New Susceptibility and Resistance HLA-DP Alleles to HBV-Related Diseases Identified by a Trans-Ethnic Association Study in Asia
et al. (2014) New Susceptibility and Resistance HLA-DP Alleles to HBV-Related Diseases
Identified by a Trans-Ethnic Association Study in Asia. PLoS ONE 9(2): e86449. doi:10.1371/journal.pone.0086449
New Susceptibility and Resistance HLA-DP Alleles to HBV-Related Diseases Identified by a Trans-Ethnic Association Study in Asia
Sang Hoon Ahn
Kwang- Hyub Han
Ferruccio Bonino, University of Pisa, Italy
Previous studies have revealed the association between SNPs located on human leukocyte antigen (HLA) class II genes, including HLA-DP and HLA-DQ, and chronic hepatitis B virus (HBV) infection, mainly in Asian populations. HLA-DP alleles or haplotypes associated with chronic HBV infection or disease progression have not been fully identified in Asian populations. We performed trans-ethnic association analyses of HLA-DPA1, HLA-DPB1 alleles and haplotypes with hepatitis B virus infection and disease progression among Asian populations comprising Japanese, Korean, Hong Kong, and Thai subjects. To assess the association between HLA-DP and chronic HBV infection and disease progression, we conducted high-resolution (4-digit) HLA-DPA1 and HLA-DPB1 genotyping in a total of 3,167 samples, including HBV patients, HBV-resolved individuals and healthy controls. Trans-ethnic association analyses among Asian populations identified a new risk allele HLA-DPB1*09:01 (P = 1.3661026; OR = 1.97; 95% CI, 1.50-2.59) and a new protective allele DPB1*02:01 (P = 5.2261026; OR = 0.68; 95% CI, 0.58-0.81) to chronic HBV infection, in addition to the previously reported alleles. Moreover, DPB1*02:01 was also associated with a decreased risk of disease progression in chronic HBV patients among Asian populations (P = 1.5561027; OR = 0.50; 95% CI, 0.39-0.65). Trans-ethnic association analyses identified Asian-specific associations of HLA-DP alleles and haplotypes with HBV infection or disease progression. The present findings will serve as a base for future functional studies of HLA-DP molecules in order to understand the pathogenesis of HBV infection and the development of hepatocellular carcinoma.
Funding: This work was supported by a Grant-in-Aid from the Ministry of Health, Labour, and Welfare of Japan H24-Bsou-kanen-ippan-011 and
H24-kanenippan-004 to Masashi Mizokami, H23-kanen-005 to Katsushi Tokunaga, H25-kanen-wakate-013 to Nao Nishida, and H25-kanen-wakate-012 to Hiromi Sawai. This
work was also supported by The Grant for National Center for Global Health and Medicine 22-shi-302 to Masashi Mizokami and 24-shi-107 to Nao Nishida. Partial
support by Grant-in-Aid from the Ministry of Education, Culture, Sports, Science of Japan [grant number 22133008] for Scientific Research on Innovative Areas to
Katsushi Tokunaga, [grant number 24790728] for Young Scientists (B) to Nao Nishida, and [grant number 25870178] for Young Scientists (B) to Hiromi Sawai, is
also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Hepatitis B virus (HBV) infection is a major global health
problem, resulting in 0.51.0 million deaths per year . The
prevalence of chronic HBV infection varies. About 75% of the
chronic carriers in the world live in Southeast Asia and East Pacific
. Due to the introduction of vaccination programs, the
prevalence of HBV infection in many countries has gradually
been decreasing with consequent decreases in HBV-related
hepatocellular carcinoma (HCC) . Although some HBV
carriers spontaneously eliminate the virus, about 1015% of
carriers develop liver cirrhosis (LC), liver failure and HCC .
