioli is wheezing N (%) 24 (19.4)
Inhaled co icos e oids 16 (12.4)
Food alle gy 30 (23.3)
A opic de ma i is 18 (14.0)
Table 2. Clinical cha ac e is ics o he b onchioli is popula ion du ing hospi aliza ion and ollow-up isi
a he mean age o 1.5 yea s.
TLR7 emales N = 70
AA
N = 43, No. (%)
AT
N = 23, No. (%)
TT
N = 4, No. (%)
Majo allele A
N = 109, No. (%)
Vi us RSV N = 52 34 (79.1) p = 0.19 15 (65.2) 3 (75.0) p = 0.73 83 (76.1) p = 0.71
Oxygen equi ed
N = 16 9 (20.9) p = 0.42 6 (26.1) 1 (25.0) p = 0.67 24 (22.0) p = 0.73
Feeding suppo
N = 28 19 (67.9) p = 0.26 7 (30.4) 2 (50.0) p = 0.53 45 (41.3) p = 0.70
LOS, mean in days
(s anda d de ia ion) 5.4 (SD 3.7) p = 0.51 5.0 (SD 3.0) 4.0 (SD 1.8) p = 0.48
TLR8 emales N = 70 GG N = 19, No. (%) GC N = 41, No. (%) CC N = 10, No. (%) Majo allele G
N = 79, No. (%)
Vi us RSV N = 52 14 (73.7) p = 0.59 30 (73.2) 8 (80.0) p = 0.50 58 (73.4) p = 0.92
Oxygen equi ed
N = 16 5 (26.3) p = 0.45 10 (24.4) 1 (10.0) p = 0.28 20 (25.3) p = 0.56
Feeding suppo
N = 28 9 (47.4) p = 0.31 15 (36.4) 4 (40.0) p = 0.64 33 (41.8) p = 0.75
LOS, mean in days
(s anda d de ia ion) 4.9 (SD 2.1) p = 0.69 5.4 (SD 3.9) 4.8 (SD 3.4) p = 0.72
Table 3. Geno ypes and majo allele equencies o TLR7 s179008 and TLR8 s2407992 genes in 70 emale
pa ien s in ela ion o clinical cha ac e is ics o b onchioli is. The leng h o hospi al s ay (LOS) was mean 5.2
days (SD 3.4, ange 1–25) in all 70 cases. Majo allele s mino allele.
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Fo me b onchioli is pa ien s wi h he homozygous a ian TLR9 s187084 geno ype CC had epea ed wheez-
ing mo e o en (30.5%, p = 0.02) han child en wi h he wild- ype geno ype TT (12.2%) du ing he ollow-up
pe iod un il he age o 1.5 yea s (Table8). The OR was 2.66 (95% CI 1.01–7.06) when adjus ed o age and sex,
and 2.73 (95% CI 1.02–7.29) when adjus ed u he o a opic de ma i is. The e we e no signi ican associa ions
be ween TLR7 s179008, TLR8 s2407992 o TLR10 s4219009 gene polymo phisms and epea ed wheezing.
Child en wi h TLR10 s4219009 geno ype AG (no child had he GG geno ype) had mo e o en had ele a ed
o al se um IgE han child en wi h TLR10 geno ype AA (Table7). The OR was 3.79 (95%CI 1.25–11.56) when
adjus ed o age and sex. The e we e no signi ican associa ions be ween TLR7 s179008, TLR8 s2407992 o TLR9
s187084 gene polymo phisms and o al se um IgE, ood alle gy o a opic de ma i is du ing he i s 1.5 yea s o li e.
