HLA-A*0206 with TLR3 Polymorphisms Exerts More than Additive Effects in Stevens-Johnson Syndrome with Severe Ocular Surface Complications
et al. (2012) HLA-A*0206 with TLR3 Polymorphisms Exerts More than Additive Effects in Stevens-
Johnson Syndrome with Severe Ocular Surface Complications. PLoS ONE 7(8): e43650. doi:10.1371/journal.pone.0043650
HLA-A*0206 with TLR3 Polymorphisms Exerts More than Additive Effects in Stevens-Johnson Syndrome with Severe Ocular Surface Complications
Yoshihiko Hoshino, National Institute of Infectious Diseases, Japan
Background: Stevens-Johnson syndrome (SJS) is an acute inflammatory vesiculobullous reaction of the skin and mucosa, often including the ocular surface, and toxic epidermal necrolysis (TEN) occurs with its progression. Although SJS/TEN is thought to be initiated by certain types of medication coupled with possible infection. In the present study we examined the multiplicative interaction(s) between HLA-A*0206 and 7 Toll-like receptor 3 (TLR3) Single-nucleotide polymorphisms (SNPs) in patients with SJS/TEN. Principal Findings: We analyzed the genotypes for HLA-A and 7 TLR3 SNPs in 110 Japanese SJS/TEN patients with severe ocular complications and 206 healthy volunteers to examine the interactions between the two loci. We found that HLAA*0206 exhibited a high odds ratio for SJS/TEN (carrier frequency: OR = 5.1; gene frequency: OR = 4.0) and that there was a strong association with TLR3 rs.5743312T/T SNP (OR = 7.4), TLR3 rs.3775296T/T SNP (OR = 5.8), TLR3 rs.6822014G/G SNP (OR = 4.8), TLR3 rs.3775290A/A SNP (OR = 2.9), TLR3 rs.7668666A/A SNP (OR = 2.7), TLR3 rs.4861699G/G SNP (OR = 2.3), and TLR3 rs.11732384G/G SNP (OR = 1.9). There was strong linkage disequilibrium (LD) between rs.3775296 and rs.5743312 and between rs.7668666 and rs.3775290. The results of interaction analysis showed that the pair, HLA-A*0206 and TLR3 SNP rs3775296T/T, which exhibited strong LD with TLR3 rs.5743312, exerted more than additive effects (OR = 47.7). The other pairs, HLA-A*0206 and TLR3 rs.3775290A/A SNP (OR = 11.4) which was in strong LD with TLR3 rs7668666A/A SNP, and TLR3 rs4861699G/G SNP (OR = 7.6) revealed additive effects. Moreover, the combination HLA-A*0206 and TLR3 rs3775296T/T was stronger than the TLR3 rs6822014G/G and TLR3 rs3775290A/A pair, which reflected the interactions within the TLR3 gene alone. Significance: By interaction analysis, HLA-A*0206 and TLR3 SNP rs3775296T/T, which were in strong LD with TLR3 SNP rs5743312T/T, manifested more than additive effects that were stronger than the interactions within the TLR3 gene alone. Therefore, multiplicative interactions of HLA-A and TLR3 gene might be required for the onset of SJS/TEN with ocular complications.
Competing Interests: The authors have declared that no competing interests exist.
Stevens-Johnson syndrome (SJS) is an acute inflammatory
vesiculobullous reaction of the skin and mucous membranes. It
was first described in 1922 by Stevens and Johnson,  both
pediatricians, who encountered 2 boys aged 8 and 7 who
manifested an extraordinary, generalized skin eruption, persistent
fever, inflamed buccal mucosa, and severe purulent conjunctivitis
resulting in marked visual disturbance. Subsequently, other
pediatricians reported that SJS was associated with infectious
agents such as Mycoplasma pneumoniae,  and a viral etiology
involving herpes simplex virus, Epstein-Barr virus,
cytomegalovirus, and varicella zoster virus . On the other hand,
dermatologists claimed that more than 100 different drugs were implicated
in eliciting SJS and its severe form, toxic epidermal necrolysis
(TEN) [4,5]. The annual incidence of SJS and TEN has been
estimated to be 0.41 and 16 cases per million persons,
respectively; [6,7] the reported mortality rate is 3% and 27%,
respectively . Although rare, these reactions have high
morbidity and mortality rates, and often result in severe and
definitive sequelae such as vision loss. SJS/TEN is one of the most
devastating ocular surface diseases leading to corneal damage and
loss of vision. The reported incidence of ocular complications in
SJS/TEN is 5068% [7,8].