Moreover, the progression of liver disease was revealed to be
associated with the presence of several distinct mutations in HBV
infections . Genetic variations in STAT4 and HLA-DQ genes
were recently identified as host genetic factors in a large-scale
genome-wide association study (GWAS) for HBV-related HCC in
With regard to the genes associated with susceptibility to
chronic HBV infection, HLA-DP and HLA-DQ genes were
identified by GWAS in Japanese and Thai populations in 2009
 and 2011 , respectively. In addition, our previous GWAS
confirmed and identified the association of SNP markers located
on HLA-DPA1 (rs3077) and HLA-DPB1 (rs9277535) genes with
susceptibility to chronic hepatitis B (CHB) and HBV clearance in
Japanese and Korean subjects. The significant associations of
HLA-DP with CHB and HBV clearance have mainly been
detected in Asian populations, such as Japanese [8,9], Thai ,
Chinese , and Korean . In 2012, the association
between HLA-DPA1 gene SNPs and persistent HBV infection was
replicated in a Germany non-Asian population for the first time;
however, this showed no association with HBV infection .
These results seem to be explained by the fact that allele
frequencies of both rs3077 (0.155, 0.587 and 0.743 for C allele,
on HapMap CEU, JPT, and YRI) and rs9277535 (0.261, 0.558
and 0.103 for G allele, on HapMap CEU, JPT, and YRI) are
markedly different between populations. Moreover, the previous
study showed that HBsAg seropositivity rates were higher in
Thailand and China (512%) than in North America and Europe
(0.20.5%) . These results suggest that comparative analyses of
HLA-DP alleles and haplotypes in Asian populations would clarify
key host factors of the susceptible and protective HLA-DP alleles
and haplotypes for CHB and HBV clearance. Here, we performed
trans-ethnic analyses of HLA-DP alleles and haplotypes in Asian
populations comprising Japanese, Korean, Hong Kong and Thai
individuals. The findings from this study will serve as a base for
future functional studies of HLA-DP molecules.
Characteristics of studied subjects
The characteristics of a total of 3,167 samples, including
Japanese, Korean, Hong Kong and Thai subjects, are shown in
Table 1. Each population included three groups of HBV patients,
resolved individuals and healthy controls. The clinical definitions
of HBV patients and resolved individuals are summarized in
Materials and Methods. Some of the Japanese and all of the
Korean samples overlapped with the subjects in our previous study
Healthy controls 467
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
We performed genotyping for HLA-DPA1 and HLA-DPB1 in all
3,167 samples, and a total of 2,895 samples were successfully
genotyped. The characteristics of successfully genotyped samples
are shown in Table S1.
Association of HLA-DPA1 and HLA-DPB1 alleles in Asian
As for a general Asian population, including 464 Japanese, 140
Korean, 156 Hong Kong, and 122 Thai subjects, five HLA-DPA1
alleles and twenty-four HLA-DPB1 alleles were observed (Table
S2). The frequencies of HLA-DPA1 and HLA-DPB1 alleles were
similar between Japanese and Korean subjects. On the other
hand, the number of alleles with frequencies of 12% was larger in
Hong Kong and Thai populations, despite the small sample size.
Although the frequencies of HLA-DP alleles varied in Asian
populations, HLA-DPB1*05:01 was the most prevalent with over
30% in all populations.
The associations of HLA-DPA1 and HLA-DPB1 alleles with
chronic HBV infection (i.e., comparison between HBV patients
and healthy controls) are shown in Table S2. To avoid false
positives caused by multiple testing, the significance levels were
corrected based on the numbers of HLA-DPA1 and HLA-DPB1
Hong Kong Thai
Abbreviation: IC, Inactive Carrier; CH, Chronic Hepatitis; AE, Acute Exacerbation;
LC, Liver Cirrhosis; HCC, Hepatocellular Carcinoma.
* Resolved individuals were HBsAg negative and HBcAb positive.
** 419 of 467 healthy controls were de-identified, without information on age.
alleles in the focal population. Briefly, the significance level was set
at 0.05/(# of observed alleles at each locus) in each population
(see Materials and Methods). With regard to high-risk alleles of
HLA-DPA1, the most prevalent allele HLA-DPA1*02:02 was
significantly associated with susceptibility to HBV infection in
Japanese (P = 3.4561024; OR = 1.39; 95% CI, 1.161.68) and
Korean subjects (P = 2.6661025; OR = 1.89; 95% CI, 1.392.58),
whereas this association was not observed in Hong Kong or Thai
subjects. The association of HLA-DPA1*02:01 with susceptibility to
HBV infection was significant only in Japanese (P = 2.6161027;
OR = 1.88; 95% CI, 1.462.41). The significant association of
HLA-DPA1*01:03 with protection against HBV infection was
commonly observed among four Asian populations (Table S2).