Discussion
Ou s udy was an explo a o y s udy on he possible associa ion be ween he TLR7 s179008, TLR8 s4207992,
TLR9 s187084 o TLR10 s4219009 gene polymo phisms and cha ac e is ics o b onchioli is in ea ly in ancy
o pos -b onchioli is ou comes. The main esul was ha he TLR9 s187084 polymo phism was associa ed wi h
epea ed wheezing un il he age o 1.5 yea s a e b onchioli is a age less han 6 mon hs. One- hi d o child en
who we e homozygous o he a ian geno ype CC had su e ed om epea ed wheezing, compa ed o 12.2%
o o he o me b onchioli is pa ien s. Ano he impo an esul was ha he TLR10 s4219009 mino allele G
was associa ed wi h ele a ed o al se um IgE in in ancy. Howe e , such associa ions we e no seen in he case o
clinical indings, i.e., ood alle gy o a opic de ma i is du ing he i s 1.5 yea s o li e. Bo h hese esul s emained
signi ican when adjus ed o he mos impo an con ounde s. In addi ion, we e ealed a p elimina y associa ion
be ween he p esence o he TLR8 s4207992 mino allele C and ecu en o i is media in males bu no in emales.
Howe e , polymo phisms TLR7 s179008, TLR8 s4207992, TLR9 s187084 and TLR10 s4219009 we e no
associa ed wi h he cha ac e is ics o b onchioli is, including hose e lec ing b onchioli is se e i y. All pa ien s
TLR7 males
N = 65
A p esen
N = 54, No. (%)
T p esen
N = 11, No. (%)
Vi us RSV N = 49 38 (70.4) p = 0.03 11 (100)
Oxygen equi ed N = 9 6 (11.1) p = 0.71 3 (27.3)
Feeding suppo N = 19 13 (24.1) p = 0.05 6 (54.5)
LOS, mean in days
(s anda d de ia ion) 4.0 (SD 2.7) p = 0.06 5.8 (SD 3.7)
TLR8 males N = 65 G p esen N = 32 (%) C p esen N = 33 (%)
Vi us RSV N = 49 26 (81.3) p = 0.21 23 (69.7)
Oxygen equi ed N = 9 6 (18.8) p = 0.22 3 (9.1)
Feeding suppo N = 19 9 (28.1) p = 0.53 10 (30.3)
LOS, mean in days
(s anda d de ia ion) 4.1 (SD 3.2) p = 0.69 4.4 (SD 2.7)
Table 4. Geno ypes and majo allele equencies o TLR7 s179008 and TLR8 s2407992 genes in 65 male
pa ien s in ela ion o he clinical cha ac e is ics o b onchioli is. The leng h o hospi al s ay (LOS) was mean
4.3 days (SD 3.0, ange 0–15) in all 65 cases.
TLR9 N = 133
TT
N = 42, No. (%)
CT
N = 55, No. (%)
CC
N = 36, No. (%)
Majo allele T
N = 139, No. (%)
Vi us RSV N = 99 33 (78.6) p = 0.78 39 (70.9) 27 (75.0) p = 0.97 105 (75.5) p = 0.87
Oxygen equi ed
N = 25 7 (16.7) p = 0.43 13 (23.6) 5 (13.9) p = 0.27 27 (19.4) p = 0.82
Feeding suppo
N = 45 14 (33.3) p = 0.55 21 (38.9) 10 (27.8) p = 0.25 49 (35.3) p = 0.72
LOS, mean in days
(s anda d de ia ion) 5.10 (SD 3.80) p = 0.85 4.45 (SD 2.41) 4.61 (SD 3.58) p = 0.34
TLR10 N = 135 AA N = 113, No. (%) AG N = 21, No. (%) GG N = 1, Majo allele A
N = 247, No. (%)
Vi us RSV N = 101 84 (74.3) p = 0.50 16 (76.2) 1184 (74.5) p = 0.
Oxygen equi ed
N = 25 22 (19.5) p = 0.38 2 (9.5) 146 (18.6) p = 0.
Feeding suppo
N = 47 40 (35.4) p = 0.48 6 (28.6) 186 (34.8) p = 0.
