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Scien i ic RepoR s | 7:41522 | DOI: 10.1038/s ep41522
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Rapid de ec ion o unc ional gene
polymo phisms o TLRs and IL-17
using high esolu ion mel ing
analysis
Johanna Te äsjä i1, An i Hakanen2, Ma i Ko ppi3, Ki si Nuoli i a4,
Ki si G öndahl-Yli-Hannuksela1, Jussi Me sola5, Ville Pel ola5 & Qiushui He1,6
Gene ic a ia ions in oll-like ecep o s (TLRs) and IL-17A ha e been widely connec ed o di e en
diseases. Associa ions be ween suscep ibili y and esis ance o di e en in ec ions and single
nucleo ide polymo phisms (SNPs) in TLR1 o TLR4 and IL17A ha e been ound. In his s udy, we aimed
o de elop a apid and high h oughpu me hod o de ec unc ional SNPs o abo e men ioned p o eins.
The ollowing mos s udied and clinically impo an SNPs: TLR1 ( s5743618), TLR2 ( s5743708), TLR3
( s3775291), TLR4 ( s4986790) and IL17 ( s2275913) we e es ed. High esolu ion mel ing analysis
(HRMA) based on eal- ime PCR combined wi h mel ing analysis o a sa u a ing double s anded-
DNA binding dye was de eloped and used. The ob ained esul s we e compa ed o he “s anda d”
sequencing me hod. A o al o 113 DNA samples wi h known geno ypes we e included. The HRMA
me hod co ec ly iden i ied all geno ypes o hese i e SNPs. Co-e icien alues o a ia ion o in a-
and in e - un p ecision epea abili y anged om 0.04 o 0.23%. The de e mined limi o quali ica ion
o es ing samples was om 0.5 o 8.0 ng/μl. The iden ical geno yping esul was ob ained om he
same sample wi h hese concen a ions. Compa ed o “s anda d” sequencing me hods HRMA is cos -
e ec i e, apid and simple. All he i e SNPs can be analyzed sepa a ely o in combina ion.
Toll-like ecep o s (TLR) a e a c i ical pa o inna e immuni y. So a , en unc ional TLRs ha e been iden i ied in
humans. TLRs ecognize a wide ange o pa hogen-associa ed molecula pa e ns (PAMPs) om mic oo ganisms
and dange -associa ed molecula pa e ns (DAMPs) eleased om damaged issue1,2. In u n, in e leukins (ILs)
a e a la ge g oup o immune modula ing p o eins ha bind o hei speci ic ecep o s and play an impo an ole
in he communica ion in he immune sys em3.
TLR2 and TLR1 a e membe s o he TLR2 sub amily which comp ises TLR1, TLR2, TLR6 and TLR10. TLR1
is co- ecep o o TLR2 and hey occu as he e odime s on a cell su ace. TLR1 and TLR2 a e capable o de ec ing
a wide ange o ligands, especially om G am-posi i e bac e ia and mycobac e ia. The main ligand o TLR2 is
pep idoglycan and ha o TLR1 is iacyla ed lipopep ide. TLR3 is loca ed on he su ace o endosome and i
ecognizes i al dsRNA, syn he ic oligonucleo ides and DAMPs. TLR4 is loca ed on he cell su ace o on he
su ace o endosome. I ecognizes lipopolysaccha ides (LPS) and PAMPs o G am-nega i e bac e ia4. Bo h TLR3
and TLR4 a e also inna e an i i al immune esponse ecep o s. IL17 (IL17A) is a p o-in lamma o y cy okine. I
is p oduced by di e en ypes o cells, including CD4+ T cells and neu ophils. IL17 o ches a es o ec ui men
o neu ophils and s imula es p oduc ion o he an imic obial pep ides3.
Gene ic a ia ions, especially single nucleo ide poin mu a ions (SNPs) can a ec de elopmen o disease in
indi iduals and esponses o pa hogens, d ugs, accines and o he agen s. Recen s udies ha e shown a num-
be o SNPs in genes coding he TLR1, TLR2, TLR3, TLR 4 and IL17A. These SNPs ha e been associa ed wi h
1Depa men o Medical Mic obiology and Immunology, Uni e si y o Tu ku, Tu ku, Finland. 2TYKS-Sapa,
Mic obiology and Gene ics Se ice A ea, Mic obiology B anch, Tu ku Uni e si y Hospi al, Tu ku, Finland. 3Cen e o
Child Heal h Resea ch, Tampe e Uni e si y and Uni e si y Hospi al, Tampe e, Finland. 4Depa men o Pedia ics,
Seinäjoki Cen al Hospi al, Seinäjoki, Finland. 5Depa men o Pedia ics and Adolescen Medicine, Tu ku Uni e si y
Hospi al, Tu ku, Finland. 6Depa men o Medical Mic obiology, Capi al Medical Uni e si y, Beijing, China.