The pooled OR and 95% CI were 0.51 and 0.410.63,
respectively in a meta-analysis (P = 3.15610210) (Fig. S1A).
As shown in Table S2, HLA-DPB1 shows higher degree of
polymorphism than HLA-DPA1. The most common allele in Asian
populations, HLA-DPB1*05:01, was significantly associated with
HBV susceptibility in both Japanese and Korean subjects.
Although HLA-DPB1*05:01 showed no significant association in
the Hong Kong and Thai populations, the same direction of
association (i.e., HBV susceptibility) was observed. Meta-analysis
of the four populations revealed a significant association between
HLA-DPB1*05:01 and susceptibility to HBV infection
(P = 1.5161024; OR = 1.45; 95% CI, 1.191.75) (Fig. S1B). The
frequency of HLA-DPB1*09:01 was significantly elevated in
Japanese HBV patients (15.7%) as compared with healthy controls
(8.7%) (P = 3.7061026; OR = 1.94; 95% CI, 1.452.62), and this
association was most significant (i.e., the smallest P value) in the
Japanese population. Because of lower allele frequencies of
HLADPB1*09:01 or lack of statistical power in the other populations,
no significant associations were observed. A common allele in Thai
subjects, HLA-DPB1*13:01, was significantly associated with
susceptibility to HBV infection (P = 2.4961024; OR = 2.17; 95%
CI, 1.403.47) with the same direction of associations in Japanese
and Hong Kong (OR = 1.52 and 1.40, respectively).
HLA-DPB1*04:02 was identified as the most protective allele for
HBV infection in Japanese (P = 1.5961027; OR = 0.37; 95% CI,
0.240.55) and Korean subjects (P = 1.2761027; OR = 0.19; 95%
CI, 0.100.38). Both HLA-DPB1*02:01 and HLA-DPB1*04:01
were also significantly associated with protection in the Japanese
population, and the former was significantly associated with
protection in Hong Kong subjects (P = 9.1761024; OR = 0.49;
95% CI, 0.320.76). This common allele among four Asian
populations, HLA-DPB1*02:01, showed a significant association
with protection against HBV infection (P = 5.2261026;
OR = 0.68; 95% CI, 0.580.81) in a meta-analysis (Fig. S1B).
The frequencies of associated HLA-DP alleles in a comparison of
HBV patients with healthy controls (Table S2) or with
HBVresolved individuals (Table S3) were similar in all four Asian
populations. In the Japanese population, the associations of
susceptible and protective HLA-DPB1 alleles to chronic HBV
infection seem weaker in the comparison of HBV patients with
HBV-resolved individuals than in the comparison of HBV patients
with healthy controls. Moreover, the results of association analyses
showed no difference in the comparison of HBV patients with
HBV-resolved individuals, including or excluding HCV positive
individuals (Table S3). In contrast, the association became
stronger in the comparison of HBV patients with HBV-resolved
individuals among the Korean subjects. The protective allele
HLADPB1*04:01 was also identified to have a strong association with
HBV clearance in Hong Kong subjects (Table S3). Moreover, in
Hong Kong subjects, the HLA-DPB1*05:01 associated with the
risk for HBV infection showed lower frequency in HBV-resolved
*Population was adjusted using dummy variables.
individuals (42.9%) than in the healthy controls (48.1%), which
accounts for a strong association in the comparison of HBV
patients with HBV-resolved individuals (P = 6.2461023;
OR = 1.64; 95% CI, 1.142.36). Although the number of samples
was insufficient, HLA-DP*100:01 showed a significant association
with protection against HBV infection in the Hong Kong
population (P = 3.0561026; OR = 0.03; 95% CI, 0.00070.20).