LOS, mean in days
(s anda d de ia ion) 4.74 SD 3.25 p = 0.94 4.52 SD 3.04 NC
Table 5. Geno ypes and majo allele equencies o TLR9 s187084 (N = 133) and TLR10 s4219009
(N = 135) genes in b onchioli is pa ien s in ela ion o he clinical cha ac e is ics o b onchioli is. The
leng h o hospi al s ay (LOS) was mean 4.7 days (SD 3.2, ange 0–22) in all 133 cases. NC no calcula ed. Majo
allele s mino allele.
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wi h b onchioli is equi ed hospi aliza ion; one- ou h ecei ed supplemen a y oxygen; one- hi d equi ed
eeding suppo du ing hospi aliza ion. Vi uses such as RSV a e ecognized a e in asion o he cells by hose
i al-sensing TLRs and o he pa e n- ecognizing p o eins, which a e loca ed wi hin endosomes21.
In p e ious s udies, TLR4 gene polymo phisms ha e been associa ed wi h RSV in ec ions11, and he e is p e-
limina y e idence ha TLR3 gene polymo phism14, and TLR9 and TLR10 gene polymo phisms as well11,13, may
be associa ed wi h RSV b onchioli is. In his explo a o y s udy, b onchioli is cases and popula ion-based con ols
did no di e signi ican ly o geno ypes o allele equencies o TLR7 s179008, TLR8 s4207992, TLR9 s187084
and TLR10 s4219009 polymo phisms.
In a ecen sys ema ic e iew and me a-analysis, TLR9 s5743836 gene polymo phism was associa ed wi h
inc eased as hma isk15. TLR9 ecognizes bac e ial and i al CpG-DNA22. In an expe imen al s udy, he 1237
T/C single nucleo ide polymo phisms (SNP) o TLR9 s5743836 gene a ed a unc ional IL-6 esponse elemen .
Mononuclea cells ca ying his SNP, when exposed o IL-6, esponded by p oducing mo e TLR9, which hen
exace ba ed he cellula esponse o he TLR9 ligand CpG23. IL-6 is a p o-in lamma o y cy okine ha may
con ibu e o impai ed lung unc ion in alle gic as hma24. In ano he s udy, he TLR9 s5743836 a ian gen-
o ype was associa ed wi h diminished lung unc ion and ch onic obs uc i e pulmona y disease (COPD)25.
This e ec was hough o be caused by al eola mac ophage dys unc ion. Howe e , he e we e no associa ions
wi h TLR9 s187084 SNPs25. None heless, TLR9 s187084 polymo phism as applied in his s udy has also been
shown o be unc ional26, being associa ed wi h immuni y agains ce ain in ec ious diseases such as ube cu-
losis and mala ia26. Mo eo e , ha polymo phism was associa ed wi h ai way hype - esponsi eness in ou
popula ion-based samples om Canada and Aus alia27. In ou p esen s udy, he TLR9 s187084 polymo phism
was associa ed wi h epea ed pos -b onchioli is wheezing. In his same coho , TLR3 s3775291 gene polymo -
phism was associa ed wi h epea ed wheezing a e b onchioli is in in ancy14.
The associa ion o he TLR10 gene wi h as hma has been documen ed in Eu opean-Ame ican popula ions28.
Two- hi ds o child en who ha e epea ed pos -b onchioli is wheezing un il he age o wo yea s a e “ ansien
wheeze s” which means ha hey will g ow ou o hei wheezing endency by o a school age29. The isk o alle -
gic as hma ha is associa ed wi h he TLR10 gene polymo phism28 was mino in ou pa ien s wi h b onchioli is
in ea ly in ancy. Howe e , we ound an associa ion be ween TLR10 s4219009 gene polymo phism and ele a ed
o al se um IgE, and based on many s udies, a opy and a opic sensi iza ion a e well-known isk ac o s o alle gic
as hma, in pos -b onchioli is coho s as well30.