Co espondence and eques s o ma e ials should be add essed o Q.H. (email. [email p o ec ed])
Recei ed: 10 Oc obe 2016
Accep ed: 19 Decembe 2016
Published: 02 Feb ua y 2017
OPEN
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Scien i ic RepoR s | 7:41522 | DOI: 10.1038/s ep41522
suscep ibili y o esis ance o di e en in ec ions4,5. In his s udy, we ook in o accoun he mos s udied and
clinically impo an SNPs o he TLRs and IL17, which a e lis ed in Table1.
One o he mos s udied SNPs in TLR2 gene is 2258G > A ( s5743708). This SNP causes amino acid subs i-
u ion om a ginine (A g) o glu amine (Gln) in he C- e minal end o he TLR2 a posi ion o 753. Indi iduals
who ca y he mino allele A, may ha e inc eased isk o a opy6, sepsis7,8 and suscep ibili y o ube culosis9,10.
Hae ynck and his colleagues ha e ound a co ela ion be ween allele A and as e decline o o ced expi a o y
olume 1 (FEV1) esul o pa ien s wi h cys ic ib osis11. Sch öde e al. ha e shown ha he e ozygous polymo -
phism (A g753Gln) can impai immune ac i a ion by Bo elia bu gdo e i and may p o ec om la e s age Lyme
disease12. The SNP 1805G > T ( s5743618) in TLR1 gene leads o subs i u ion o amino acid a he posi ion 602
om se ine (Se ) o isoleucine (Ile) on he cy oplasmic side o he ansmemb ane domain o he ecep o . I
a ec s he cell su ace a icking in se e al cell ypes4. Allele G, which is a mino allele in all o he popula ions,
excep Eu opeans, is associa ed wi h inc eased suscep ibili y o as hma and alle gic hini is a e hospi aliza ion
due o b onchioli is in in ancy13 and is also a isk ac o o ube culosis14–16. The SNP 1234C > T ( s3775291) in
TLR3 gene causes an amino acid change om leucine (Leu) o phenylalanine (Phe) a he posi ion 412 in he
ex acellula domain o his p o ein. The dominan allele C was ound o be a isk ac o o ick-bo ne encephali-
is i us (TBEV)17–19 and b onchioli is in ea ly in ancy20. TLR4 SNP 896A > G ( s4986790) causes an amino acid
change om aspa ic acid (Asp) o glycine (Gly) a he posi ion o 299. I is loca ed in he ex acellula domain
o his p o ein. In he p e ious s udies we ha e shown an associa ion be ween he mino allele G and inc eased
isk o epea ed coloniza ion o Mo axella ca a halis21. The allele G has also been connec ed o an inc eased isk
o di e en diso de s caused by bac e ia and i uses, such as sep ic shock due o in ec ion by G am-nega i e
bac e ia22,23, inc eased mo ali y o child en wi h in asi e meningococcal disease24, ch onic ca i a y pulmona y
aspe gillosis25 and suscep ibili y o espi a o y syncy ial i us (RSV) in ec ion26. The IL17A SNP − 197G > A
( s2275913) is loca ed in he p omo e o IL17A gene and has an e ec on i s ansc ip ional egula ion. Child en
who ca y geno ype AA o IL17A ha e usually low se um le el o IL17A. We ha e p e iously epo ed an asso-
cia ion be ween he mino allele A and inc eased coloniza ion o S ep ococcus pneumoniae in young Finnish
child en27. O he s udies ha e shown associa ions o he allele A wi h gas oin es inal diseases28 and childhood
as hma29. The SNPs s udied in TLRs and IL17A and po en ial e ec s on suscep ibili y and esis ance o di e en
diseases a e summa ized in Table1.