As for disease progression in CHB patients among Asian
populations, a protective effect of HLA-DPB1*02:01 on disease
progression was observed in the Japanese (P = 4.2661025;
OR = 0.45; 95% CI, 0.300.67) and Korean populations
(P = 8.7461024; OR = 0.47; 95% CI, 0.290.75) (Table S4).
Multivariate logistic regression analysis adjusted for age and sex
revealed that the number of DPB1*02:01 alleles (i.e., 0, 1, or 2) was
significantly associated with disease progression in CHB patients in
Japanese (P = 1.7761024; OR = 0.47; 95% CI, 0.320.70)
(Table 2). Moreover, protective effects of DPB1*02:01 on disease
progression in Asian populations (P = 1.5561027; OR = 0.50;
95% CI, 0.390.65) were detected in a multivariate logistic
regression analysis adjusted for age, gender, and population
Associations of DPA1-DPB1 haplotypes in Asian
The estimated frequencies of HLA DPA1-DPB1 haplotypes are
shown in Table S5. The most frequent haplotype among the four
Asian populations was DPA1*02:02-DPB1*05:01. The number of
haplotypes with low frequencies of 12% was 10 in both Japanese
and Korean subjects, whereas more haplotypes appeared with
frequencies of 12% in Hong Kong and Thai subjects. The
associations of DPA1-DPB1 haplotypes with HBV infection are
shown in Table S5. In the Japanese population,
DPA1*02:01DPB1*09:01 showed the most significant association with
susceptibility to HBV infection (P = 3.3861026; OR = 1.95; 95% CI,
1.462.64). The most common haplotype in the four Asian
populations, DPA1*02:02-DPB1*05:01, was found to be
significantly associated with susceptibility to HBV infection in the
Japanese and Korean subjects (P = 7.4061024; OR = 1.37; 95%
CI, 1.141.66 for Japanese, and P = 4.5061026; OR = 2.02; 95%
CI, 1.482.78 for Korean). In the Thai subjects, HLA-DPB1*13:01
was the most significant risk allele for HBV infection (Table S2);
however, no significant associations were found for the three
different haplotypes bearing HLA-DPB1*13:01:
DPA1*02:01DPB1*13:01, DPA1*02:02-DPB1*13:01, and
DPA1*04:01DPB1*13:01,indicating that the association of HLA-DPB1*13:01
with susceptibility to HBV infection did not result from a specific
DPA1-DPB1 haplotype or combination with a specific DPA1 allele.
In the Japanese population, both haplotypes
DPA1*01:03DPB1*04:01 and DPA1*01:03-DPB1*04:02 showed significant
associations with protection against HBV infection
(P = 1.1761025; OR = 0.32; 95% CI, 0.180.56 for
DPA1*01:03-DPB1*04:01 and P = 1.9561027; OR = 0.37; 95%
CI, 0.240.55 for DPA1*01:03-DPB1*04:02). In the Korean
subjects, a significant association of DPA1*01:03-DPB1*04:02
was also demonstrated; however, no association was observed for
DPA1*01:03-DPB1*04:01. Because the observed number of each
haplotype was small, none of the other haplotypes showed a
significant association with protection against HBV infection.
In order to identify trans-ethnic DPA1-DPB1 haplotypes
associated with HBV infection, a meta-analysis was performed.
A meta-analysis further revealed that the
DPA1*01:03DPB1*02:01 haplotype was significantly associated with protection
against HBV infection (P = 1.4561025; OR = 0.69; 95% CI, 0.58
0.82) (Fig. S1C).
Among 2.2 billion individuals worldwide who are infected with
HBV, 15% of these are chronic carriers. Of chronic carriers, 10
15% develops LC, liver failure and HCC, and the remaining
individuals eventually achieve a state of nonreplicative infection,
resulting in HBsAg negative and anti-HBc positive, i.e.
HBVresolved individuals. To identify host genetic factors associated
with HBV-related disease progression may lead HBV patients to
discriminate individuals who need treatment.