TLR7 s179008 and TLR8 s4207992 gene polymo phisms, loca ed on he X ch omosome, we e no associa ed
wi h pos -b onchioli is ou come measu es in ei he boys o gi ls. In ea lie s udies, TLR7 and TLR8 polymo -
phisms we e associa ed wi h as hma and alle gic hini is16,17. Ou inding is in disag eemen wi h he esul s o a
p e ious s udy16, in which he TLR8 s4207992 gene polymo phism, also used in he p esen s udy, was associa ed
wi h as hma, a opic de ma i is, alle gic hini is and ele a ed se um alle gen-speci ic IgE. In expe imen al s udies,
concomi an TLR7 gene de iciency and ea ly Pneumo i us in ec ion p edisposed mice owa d he de elopmen
o as hma-like pa hology20. In he p esen coho , 19.4% o child en su e ed om epea ed pos -b onchioli is
i us-induced wheezing un il 1.5 yea s o age. Howe e , alle gic as hma may de elop la e in childhood, e en
a e many symp om- ee yea s29.
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 a gene ic
s udy. The small numbe o pa ien s mean a isk o ype-2 s a is ical e o s. On he o he hand, we ca ied
ou mul iple analyses o polymo phisms o ou di e en TLR encoding genes, which isked ype-1 s a is ical
e o s. Because he da a on TLR7, TLR8, TLR9 and TLR10 genes as isk ac o s o b onchioli is, ea ly-childhood
wheezing and childhood as hma a e mainly lacking, ou s udy was an explo a o y one wi h no pu pose o es
TLR7 males Ou come a iables,
N = 62
A p esen
N = 54, No. (%)
T p esen
N = 11, No. (%)
Repea ed (≥ 2 episodes) wheezing
N = 15 14 (27.4) p = 0.19 1 (9.1)
Inhaled co icos e oids N = 11 10 (19.6) p = 0.37 1 (9.1)
A opic de ma i is N = 10 10 (19.6) p = 0.12 0
Food alle gy N = 15 11 (21.6) p = 0.25 4 (36.4)
IgE ≥ 60 N = 10/59 8 (15.7) p = 0.60 2 (18.2)
Recu en (≥ 3) o i is media N = 35 26 (50.9) p = 0.06 9 (81.8)
TLR8 males Ou come a iables,
N = 62
G p esen
N = 30, No. (%)
C p esen N = 32,
No. (%)
Repea ed (≥ 2 episodes) wheezing
N = 15 5 (16.7) p = 0.15 10 (31.3)
Inhaled co icos e oids N = 11 3 (10.0) p = 0.11 8 (25.0)
A opic de ma i is N = 10 2 (6.7) p = 0.05 8 (25.0)
Food alle gy N = 15 7 (23.3) p = 0.56 8 (25.0)
IgE ≥ 60 N = 10/59 3 (10.0) p = 0.19 7 (21.9)
Recu en (≥ 3) o i is media N = 35 13 (39.4)
p = 0.04 22 (68.8)
Table 6. Geno ypes and majo allele equencies o TLR7 s179008 and TLR8 s2407992 genes in 62 male
b onchioli is pa ien s in ela ion o ou come a iables du ing he pos -b onchioli is ollow-up un il he age
o 1.5 yea s.

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hypo heses, bu a he o p oduce hypo heses o la e con i ma o y s udies31,32. The e o e, we did no make any
s a is ical mul iplici y co ec ions. The main esul s we e con i med wi h mul i a ia e es s adjus ed o he mos
impo an con ounding ac o s.
The s eng hs o he p esen s udy we e he p ospec i e design, an ex ensi e i ological es panel a ailable
du ing hospi aliza ion o b onchioli is and ca e ul da a collec ion du ing b onchioli is and a he subsequen
con ol isi including e.g. daily dia ies eco dings by pa en s. The homogenei y o s udy popula ions, as in he
p esen s udy, is a clea bene i in gene ic s udies.