In the acute stage, patients manifest vesiculobullous lesions of
the skin and mucosa, especially that of the eyes and mouth, and
severe conjunctivitis. The loss of finger nails in the acute or
subacute stage due to paronychia was observed, has been observed
in almost all SJS/TEN patients with severe ocular surface
In the chronic stage, despite healing of the skin lesions, ocular
surface complications such as conjunctival invasion into the cornea
[10,11,12,13,14,15,16,17,18]. It is also reported that lid margin
keratinization and tarsal scarring, together with lipid tear
deficiency, contributes to corneal complications because of
blinkrelated microtrauma .
Elsewhere we reported that the frequency of carriers of the
HLA-A*0206 antigen is significantly higher among Japanese
patients with severe ocular surface complications than in other
populations [18,20]. Our single nucleotide polymorphism (SNP)
association analysis of candidate genes documented the associated
polymorphisms of several immune-related genes including TLR3,
[12,17] IL4R, [14,16] IL13,  and FasL in Japanese SJS/
TEN patients with severe ocular surface complications. To
elucidate the detailed pathophysiology of SJS/TEN we performed
a genome-wide association study of SJS/TEN patients and found
associations between 6 SNPs in the prostaglandin E receptor 3
(EP3) gene (PTGER3) and SJS/TEN accompanied by severe
ocular surface complications . Moreover, gene-gene
interaction analysis in SJS/TEN patients with severe ocular surface
complications revealed that the interaction between TLR3 and
PTGER3 exerted SJS/TEN susceptibility effects, and there was
a functional interaction between TLR3 and EP3 in a murine
experimental allergic conjunctivitis model. .
In the present study we examined the multiplicative
interaction(s) between HLA-A*0206 and 7 TLR3 SNPs (rs3775296
(uSNP), rs5743312 (iSNP), rs6822014 (gSNP), rs3775290 (sSNP),
rs7668666 (iSNP), rs11732384 (iSNP), and rs4861699 (gSNP))
associated with the SJS/TEN patients [12,17] as the onset of SJS/
TEN was associated not only with the administration of drugs but
also with putative viral syndromes [10,11,12,17]. HLA-A is
a component of HLA class I, which resides on the surface of all
nucleated cells and alerts the immune system that the cell may be
infected by a virus, thereby targeting the cell for destruction.
TLR3 recognises viral double-stranded RNA .
We analyzed the genotypes for HLA-A and 7 TLR3 SNPs in
110 Japanese SJS/TEN patients with severe ocular complications
and 206 healthy volunteers to examine the interactions between
the two loci.
We found that HLA-A*0206 exhibited a high odds ratio for
SJS/TEN (carrier frequency: p = 6.9610210, OR = 5.1; gene
frequency: p = 2.561029, OR = 4.0) (Table 1).
We also found that there was a strong association with TLR3
rs.5743312T/T SNP (T/T vs T/C+C/C: p = 2.561026,
OR = 7.4), TLR3 rs.3775296T/T SNP (T/T vs T/G+G/G:
p = 8.261026, OR = 5.8), TLR3 rs.6822014G/G SNP (G/G vs
G/A+A/A: p = 1.261024, OR = 4.8), TLR3 rs.3775290A/A SNP
(A/A vs A/G+G/G: p = 7.161024, OR = 2.9), TLR3
rs.7668666A/A SNP (A/A vs A/G+G/G: p = 1.261023,
OR = 2.7), TLR3 rs.4861699G/G SNP (G/G vs G/A+A/A:
Case (N = 110)
Control (N = 206)
Genotype 11 vs.
Allele 1 vs. Allele 2 12+22
Genotype 11+12 vs. 22
aP-value for allele or genotype frequency comparisons between cases and controls using the chi-square test.
bOR, odds ratio.
cCI, confidence interval.
p = 4.261024, OR = 2.3), and TLR3 rs.11732384G/G SNP (G/
G vs G/A+A/A: p = 8.561023, OR = 1.9) (Table 2). All SNPs
were in Hardy-Weinberg equilibrium (p.0.01) in the samples
from patients and the controls. Based on the squared correlation
coefficient r2, we investigated the linkage disequilibrium (LD)
among the TLR3 SNPs. We found strong LD between rs.3775296
and rs.5743312 (D = 1, r2 = 0.911), and between rs.7668666 and
rs.3775290 (D = 0.973, r2 = 0.934) (Fig. 1).