The HLA-DPA1 and HLA-DPB1 genes were identified as host
genetic factors significantly associated with CHB infection, mainly
in Asian populations , and not in European populations
. In the previous association analyses of HLA-DPB1 alleles with
HBV infection, one risk allele HLA-DPB1*05:01 (OR = 1.52; 95%
CI, 1.311.76), and two protective alleles, HLA-DPB1*04:01
(OR = 0.53; 95% CI, 0.340.80) and HLA-DPB1*04:02
(OR = 0.47; 95% CI, 0.34.64), were identified in the Japanese
population . In this study, we further identified a new risk allele
HLA-DPB1*09:01 (OR = 1.94; 95% CI, 1.452.62) for HBV
infection and a new protective allele HLA-DPB1*02:01
(OR = 0.71; 95% CI, 0.560.89) in the Japanese population, in
addition to the previously reported alleles (Table S2) . The
discrepancy in the association of HLA-DPB1*09:01 allele with risk
for HBV infection in a previous study  results from the elevated
frequency of HLA-DPB1*09:01 in the controls (12.2%), which is
higher than our controls (8.7%). In this study, healthy subjects
were recruited as controls. In contrast, individuals that were
registered in BioBank Japan as subjects with diseases other than
CHB were recruited as controls in the previous study , which
may have included patients with diseases with which
HLADPB1*09:01 is associated. Although no significant association of
HLA-DPB1*09:01 with risk for HBV infection was observed in the
Korean subjects, HLA-DPB1*09:01 appears to have a susceptible
effect on HBV infection, as it showed the same direction of
association. When the association analyses in Japanese and
Korean subjects were combined in meta-analysis, the association
was statistically significant (P = 1.3661026; OR = 1.97; 95% CI,
1.502.59). Thus, HLA-DPB1*09:01 may be a Northeast
Asianspecific allele associated with risk for HBV infection.
Moreover, a significant association of HLA-DPB1*13:01 with
risk of HBV infection (OR = 2.17; 95% CI, 1.403.47) was
identified in the Thai subjects. However, the frequency of
HLADPB1*13:01 in Thai healthy controls (11.5% in the present study)
reportedly varies, ranging from 15.4% to 29.5%, due to the
population diversity . Therefore, a replication analysis is
required to confirm the association of HLA-DPB1*13:01 with
HBV infection in the Thai subjects. There were four other
marginally associated HLA-DPB1 alleles with low allele frequencies
below 5% in HBV patients and healthy controls, including
HLADPB1*28:01, -DPB1*31:01, -DPB1*100:01, and -DPB1*105:01, in
the Hong Kong and Thai subjects. Because these infrequent alleles
may have resulted from false positive associations, the association
needs to be validated in a large number of subjects.
HLA-DPB1*02:01 showed a significant association with
protection against HBV infection in both Japanese and Hong Kong
populations (Table S2); however, the HLA-DPB1*02:01 allele was
not associated with HBV infection in the previous study .
Although HLA-DPB1*02:01 showed no association in either
Korean or Thai populations, a significant association of
HLADPB1*02:01 with protection against HBV infection among four
Asian populations was detected in meta-analysis (P = 5.2261026;
OR = 0.68; 95% CI, 0.580.81) (Fig. S1B). We therefore conclude
that the present finding is not a false positive.
A recent report showed that HLA-DPB1*02:01:02, *02:02,
*03:01:01, *04:01:01, *05:01, *09:01, and *14:01 were
significantly associated with response to booster HB vaccination in
Taiwan neonatally vaccinated adolescents . The
HLADPB1*02:01:02, *02:02, *03:01:01, *04:01:01, and *14:01 were
significantly more frequent in recipients whose post-booster titers
of antibodies against HBV surface antigen (anti-HBs) were
detectable, on the other hand, HLA-DPB1*05:01 and *09:01 were
significantly more frequent in recipients who were undetectable.
Moreover, the HLA-DPB1*05:01 and *09:01 significantly increase
the likelihoods of undetectable pre-booster anti-HBs titers. These
results seem consistent with our findings, in which
HLADPB1*05:01 and *09:01 are associated with susceptibility to
chronic hepatitis B infection.