In conclusion, we ound p elimina y e idence ha TLR9 polymo phisms may be associa ed wi h
pos -b onchioli is wheezing. This inding needs o be con i med o uled ou in subsequen con i ma o y s udies.
Me hods
B onchioli is pa ien s. P e iously heal hy, ull- e m in an s hospi alized o b onchioli is a less han 6
mon hs o age du ing he pe iod Decembe 1, 2001 h ough May 31, 2002 and du ing he pe iod om Oc obe
28, 2002 h ough May 31, 2004 we e en olled in he s udy. In all, 187 eligible in an s we e hospi alized o he i s
ime du ing he wo s udy pe iods. App op ia e clinical da a including i al indings and gene polymo phisms
we e a ailable om 135 in an s33. B onchioli is was de ined as an acu e LRTI cha ac e ized by hino hea, cough,
and di use wheezes and/o c ackles1. The i al ae iology o b onchioli is was s udied by an igen de ec ion and
polyme ase chain eac ion (PCR) in nasopha yngeal aspi a es (NPA), as desc ibed ecen ly33. The s udied i uses
we e RSV, human hino i us (hRV), human me apneumo i us (hMPV), In luenzaA i us, Pa ain luenza3 i us
(PIV3), adeno i us and human boca i us (hBoV). Da a on disease se e i y, such as he need o supplemen a y
oxygen and eeding suppo , and he leng h o hospi al s ay (LOS) we e eco ded du ing he inpa ien ca e33.
Supplemen a y oxygen was gi en when oxygen sa u a ion (SaO2) measu ed wi h pulse-oxime y was less han
94%, and eeding suppo was de ined as a need o in a enous luids o eeding ia a nasogas ic ube.
A e hospi aliza ion o b onchioli is, he child en we e in i ed o a ollow-up isi a 1.5 yea s o age (on a e age),
and 129 (92.8% o hose in i ed; 69.0% o hose hospi alized) a ended. A he ollow-up isi , he pa en s we e
in e iewed using a s uc u ed ques ionnai e on he occu ence o o i is media, and wheezing episodes, he p e-
sc ip ion o an ibio ics, and he use o co icos e oids o wheezing a e hospi aliza ion o b onchioli is34. The
pa en s had eco ded a dia y du ing he 1.5 yea s pos -b onchioli is ollow-up pe iod, including desc ip ions o
all in ec ions and wheezing pe iods e i ied by a amily doc o o a paedia ician. The s udy physician and he
pa en s checked oge he he dia ies a he con ol isi . The doc o -diagnosed episodes we e eco ded o he
analyses.
Food alle gy and a opic de ma i is, al hough epo ed by pa en s, we e egis e ed du ing hospi aliza ion and
a he con ol isi on a e age 1.5 yea s la e —only doc o -diagnosed cases we e included. A blood sample o
immunoglobulin E (IgE) measu emen was aken a he ollow-up isi a he age o 1.5 yea s. To al se um IgE was
de e mined by elec o-chemiluminescence (ECLIA, Roche Diagnos ics GmbH, Mannheim, Ge many) acco ding
o labo a o y p ac ice. Se um IgE was conside ed o be ele a ed i he concen a ion was mo e han + 2SD abo e
he mean o non-a opic Finnish child en (> 60 IU/ml)35. Co icos e oid ea men mean main enance medica-
ion pe iods wi h inhaled co icos e oids (ICSs) o epea ed wheezing o suspec ed as hma. Repea ed wheezing
was de ined as wo o mo e wheezing episodes du ing he pos -b onchioli is ollow-up pe iod.
F ozen whole-blood samples we e a ailable o TLR7 s179008, TLR8 s2407992, and TLR10 s4129009 geno-
yping om all 129 child en. Samples o TLR9 s187084 geno yping we e a ailable om 127 child en.