Results of interaction analysis showed that the pair,
HLAA*0206 and TLR3 SNP rs3775296T/T, which exhibited strong
LD with TLR3 rs.5743312, exerted more than additive effects. We
found that while 11 of the 110 patients (10%) manifested both
HLA-A*0206 and TLR3 rs3775296T/T SNP, none of the 206
controls did (p = 6.561026, OR = 47.7, Woolfs correction). The
other pairs, HLA-A*0206 and TLR3 rs.3775290A/A SNP, which
was in strong LD with TLR3 rs.7668666, or TLR3 rs4861699G/
G SNP revealed additive effects: 16 of the 110 patients (14.5%) but
only 3 of the 206 controls (1.5%) had both HLA-A*0206 and
TLR3 rs.3775290A/A SNP (p = 7.461026, OR = 11.4). In
addition, 33 of the 110 patients (30%), compared to 11 of the
206 controls (5.3%), had both HLA-A*0206 and TLR3
rs.4861699G/G SNP (p = 1.661029, OR = 7.6) (Table 3).
Moreover, to examine the interactions within the TLR3 gene
alone we analyzed interactions between 2 each of 5 TLR3 SNPs
(rs3775296, rs6822014, rs3775290, rs11732384, rs4861699).
Combinations of high risk genotypes, on which the observed
numbers in cases were greater than of the controls and greater
than five, were analyzed. One of the 9 combinations, TLR3
rs6822014G/G and TLR3 rs3775290A/A, exerted more than
additive effects (OR 16.1, p = 2.061026) (Table 4). However, the
combination HLA-A*0206 and TLR3 rs3775296T/T produced
a stronger additive effect than it. In addition, we performed
haplotype association analysis with the 7 TLR3 SNPs (rs4861699,
rs6822014, rs11732384, rs3775296, rs5743312, rs7668666,
rs3775290) and the 5 TLR3 SNPs (rs4861699, rs6822014,
rs11732384, rs3775296, rs3775290), and found that no haplotype
showed strong association (p,0.001) (Table S1). Thus, the
haplotype associations appear to contribute little to the observed
To our knowledge, ours is the first report documenting the
additive effects of HLA-A*0206 and TLR3 polymorphisms. Our
interaction analysis showed that the pair HLA-A*0206 and TLR3
SNP rs3775296T/T, which was in strong LD with TLR3
rs.5743312, exerted more than additive effects, and that other
pairs, HLA-A*0206 and TLR3 rs.3775290A/A SNP in strong LD
with TLR3 rs.7668666, and TLR3 rs4861699G/G SNP exerted
additive effects. Moreover, the combination HLA-A*0206 and
TLR3 rs3775296T/T was stronger than the combination with
TLR3 rs6822014G/G or TLR3 rs3775290A/A, the interactions
within the TLR3 gene alone.
HLA-A, a component of HLA class I, alerts the immune
system that the cell may be infected with a virus; TLR3
recognizes viral double-stranded RNA . It is worth noting
that about 80% of our SJS patients developed SJS after
receiving treatment for the common cold with antibiotics, cold
remedies, and/or NSAIDs; only about 5% of our SJS patient
progressed to SJS after drug treatment to prevent the
occurrence of convulsions [11,12]. Moreover, our review of
medical records revealed that 9 of the 11 patients with both
HLA-A*0206 and TLR3 SNP rs3775296T/T (and
rs.5743312T/T) developed SJS after receiving cold medicine,
leading us to suspect that they already had a viral infection
before taking the cold medicine. Particulars on the other 2
patients are unknown because they developed SJS during
Although the TLR3 SNPs exerting additive- or more than
additive effects with HLA-A*0206 were u-, i-, or gSNPs and
without amino acid changes, it is possible that TLR3 SNPs and
HLA-A*0206 were involved in the onset of SJS with severe ocular
surface complications. Moreover, their interaction might influence
the host immune response against viral infection with drug
Earlier reports indicated regional differences in HLA
associations. Although in Japanese SJS patients we were unable to detect
the HLA-Bw44 antigen, a subgroup of HLA-B12 [19,23], it was
significantly increased in Caucasian SJS patients with ocular
On the other hand, the HLA-A*0206 antigen, which is not
found in Caucasians [18,19] was significantly increased in our
SJS patients (N = 110)
Controls (N = 206)