We also identified a protective effect of HLA-DPB1*02:01 allele
on disease progression in Asian populations. Previous studies
identified the association of HLA class II genes including HLA-DQ
and HLA-DR with development of HBV related hepatocellular
carcinoma in the Chinese population [6,19,20]. In this study using
Japanese and Korean samples, we identified significant
associations between HLA-DPB1*02:01 and disease progression in CHB
patients (P = 4.2661025; OR = 0.45; 95% CI, 0.300.67, for
Japanese and P = 8.7461024; OR = 0.47; 95% CI, 0.290.75 for
Korean) (Table S4). Although the association of HLA-DPB1*02:01
with disease progression was weaker after adjustment for age and
gender in Korean subjects (P = 2.5461022; OR = 0.55; 95% CI,
0.330.93), the same direction of association was observed (i.e.
protective effect on disease progression) (Table 2). The protective
effects of HLA-DPB1*02:01 on disease progression showed a
significant association after adjustment for age and gender in the
Japanese population (P = 1.7761024; OR = 0.47; 95% CI, 0.32
0.70); moreover, a significant association between
HLADPB1*02:01 was observed among four Asian populations, under
which population was adjusted by using dummy variables in a
multivariate logistic regression analysis (P = 1.5561027;
OR = 0.50; 95% CI, 0.390.65) (Table 2).
The HLA-DPA1 and HLA-DPB1 belong to the HLA class II
alpha and beta chain paralogues, which make a heterodimer
consisting of an alpha and a beta chain on the surface of antigen
presenting cells. This HLA class II molecule plays a central role in
the immune system by presenting peptides derived from
extracellular proteins. We identified two susceptible haplotypes
(DPA1*02:02-DPB1*05:01 and DPA1*02:01-DPB1*09:01) and
three protective haplotypes (DPA1*01:03-DPB1*04:01,
DPA1*01:03-DPB1*04:02, and HLA-DPA1*01:03-DPB1*02:01) to
chronic hepatitis B infection, which may result in different binding
affinities between HLA-DP subtypes and extracellular antigens.
Although functional analyses of HLA-DP subtypes to identify
HBV-related peptides are not fully completed, identification of
susceptible and protective haplotypes as host genetic factors would
lead us to understand the pathogenesis of HBV infection including
In summary, we identified a new risk allele HLA-DPB1*09:01,
which was specifically observed in Northeast Asian populations,
Japanese and Korean. Moreover, a new protective allele
HLADPB1*02:01 was identified among four Asian populations:
Japanese, Korean, Hong Kong and Thai. The protective allele
HLA-DPB1*02:01 was associated with both chronic HBV infection
and disease progression in chronic HBV patients. Identification of
a total of five alleles, including two risk alleles (DPB1*09:01 and
DPB1*05:01) and three protective alleles (DPB1*04:01,
DPB1*04:02 and DPB1*02:01), would enable HBV-infected
individuals to be classified into groups according to the treatment
requirements. Moreover, the risk and protective alleles for HBV
infection and disease progression, identified in this study by means
of trans-ethnic association analyses, would be key host factors to
recognize HBV-derived antigen peptides. The present results may
lead to subsequent functional studies into HLA-DP molecules and
viral factors in order to understand the pathogenesis of HBV
infection and development of hepatocellular carcinoma.
Materials and Methods
All study protocols conform to the relevant ethical guidelines, as
reflected in the a priori approval by the ethics committee of
National Center for Global Health and Medicine, and by the
ethics committees of all participating universities and hospitals,
including The University of Tokyo, Japanese Red Cross
KantoKoshinetsu Block Blood Center, The University of Hong Kong,
Chulalongkorn University, Yonsei University College of Medicine,
Nagoya City University Graduate School of Medical Sciences,
Musashino Red Cross Hospital, Tokyo Medical and Dental
University, Teine Keijinkai Hospital, Hokkaido University
Graduate School of Medicine, Kurume University School of Medicine,
Okayama University Graduate School of Medicine, Yamaguchi
University Graduate School of Medicine, Tottori University,
Kyoto Prefectural University of Medicine, Osaka City University
Graduate School of Medicine, Nagoya Daini Red Cross Hospital,
Ehime University Graduate School of Medicine, Kanazawa
University Graduate School of Medicine, National Hospital
Organization Osaka National Hospital, Iwate Medical University,
Kawasaki Medical College, Shinshu University School of
Medicine, Saitama Medical University, Kitasato University School of
Medicine, Saga Medical School, and University of Tsukuba.