TLR7 emales Ou come a iable,
N = 67
AA
N = 42, No. (%)
AT
N = 21, No. (%)
TT
N = 4, No. (%)
Majo allele A
N = 105, No. (%)
Repea ed (≥ 2 episodes) wheezing
N = 9 4 (9.5) p = 0.20 5 (23.8) 0 p = 0.55 13 (12.4) p = 1.0
Inhaled co icos e oids N = 5 2 (4.8) p = 0.27 3 (14.3) 0 p = 0.73 7 (6.7) p = 0.69
A opic de ma i is N = 8 4 (9.5) p = 0.34 2 (9.5) 2 (50.0) p = 0.07 10 (9.5) p = 0.16
Food alle gy N = 15 12 (28.6) p = 0.10 3 (14.3) 0 p = 0.35 27 (25.7) p = 0.20
IgE ≥ 60 N = 10/65 4 (9.5) p = 0.12 6 (28.6) 0 p = 0.60 14 (14.3) p = 0.41
Recu en (≥ 3) o i is media
N = 28 21 (50.0) p = 0.07 6 (28.6) 1 (25.0) p = 0.44 48 (46.7) p = 0.24
TLR8 emales Ou come a iable,
N = 67 GG N = 18, No. (%) GC N = 39, No. (%) CC N = 10, No. (%) Majo allele G
N = 75, No. (%)
Repea ed (≥ 2 episodes) wheezing
N = 9 2 (11.1) p = 0.55 7 (17.9) 0 p = 0.21 10 (13.3) p = 0.62
Inhaled co icos e oids N = 5 2 (11.1) p = 0.41 3 (7.7) 0 p = 0.43 7 (9.3) p = 0.52
A opic de ma i is N = 8 2 (11.1) p = 0.63 6 (15.4) 0 p = 0.25 10 (13.3) p = 0.62
Food alle gy N = 15 3 (16.7) p = 0.37 11 (28.2) 1 (10.0) p = 0.29 17 (22.7) p = 0.94
IgE ≥ 60 N = 10/65 3 (16.7) p = 0.57 7 (17.9) 0 p = 0.20 13 (17.3) p = 0.45
Recu en (≥ 3) o i is media
N = 28 8 (44.4) p = 0.50 14 (35.9) 6 (60.0) p = 0.18 30 (40.0) p = 0.76
Table 7. Geno ypes and majo allele equencies o TLR7 s179008 and TLR8 s2407992 genes in 67 emale
b onchioli is pa ien s in ela ion o ou come a iables du ing he pos -b onchioli is ollow-up un il he age
o 1.5 yea s. Majo allele s mino allele.
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Gene ic s udies. The geno yping o TLR8 s2407992 (2040 C/G) was pe o med by py osequencing as
desc ibed o TLR3 s3775291 (1234 C/T)14. 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 μ l o elu ion bu e . A e pu i i-
ca ion, he PCR p oduc s we e pipe ed o a 96-well pla e (5 μ l) oge he wi h TLR7 s179008 (171 A/T) o wa d
p ime (1.6 μ l). The 96-well pla e was sen o he Ins i u e o Molecula Medicine labo a o y in Helsinki, Finland
o sequencing. TLR9 s187084 (1486 T/C) geno yping was pe o med using BspTI es ic ion enzyme (The mo
Fishe Scien i ic, Wal ham, USA) o he diges ion o PCR p oduc 36. High- esolu ion mel ing analysis (HMR)
(Roche Diagnos ics Ligh Cycle 480, Basel, Swi ze land), was used o geno ype TLR10 s4129009 (2322 A/G)
as desc ibed ea lie 36. The me hods we e desc ibed ecen ly in mo e de ails36. The PCR and sequencing p ime s
used o he TLR7, TLR8, TLR9, and TLR10 genes a e desc ibed in Table9. All he p ime s we e pu chased om
Sigma-Ald ich, Finland.