Di ec DNA sequencing is conside ed a “golden s anda d” o iden i ica ion o he SNPs. Howe e , he mel ing
p ope ies o DNA ha e enabled he de elopmen o many apid and high h oughpu me hods30, high esolu ion
mel ing analysis (HRMA) being one o hem. The me hod is based on analysis o eleased luo escence om
binding dye o he double s anded DNA (dsDNA) and is p o en o be a simple, ela i ely apid and low-cos
ool o SNP scanning. PCR is he i s s ep o HRMA ollowed by binding o a dye. The dye binds o dsDNA,
when amplicons a e dena u ed and hen apidly e-annealed. Mel ing analysis s a s when dsDNA amplicons
wi h sa u a ed dye a e g adually hea ed and elease he luo escence dye. The da a can hen be analyzed based
on he mel ing empe a u e (Tm) and on he shape o he no malized mel ing cu es. HRMA is ideal o clinical
use because PCR ampli ica ion and mel ing cu e analysis can be pe o med on he same pla e and wi hou any
pos -PCR p ocessing. This sa es ime and dec eases he isk o con amina ion.
HRMA me hod has been used o geno yping o Ye sinia en e ocoli ica31, o sc eening o oncogene mu a ions32
and o iden i ying o common a ian s in me hylene e ahyd o ola e educ ase (MTHFR) gene33. A me a analysis
has shown ha HRMA me hod is a highly sensi i e, simple and low-cos es o de ec human disease-associa ed
mu a ions, especially o samples wi h mu a ions o low incidence34. Recen ly HRMA me hod has been applied
o de ec ion o TLR1 ( s5743618), TLR2 ( s5743708) and TLR4 ( s4986790) SNPs35–37.
In ou p e ious s udies, we ha e used PCR-based py osequencing, Sange sequencing and Sequenom mas-
sARRAY iPlex Gold me hod o de ec ing he unc ional SNPs o TLRs21 and IL1727. These me hods a e gene ally
ime consuming and expensi e, especially o a la ge numbe o samples. Inc eased knowledge o unc ion and
impo ance o TLRs and ILs ha e a oused he in e es in clinicians o a ia ions o unc ional SNPs. In his p esen
s udy we aimed o de elop a high h oughpu me hod based on HRMA o apid de ec ion o TLR1 ( s5743618),
TLR2 ( s5743708), TLR3 ( s3775291), TLR4 ( s4986790) and IL17A ( s2275913). The knowledge o he gene ic
a ia ions in hese impo an molecules will inc ease ou unde s anding o why some indi iduals ha e highe
Ta ge SNP id
Nucleo ide
change
Amino acid
change
Allele F equency
(1000 Genome p ojec )*
Allele F equency
o his s udy
Po en ial e ec and epo ed
associa ions
TLR1 s5743618 1805G > T Se > Ile A: 0.172 C: 0.828 A: 0.208 C: 0.792 Comp omised signaling; As hma,
Alle gic hini is and TB
TLR2 s5743708 2258G > A A g > Gln G: 0.970 A: 0.030 G: 0.948 A: 0.052 Comp omised signaling; Sepsis, TB,
A opy, Cys ic Fib osis decline
TLR3 s3775291 1234C > T Leu > Phe C: 0.667 T: 0.333 C: 0.578 T: 0.422 Comp omised signaling; Tick-bo ne
encephali is i us (TBE), B onchioli is
TLR4 s4986790 896A > G Asp > Gly A: 0.884 G: 0.116 A: 0.848 G: 0.155
Hypo esponsi eness; M.ca a halis and
H.in luenzae coloniza ion, Sep ic shock,
Respi a o y syncy ial i us (RSV)
IL17A s2275913 − 197G > A —G: 0.571 A: 0.429 G: 0.530 A: 0.470
Reduced p oduc ion; S.pneumoniae
coloniza ion, Childhood as hma,
Gas oin es inal diseases
Table 1. Func ion o he s udied SNPs and he allele equencies epo ed in Finnish popula ion (1000
Genome p ojec ) and in his s udy. *Re e ence 42.
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Scien i ic RepoR s | 7:41522 | DOI: 10.1038/s ep41522
likelihood o in ec ions and di e en ou comes a e he diseases. In he u u e his knowledge may help plan a
mo e pe sonalized ca e chain.
No mally, he alida ion and e i ica ion o a me hod ollow ISO s anda ds o O ganiza ion o Economic
Coope a ion and De elopmen guidelines. Fo HRMA alida ion he e a e no such s anda ds a ailable. We used
selec ed alida ion pa ame e s, which a e desc ibed in p e ious li e a u e30,33.