**Fishers exact test.
Japanese SJS patients with ocular complications. While there
might be ethnic differences in the association of SJS/TEN with
HLA,[18,19] specific combinations of genes and certain
environmental factors may be required for the manifestation of this rare
Elsewhere  we reported that the epistatic interaction
between TLR3 and PTGER3 confers an increased risk for SJS
with ocular complications. Since SJS/TEN is a rare condition that
probably has a complex genetic background, it is reasonable to
posit that multiplicative interactions of genes such as HLA-A &
TLR3, and TLR3 & PTGER3, are required for the phenotypic
In summary, we show that HLA-A*0206 with TLR3
polymorphisms exerts more than additive effects in SJS with severe
Combination of 2 TLR3 SNPs
SJS (N = 110)
Controls (N = 206)
ocular surface complications and we suggest that gene-gene
interactions should be considered in addition to major single-locus
Materials and Methods
This study was approved by the institutional review board of
Kyoto Prefectural University of Medicine and the University of
Tokyo, Graduate School of Medicine. All experimental
procedures were conducted in accordance with the principles of the
Helsinki Declaration. The purpose of the research and the
experimental protocols were explained to all participants, and
their prior written informed consent was obtained.
Diagnosis of SJS/TEN was based on a confirmed history of
acute onset of high fever, serious mucocutaneous illness with skin
eruptions, and involvement of at least 2 mucosal sites including the
ocular surface [9,11,12,17,18].
To investigate the gene-gene interaction between HLA-A*0206
and TLR3, we enrolled 110 SJS/TEN patients in the chronic or
subacute phase; all presented with symptoms of ocular surface
complications. None of the patients were relatives. The controls
were 206 healthy volunteers. All participants and volunteers were
Japanese residing in Japan. The average age of the 110 patients
and 206 controls was 43.6618.0 (SD) and 35.4611.1 (SD) years,
respectively. The male:female ratios in the patient and control
groups were 42:68 and 82:124, respectively. Some of the SJS/
TEN patients and controls in this study were subjects in our earlier
TLR3 SNPs Genotyping
Genomic DNA was isolated from human peripheral blood at
SRL Inc. (Tokyo, Japan). Genotyping for 2 SNPs of TLR3
(rs3775290, 3775296) was performed by PCR-direct sequencing as
reported previously . For direct sequencing, PCR
amplification was conducted with AmpliTaq Gold DNA Polymerase
(Applied Biosystems) for 35 cycles at 94uC for 1 min, annealing
at 60uC for 1 min, and 72uC for 1 min on a commercial PCR
machine (GeneAmp; Perkin-Elmer Applied Biosystems). The PCR
products were reacted with BigDye Terminator v3.1 (Applied
Biosystems) and sequence reactions were resolved on an ABI
PRISM 3100 Genetic Analyzer (Applied Biosystems).
Genotyping for 5 SNPs of TLR3 (rs4861699, rs6822014,
rs11732384, rs5743312, rs7668666) as performed using DigiTag2
assay . Multiplex PCR was performed in 10 ml of Multiplex
PCR buffer containing 25 ng genomic DNA, 25 nM of each
multiplex primer mix, 200 mM of each dNTP, 2.25 mM MgCl2,
and 0.4 U KAPA2G Fast HotStart DNA polymerase (Kapa
Biosystems). Cycling was performed at 95uC for 3 min, followed
by 40 cycles of 95uC for 15 s and 68uC for 2 min. The primers and
probes used in this study previously were reported [12,17].
For HLA-A genotyping, we performed polymerase chain
reaction amplification followed by hybridization with
sequencespecific oligonucleotide probes (PCR-SSO) using commercial
bead-based typing kits (WAK Flow, Wakunaga, Hiroshima,
Japan), as described previously [18,19].
Statistical significance of the association with each SNP was
assessed using Chi-square test or Fishers exact test on two-by-two
contingency tables. When the value obtained for the control was
0 the odds ratio was calculated using Woolfs correction.
Haploview software (ver. 4.2) was used to infer the linkage
disequilibrium structure of the 7 TLR3 SNPs and to perform
a haplotype analysis of TLR3 gene.
Table S1 Haplotype analysis of TLR3 gene. Haplotype
association analysis with the 7 TLR3 SNPs (rs4861699, rs6822014,
rs11732384, rs3775296, rs5743312, rs7668666, rs3775290) and
the 5 TLR3 SNPs (rs4861699, rs6822014, rs11732384, rs3775296,
Conceived and designed the experiments: MU. Performed the
experiments: MU KT HS. Analyzed the data: MU KT HS GT. Contributed
reagents/materials/analysis tools: MU CS TI SK. Wrote the paper: MU.
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