Con ols. TLR7 s179008 and TLR8 s2407992 polymo phisms we e examined in wo-mon h-old heal hy
Finnish in an s, who pa icipa ed in he S eps o Child en’s Heal hy De elopmen and Wellbeing s udy (STEPS),
which is a p ospec i e bi h coho s udy o app oxima ely 1,800 child en37. No selec ion c i e ia we e applied
in he ec ui men o child en o he STEPS S udy o o inclusion in he subg oup wi h TLR geno yping. DNA
was ex ac ed om blood collec ed a he age o wo o h ee mon hs in 412 subjec s, who isi ed he s udy clinic
in Tu ku, Finland37. Only 328 child en we e included as con ols in he p esen s udy o TLR7 s 179008, TLR8
s2407992 and TLR10 s 4129009 polymo phisms, due o he limi ed amoun o DNA a ailable om he in an s
who we e no included. Fo TLR 9 s187084, he e was enough DNA o analyze only 270 samples. The geno yping
p ocesses we e iden ical in he b onchioli is coho and in he con ols, excep o he TLR10 s4129009 gene.
De e mina ion o ha polymo phism was pe o med by diges ion simila ly o TLR9 s187084 in he con ol
g oup, and by HRMA in he b onchioli is coho .
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. 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, bo h du ing hospi aliza ion and a he con ol isi . 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. 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, and he Ins i u e o Molecula Medicine,
Helsinki, Finland. The geno yping o con ol child en was done wi hin he STEPS s udy, which 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 was app o ed by
he E hics Commi ee o he Hospi al Dis ic o Sou hwes Finland, Tu ku, Finland. The pa en s o pa icipa ing
child en p o ided hei w i en, in o med consen .
S a is ical analyses. Da a we e analyzed using SPSS package e sion 23.0 (IBM Co p. NY, USA). Chi squa e
and Fishe ’s exac es s we e used o ca ego ized a iables. S uden ’s - es was used o no mally dis ibu ed
a iables. The Mann–Whi ney es was used o non-no mally dis ibu ed con inuous a iables. The esul s we e
exp essed as equencies, p opo ional equencies, means, medians and s anda d de ia ions (SD).
TLR9 Ou come a iables,
N = 127
TT
N = 41, No. (%)
TC
N = 53, No. (%)
CC
N = 33, No.(%)
Majo allele T
N = 135, No.(%)
Repea ed (≥ 2 episodes) wheezing
N = 24 5 (12.2) p = 0.14 8 (15.1) 11 (30.5) p = 0.02 18 (13.3) p = 0.047
Inhaled co icos e oids N = 16 3 (7.3) p = 0.17 7 (13.2) 6 (18.2) p = 0.20 13 (9.6) p = 0.18
A opic de ma i is N = 18 9 (22.0) p = 0.07 4 (7.5) 5 (15.2) p = 0.53 22 (16.3) p = 0.37
Food alle gy N = 30 7 (17.1) p = 0.17 14 (26.4) 9 (27.3) p = 0.36 28 (20.7) p = 0.37
IgE ≥ 60 N = 20/123 7 (17.1) p = 0.46 7 (13.2) 6 (18.2) p = 0.46 21 (15.6) p = 0.94
Recu en (≥ 3) o i is media
N = 62 22 (53.7) p = 0.29 21 (39.6) 19 (57.6) p = 0.17 65 (48.1) p = 0.89
TLR10 Ou come a iables,
N = 129 AA N = 109, No. (%) AG N = 19, No. (%) GG N = 1 Majo allele A
N = 237, No. (%)
Repea ed (≥ 2 episodes) wheezing
N = 24 20 (18.3) p = 0.54 4 (21.1) 044 (18.6) p = 0.78
Inhaled co icos e oids N = 16 13 (11.9) p = 0.47 3 (15.8) 029 (12.2) p = 0.73
A opic de ma i is N = 18 16 (14.7) p = 0.44 2 (10.5) 034 (14.3) p = 1.0
Food alle gy N = 30 27 (24.8) p = 0.26 3 (15.8) 057 (24.1) p = 0.59
IgE ≥ 60 N = 20/124 13(11.9) p = 0.015 7 (36.8) 033 (13.9) p = 0.08
Recu en (≥ 3) o i is media
N = 63 54 (49.5) p = 0.45 9 (47.4) 0117 (49.4) p = 0.84
Table 8. Geno ypes and majo allele equencies o TLR9 s187084 (N = 127) and TLR10 s4219009
(N = 129) genes in b onchioli is pa ien s in ela ion o ou come a iables du ing he pos -b onchioli is
ollow-up un il he age o 1.5 yea s. Majo allele s. mino allele.