Resul s
PCR and HRM pa ame e s op imiza ion. Th ee samples wi h known geno ypes we e used o de e mine
he p ope concen a ion o MgCl2 and annealing empe a u e (Ta °C) o each indi idual assay, shown in Table2.
In o de o he me hod o be sui able o diagnos ic use i would be necessa y o analyze all he SNPs on one pla e
du ing he same un. The e o e, he second s ep on he op imiza ion p ocess was o ind ou he op imal annealing
empe a u e (Ta) which wo ks o all used p ime se s. The op imal Ta a ied be ween 60.2 °C and 57 °C in indi id-
ual assays. Two es uns we e made and he annealing empe a u e was se a 58 °C on he i s un and a 59 °C
on he second un. When compa ing he annealing cu es and mel ing p o iles, he highe annealing empe a u e
gi es mo e ep esen a i e esul s, hus 59 °C is used as he annealing empe a u e in he assay.
Valida ion. Based on he op imiza ion uns, wo samples wi h he bes mel ing cu e, we e chosen o ep e-
sen each a ian and la e hese samples we e used as he con ols. Mel ing cu es o di e en geno ypes o TLR1,
TLR2 and TLR3 used as con ols a e shown in Fig.1 and hose o TLR4 and IL17 a e shown in Fig.2. Sequences
o he con ols we e con i med by Sange sequencing (da a no shown).
To al o 77 samples we e used o es he me hod accu acy o TLR1 ( s5743618), TLR2 ( s5743708) and TLR3
( s3775291). The esul s ob ained by HRMA we e compa ed o py osequencing esul s. Resemblance be ween
HRMA and sequencing geno yping esul s was 100%. To al o 60 samples we e used o es he me hod accu-
acy o TLR4 ( s4986790) and IL17 ( s2275913). The esul s ob ained by HRMA we e compa ed o hose by
py osequencing (TLR4) and Sequenom massARRAY iPlex Gold me hod (IL17). Iden ical geno yping esul s
we e ob ained.
Fo analysis o mel ing cu es, all es ed samples we e g ouped oge he wi h ega d o he con ols, and
mel ing p o iles we e iden ical o DNA samples wi h he same geno ype. The coe icien o a ia ion (CV) o
in a- and in e - un p ecision- epea abili y a ied be ween 0.04 and 0.23%. In TLRs and IL17A geno ypes he e
we e no conside able di e ences in CV% o mel ing p o iles.
The limi o quan i ica ion was es ed o he i e s udied SNPs using a DNA empla e. Two old se ial dilu ions
o he sample we e used, anging om 0.5 o 8.0 ng/μ l. The iden ical geno yping esul was ob ained om he
same sample wi h di e en concen a ions o all i e s udied SNPs. Only a ma ginal change in mel ing cu e p o-
iles and mel ing empe a u es was obse ed be ween di e en DNA concen a ions o he same sample (Fig.3).
Con i ma ion o TLR1 SNP. A he beginning o his s udy we no iced ha HRMA mel ing cu es and
sequencing diag ams we e a ypical o he TLR1 ( s5743618). HRMA clea ly di ided he six es ing samples
wi h known h ee geno ypes ( wo o each wild ype, he e ozygo e a ian and homozygo e a ian ) in h ee
di e en g oups (Fig. 5a–c). Al hough he sequencing diag am (Fig. 4a) showed ha he e was a nucleo ide
T-peak in each sample, e en in hose which we e supposed o be he homozygous wild ype G geno ype. We
hus pe o med a Genbank sea ching and ound ha TLR1 p ime se used o HRMA was no speci ic only
o TLR1 s5743618 (1805G < T), bu he p ime s we e also capable o binding o TLR6 gene. The close exam-
ina ion unco e ed a ~300 bp a ea in TLR1, wi h 97% simila i y o TLR6. The backg ound peak T was ac ually
de i ed om co-ampli ica ion o he TLR6 and TLR1 genes. In o de o di e en ia e he h ee geno ypes o TLR1
s5743618 we edesigned a se o p ime s only speci ic o TLR1. The backg ound peak T in he TLR1 sequence
disappea ed a e he p e-PCR s ep (Fig.4b) and bo h no malized HRMA cu es became ypical (Fig.5e, ). The
h ee geno ypes o TLR1 s5743618 (1805G < T) we e clea ly dis inguished by HRMA no ma e whe he he
p e-PCR s ep was pe o med o no (Fig.5).
Ta ge SNP id P ime s
P oduc size
(bp)
Annealing empe a u e
(⁰C)
MgCl2 concen a ion
(mM)
TLR1 s5743618 F:5′ -CTGGCACACCATCCTGAGAT-3′
R:5′ -GTTGGCTGTGACTGTGACCT-3′ 70 57 2.5
TLR2 s5743708 F:5′ -TCTCAATTCTTCTGGAGCCCAT-3′
R:5′ -GGCCACTCCAGGTAGGTCT-3′ 96 57 2.5
TLR3 s3775291 F:5′ -ACTTGCTCATTCTCCCTTACACAT-3′
R:5′ -GCCCAATTTCATTAAGGCCCA-3′ 125 60 3
TLR4 s4986790 F:5′ -ACCATTGAAGAATTCCGATTAGCA-3′
R:5′ -CCAGGGAAAATGAAGAAACATTTG-3′ 95 59 2.5
IL17A s2275913 F:5′ -TCTGCCCTTCCCATTTTCCTTC-3′
R:5′ -GGTTAAAATTTCCGCCCCCAATT-3′ 70 59 3
Table 2. P ime s and pa ame e s used in he High Resolu ion Mel ing Analyze in each indi idual assay*.
*Annealing empe a u e 59 °C was used when analyzing all he i e SNPs in same un.
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Discussion
The pu pose o he s udy was o de elop a apid, less expensi e and high h oughpu geno yping me hod o
unc ional polymo phism o impo an inna e immune genes such as TLRs and IL-17. A se o samples we e
selec ed om wo o ou ea lie s udies: b onchioli is and STEPS s udies conduc ed in Finland in which unc-
ional SNPs o TLRs and IL-17 we e analyzed o ind ou hei associa ions wi h de elopmen o subsequen in ec-
ions and pos -b onchioli is ou comes13,20,21,27,38. The geno yping me hod was based on eal- ime PCR combined
wi h HRMA. The esul s we e compa ed o he PCR-based sequencing me hods such as py osequencing and
Sange sequencing. All s udied geno ypes o hese ou TLRs and IL-17A we e co ec ly iden i ied wi h HRMA.
Compa ed o he “ adi ional” sequencing me hods HRMA is cos -e ec i e, apid and simple. All he i e SNPs
can be analyzed sepa a ely o oge he which make i well-adap ed analysis ool o diagnos ic use.
In gene al, pos -PCR mel ing analyses a e used o sepa a e homozygo e and he e ozygo e a ian s. Sepa a ion
be ween homozygo e wild ype and homozygo e a ian s is mo e di icul , because hey di e only in be ween
mel ing empe a u es (Tm) bu he shapes o no malized and shi ed mel ing cu es a e gene ally simila (Fig.1h).
In ou case, he di e ence be ween a homozygo e wild ype and a homozygo e a ian is > 0,5 °C. These ypes
o SNPs a e ela i ely easy o be di e en ia ed, bu when analyzing all he SNPs on he same pla e a he same
ime, PCR and mel ing condi ions equi e ce ain comp omises. In his case, he annealing condi ions o PCR
a e no ully op imized o all he SNPs. In ou s udy, annealing empe a u e was op imal o IL-17 and TLR4
(Table2). Because o his, he shapes o he mel ing cu es o he TLR1–3 we e no as sha p and ep esen a i e
as hey we e when es ed sepa a ely. Howe e , simul aneous ampli ica ions o he i e PCRs we e p o en o be
speci ic and we we e able o dis inguish geno ypes o all hese i e SNPs. Mo eo e , bo h in a- and in e - un
p ecision- epea abili y CV we e e y low (< 0,2%), showing ha HRMA is a eliable me hod o he SNP analysis
o TLR1-4 and IL17A.
TLR1, TLR2, TLR6 and TLR10 comp ise he TLR2 sub amily. These TLRs ha e simila i ies in hei s uc u e
and sequence. The sequence simila i ies may become a p oblem when using HRMA o de e mine he TLR SNPs
o his sub amily. We me his kind o p oblem wi h TLR1 SNP s5743618. The SNP is loca ed in he middle o
~300 bp a ea wi h 97% simila i y o TLR6 gene. We used he p e-PCR s ep o con i m he iden i y o each TLR1
a ian . In he TLR6 gene he e is also a SNP ( s62622399) in he same loca ion as TLR1 SNP s5743618. The
SNP in TLR6 gene has been ound only in one small Ban hu popula ion in A ica. Since TLR6 SNP s62622399 is
ex emely a e (only epo ed in wo cases) and he p e-PCR s ep is qui e labo ious o he ou ine diagnos ic es ,
TLR1 SNP s5743618 can be analyzed wi hou he p e-PCR s ep, as clea ly shown in Fig.5.