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Logis ic eg ession was used o he mul i a ia e analyses, adjus ed o age, sex and a opic de ma i is du ing
he i s 1.5 yea s o li e when app op ia e. The esul s we e exp essed as odds a ios (OR) and 95% con idence
in e als (95% CI). The FINETTI p og am was used o e alua e he Ha dy–Weinbe g equilib ium (HWE) o he
s udied TLR9 and TLR10 alleles s udied.
Re e ences
1. AAP. 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).
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TLR7 11Gln > Leu (171A/T) ( s179008)
o 5′ -AGATGTCTGGTATGTGGTT-3′
e 5′ -TGATTCTTGGTATGTTTTAGA-3′
TLR8 651Leu > Leu (2040C/G) ( s2407992)
o 5′ -TGGAAAGCAAGTCCCTGGTA-3′
e 5′ -Bio in-AGTGAGACTCGCTGGCAAAT-3′
seq 5′ -ATCCCTTAATAGGCT-3′
TLR9 (silen mu a ion) (1486T/C) ( s187084)
o 5′ -ACTATGGAGCCTGCCTGCCATGATACC-3′
e 5′ -ATCCAGCCTTCTTACAAACCTCCCACC-3′
es ic ion enzyme BspTI
TLR10 775Ile > Val (2322A/G) ( s4129009)
o 5′ -CTTACTGGAACCCATTCCATTCTATTGC-3′
e 5′ -TCAATGTACATCCCAACAGTGTATGTGG-3′
es ic ion enzyme VspI
TLR10 775Ile > Val (2322A/G) ( s4129009)
o 5′ -AGTTCATACATTTCTCTGGTGGCT-3′
e 5′ -GTGGGCTTTTCTGGGCAAAC-3′
Table 9. P ime sequences, allele and amino acid changes in oll-like ecep o (TLR) genes TLR7 s179008
(171A/T), TLR8 s2407992 (2040C/G), TLR9 s187084 (1486T/C) and TLR10 s4129009 (2322A/G). TLR7
SNP was de ec ed by common sequencing, TLR8 SNP by py osequencing, TLR9 by diges ion and TLR10 by
high- esolu ion analysis.
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Scien i ic RepoR s | 6:31165 | DOI: 10.1038/s ep31165
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Au ho Con ibu ions
K.N. had esponsibili y o p o ocol de elopmen , pa ien sc eening, da a analysis and w i ing he manusc ip .
S.T. and P.K. had pa icipa ed w i ing in 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 w i ing
he manusc ip . V.P. had esponsibili y o he STEPS s udy, en olling he con ol pa ien s and a anging he
labo a o y analyses. He also pa icipa ed in w i ing he manusc ip . M.K. was esponsible o he planning and
in e p e a ion o he analyses and pa icipa ed in w i ing he manusc ip .
Addi ional In o ma ion
Compe ing inancial in e es s: The au ho s decla e no compe ing inancial in e es s.
How o ci e his a icle: Nuoli i a, K. e al. Pos -b onchioli is wheezing is associa ed wi h oll-like ecep o 9
s187084 gene polymo phism. Sci. Rep. 6, 31165; doi: 10.1038/s ep31165 (2016).
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