Figu e 1. Analysis o TLR1 SNP 1805G > T ( s5743618), TLR2 SNP 2258 G > A ( s5743708) and TLR3
SNP 1234C > T ( s3775291). The uppe h ee pic u es ep esen PCR ampli ica ion o TLR1 (a), TLR2 (d) and
TLR3 (g). No malized mel ing p o iles o he SNPs a e shown in middle h ee pic u es (b,e and h). In he pic u e
h can pe cei e how bo h homozygo e geno ypes (TLR3 C/C and T/T) o ms simila no malized and shi ed
mel ing cu e and he e ozygous mel ing cu e (TLR3 C/T) se les be ween he homozygo e cu es. The las
pic u es (c, and i) ep esen no malized and emp-shi ed di e ence plo o he TLR1, TLR2 and TLR3 SNPs.
In igu e c TLR1 SNP ( s5743618) majo geno ype in Finnish popula ion, was chosen o be as a baseline and in
igu es and i wild ype geno ype was chosen o be as a baseline.
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Scien i ic RepoR s | 7:41522 | DOI: 10.1038/s ep41522
Based on he p e ious s udies39–41 i is known ha high- esolu ion DNA mel ing analysis has some limi a-
ions bu in his s udy we ha e aken accoun hese limi a ions and shown ha HRMA is a eliable and accu a e
me hod o he SNP analysis o TLR 1-4 and IL17A. The bes esul s a e ob ained when he con ol DNAs a e o
high-quali y and he concen a ions o hese con ol DNAs and s udied samples a e closed o each o he .
I is known ha quan i a i e PCR is p one o ha e a ia ion in he yield o ampli ied p oduc s, when di e en
concen a ions o a ge DNAs a e used. In his p esen s udy, wo- old se ial dilu ions o a DNA sample anging
om 0.5 o 8.0 ng/μ l we e used o es he e ec . Fo all i e s udied SNPs, he iden ical geno yping esul was
ob ained om he same DNA sample wi h di e en concen a ions. Only a ma ginal change in mel ing cu e p o-
iles and mel ing empe a u es was obse ed be ween di e en DNA concen a ions o he same sample (Fig.3).
Howe e , i should be kep in mind ha he di e ence in DNA concen a ions es ed was only less han 16 olds.
TLRs and IL-17 a e key molecules in inna e and adap i e immuni y. Gene ic a ia ions in TLRs and ILs ha e
been widely connec ed o di e en diseases like cance , au oimmuni y diso de s and in ec ions. Fu he mo e,
many s udies ha e shown he associa ions be ween in ec ions and SNPs in TLR2, TLR3, TLR4 and IL17A. These
SNPs ha e been associa ed wi h suscep ibili y o esis ance o di e en in ec ions3–5. The me hods de eloped
in his s udy could be used o sc eening o pa ien s wi h di e en disease condi ions and he in o ma ion will
inc ease ou unde s anding abou he connec ions be ween di e en diseases and he SNPs and could p o ide a
ool o clinicians o design mo e e ec i e p ecision medicine.
Ma e ials and Me hods
Samples and DNA ex ac ion. To al o 113 anonymously selec ed samples om wo o ou ea lie s udies13,20,21,27
we e used in his s udy. The i s 53 DNA samples we e isola ed om he whole blood samples which we e col-
lec ed om ull- e m in an s hospi alized o b onchioli is a < 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, 200413. These samples
we e s o ed a − 70 °C un il DNA ex ac ion. Res o 113 samples we e andomly selec ed DNA samples om
a Finnish bi h coho s udy, called “S eps o he heal hy de elopmen and well-being o child en” ( he STEPS
s udy)38. The blood samples o he STEPS S udy we e aken be ween Augus 2008 and June 2010, when heal hy
in an s isi ed he s udy clinic a he age o 2.6 mon hs. These samples we e immedia ely anspo ed o he lab-
o a o y, whe e he DNA ex ac ion was pe o med. The DNA samples a e s o ed a − 20 °C. The p o ocols o he
Figu e 2. Analysis o TLR4 SNP 896A > G ( s4986790) and IL17 SNP − 197G > A ( s2275913). The i s
pic u es ep esen annealing s ep o TLR4 (a), and IL17 (d) PCR. The no malized mel ing p o iles o he SNPs
a e shown in he wo la e pic u es (b and e). The las pic u es (c and ) ep esen no malized and emp-shi ed
di e ence plo o he TLR4 and IL17 SNPs. Wild ype geno ype was chosen o be as a baseline.

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wo s udies we e app o ed by he E hics Commi ees o Tampe e Uni e si y Hospi al Dis ic , Tampe e and 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 . The pe sonal da a o he s udy subjec s we e no gi en o he labo a o y pe o ming
he gene ic s udies. The me hods we e ca ied ou in acco dance wi h he ele an guidelines, including any el-
e an de ails.
In bo h cases, 200 μ l o he whole blood was used o DNA isola ion. Ex ac ion was done wi h QIAGEN
QIAamp DNA Blood Mini Ki 250 (Qiagen, Hilden, Ge many) acco ding o he manu ac u e ’s p o ocol. DNA
quali y and concen a ion we e measu ed by a spec opho ome e (NanoD op ND-1000, The mo Scien i ic,
Wal ham, Massachuse s, USA). The abo e men ioned i e SNPs o TLR1, TLR2, TLR3 and IL17A we e de ec ed
and he esul s ha e been p e iously published13,20,21,27,38.
P ime design. To maximize he di e ence be ween mel ing peaks (Tm) in a ian geno ypes, p ime s we e
designed o de ine a small agmen (< 150 bp) su ounding he SNP in in e es . The manu ac u e ’s ecommen-
da ions o CG (%) con en and p ime mel ing empe a u es we e aken in o accoun . P ime s (Table2) we e
designed wi h P ime -Blas design ool (h p://www.ncbi.nlm.nih.go / ools/p ime -blas /).
TLR1 HRMA p ime s we e se speci ic o SNP s5743618 (1805G < T), which is loca ed in he middle o ~300
bp a ea wi h 97% simila i y o TLR6. In o de o sepa a e he wo TLRs, a P e-PCR was used o con i m speci ic
annealing and mel ing in HRMA. The sequences o he wo p ime s used in he TLR1 p e-PCR we e o wa d:
5′ -GAAAGGAGACTGTTCTTGG-3′ and e e se: 5′-CTTTTGAGCTTGTGATAACTGCTAG -3′ .
All p ime s we e o de ed om Sigma-Ald ich Company (Sain Louis, Missou i, USA). The P ime s used in
HRMA we e wi h HPCL quali y.
Op imiza ion o PCR and HRM pa ame e s. All HRM analyses we e pe o med by Ligh Cycle 480
e sion 5.1 (Roche, Basal, Swi ze land). Roche’s o iginal mel ing mas e ki (Ligh Cysle 480 High Resolu ion
Mel ing Mas e ) and 96-well pla es (Ligh Cycle 480 Mul iwell 96, Whi e) we e used. Th ee samples wi h known
geno ypes we e used o de e mine he p ope concen a ion o MgCl2 and annealing empe a u e (Ta°C) o each
indi idual assay (Table2). Se ial dilu ions om 1 o 3.5mM o MgCl2 we e es ed. De e mining Ta°C was ca ied
ou by a g adien PCR. In each un eac ion olume was 20 μ l consis ing o 3 μ l genomic DNA (8.0 ng/μ l) and 17 μ l
o mas e mix including 10 μ l mel ing mas e dye wi h he addi ional concen a ion o MgCl2 (se ial dilu ion) and
Figu e 3. E ec o he empla e DNA concen a ion on he annealing cu es and mel ing p o iles o he
amplicons.
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0.2 μ M o o wa d and e e se p ime s. The g adien PCR eac ion p og am s a ed wi h an ini ial dena u a ion
o 10 min a 95 °C ollowed by 45 cycles ampli ica ion o 10s a 95 °C and annealing empe a u e g adien om
57 °C o 62 °C and 15 s a 72 °C. A e he PCR s ep, HRM mel ing cycle condi ions we e as 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.
Fo TLR1 op imiza ion bo h pu e DNA and he PCR p oduc om p e-PCR we e used. The p e-PCR s ep was
pe o med wi h Applied Biosys ems The mal Cycle (The mo Scien i ic, Wal ham, Massachuse s, USA), using
Figu e 4. TLR1 SNP 1805 G > T ( s574318) sequence be o e and a e he p e-PCR. In he uppe -pa o he
pic u e (a) is clea ly o seen how he nucleo ide T om TLR6 o e laps wi h TLR1 1805G > T (a ow). In he
lowe pa o he igu e (b) is shown he TLR1 SNP sequence a e he p e-PCR s ep. A e he p e-PCR s ep he
HRMA p ime se ampli ied only o TLR1 and he T-peak om he TLR6 SNP is no pe cei ed in he sequence
(a ows in lowe pa o igu e).
Figu e 5. Mel ing Peaks (a and d), No malized and Shi ed mel ing Cu es (b and e) and No malized and
Temp-Shi ed Di e ence Plo s (c and ) be o e and a e he p e-PCR s ep. In bo h cases, be o e and a e he
p e-PCR s ep HRMA is capable o ecognize all h ee geno ypes. The cu es be o e p e-PCR s ep a e a ypical
(a,b and c), homozygo e geno ype GG exis like he e ozygo e a ian . In lowe pa o he igu e (d,e and )
cu es a e ypical o each geno ype.
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he ollowing pa ame e s; dena u a ion 10 min a 94 °C, 40 cycles a 94 °C, annealing a 55 °C o 40s and 72 °C o
60s, ollowed by one cycle a 72 °C o 5 min.
The second s ep in he op imiza ion p ocess was o ind ou he op imal condi ions whe e all he SNPs can be
un on he same pla e a he same ime. Op imal MgCl2 concen a ions a e shown in Table2. Op imal Ta a ied
be ween 57 °C and 60.2 °C. Two es uns we e made. PCR and HRM condi ions we e he same as desc ibed p e-
iously, excep he annealing empe a u e, which was on he i s un 58 °C and on he second un 59 °C.
Aga ose gel (1.5%) elec opho esis was used o con i m speci ic PCR ampli ica ions.
Valida ion o HRM pa ame e s. A o al o 77 DNA samples we e analyzed o TLR1–3 and 60 samples
o TLR4 and IL17A SNPs (Table2) by HRMA. Based on HRMA mel ing p o iles o he SNPs wo samples we e
selec ed o ep esen o each geno ype. The samples we e sen o he Finnish Ins i u e o Molecula Medicine,
Helsinki, Finland o sequencing o con i m he iden i y o he sequence. These samples we e la e used as he
con ols in each un.
Be o e he High Resolu ion Mel ing analysis, all samples we e geno yped wi h an addi ional me hod, ei he
py osequencing21, o Sequenom massARRAY iPlex Gold pla o m (IL17A)27. Resemblance be ween mel ing anal-
ysis and sequencing was de e mined and p esen ed in % conco dance.
In a-assay p ecision e lec s a iabili y among eplica e de e mina ions wi hin he same assay un. In u n,
in e - un p ecision is he compa ison o he esul s be ween he se ies un on di e en days. The same con ol
samples we e used in bo h assays. Fo in a- un assay he samples we e p epa ed in i e eplica es a one con-
cen a ion le el in one ial un and o he in e - un assay he same DNA samples we e analyzed in one ial
pe o med on h ee di e en days using eshly p epa ed eagen s. In a- and in e - un p ecision- epea abili y
was p esen ed as coe icien o a ia ion (%).
Limi o Quan i ica ion was de ec ed using a DNA empla e dilu ion anging om 0.5 o 8.0 ng/μ l.
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Acknowledgemen s
This s udy was pa ly suppo ed by TYKS-SAPA (A.H and Q.H (M4801).
Au ho Con ibu ions
Q.H. and J.T. concei ed and designed he expe imen s and w o e he manusc ip . J.T. and K.G. pe o med he
expe imen s. A.H. and J.M. pa icipa ed in he p o ocol de elopmen and w i ing he manusc ip . M.K. and K.N.
had esponsibili y o he b onchioli is s udy conduc ed in Tampe e and pa icipa ed in w i ing he manusc ip .
V.P. had esponsibili y o he STEPS s udy 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: Te äsjä i, J. e al. Rapid de ec ion o unc ional gene polymo phisms o TLRs and
IL-17 using high esolu ion mel ing analysis. Sci. Rep. 7, 41522; doi: 10.1038/s ep41522 (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.
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