Written informed consent was obtained from each patient who
participated in this study and all samples were anonymized. For
Japanese healthy controls, 419 individuals were de-identified with
information about gender, and all were recruited after obtaining
verbal informed consent in Tokyo prior to 1990. For the 419
Japanese healthy individuals, written informed consent was not
obtained because the blood sampling was conducted before the
Ethical Guidelines for Human Genome and Genetic Sequencing
Research were established in Japan. Under the condition that
DNA sample is permanently de-linked from the individual, this
study was approved by the Research Ethics Committee of
National Center for Global Health and Medicine.
Characteristics of studied subjects
All of the 3,167 genomic DNA samples were collected from
individuals with HBV, HBV-resolved individuals (HBsAg-negative
and anti-HBc-positive) and healthy controls at 26 multi-center
hospitals throughout Japan, Korea, Hong Kong, and Thailand
(Table 1). In a total of 1,291 Japanese and 586 Korean samples,
1,191 Japanese individuals and all 586 Korean individuals were
included in our previous study . With regard to additional
Japanese individuals, we collected samples from 48 healthy
controls at Kohnodai Hospital, and 52 HBV patients at Okayama
University Hospital and Ehime University Hospital, including 26
individuals with LC and 26 individuals with HCC. A total of 661
Hong Kong samples and 629 Thai samples were collected at
Queen Mary Hospital and Chulalongkorn University, respectively.
HBV status was measured based on serological results for
HBsAg and anti-HBc with a fully automated chemiluminescent
enzyme immunoassay system (Abbott ARCHITECT; Abbott
Japan, Tokyo, Japan, or LUMIPULSE f or G1200; Fujirebio,
Inc., Tokyo, Japan). For clinical staging, inactive carrier (IC) state
was defined by the presence of HBsAg with normal ALT levels
over 1 year (examined at least four times at 3-month intervals) and
without evidence of liver cirrhosis. Chronic hepatitis (CH) was
defined by elevated ALT levels (.1.5 times the upper limit of
normal [35 IU/L]) persisting over 6 months (by at least 3
bimonthly tests). Acute exacerbation (AE) of chronic hepatitis B
was defined as an elevation of ALT to more than 10 times the
upper limit of normal (ULN, 58 IU/L) and bilirubin to at least
three times ULN (15 mmol/L). LC was diagnosed principally by
ultrasonography (coarse liver architecture, nodular liver surface,
blunt liver edges and hypersplenism), platelet counts,100,000/
cm3, or a combination thereof. Histological confirmation by
fineneedle biopsy of the liver was performed as required. HCC was
diagnosed by ultrasonography, computerized tomography,
magnetic resonance imaging, angiography, tumor biopsy or a
The Japanese control samples from HBV-resolved subjects
(HBsAg-negative and anti-HBc-positive) at Nagoya City
University-affiliated healthcare center were used by comprehensive
agreement (anonymization in a de-identified manner) in this
study. Some of the unrelated and anonymized Japanese healthy
controls were purchased from the Japan Health Science Research
Resources Bank (Osaka, Japan). One microgram of purified
genomic DNA was dissolved in 100 ml of TE buffer (pH 8.0)
(Wako, Osaka, Japan), followed by storage at 220uC until use.
Genotyping of HLA-DPA1 and HLA-DPB1 alleles
High resolution (4-digit) genotyping of HLA-DPA1 and -DPB1
alleles was performed for HBV patients, resolved individuals, and
healthy controls in Japan, Korea, Hong Kong, and Thailand.
LABType SSO HLA DPA1/DPB1 kit (One Lambda, CA) and a
Luminex Multi-Analyte Profiling system (xMAP; Luminex, Austin,
TX) were used for genotyping, in according with the
manufacturers protocol. Because of the small quantity of genomic DNA in
some Korean samples, we performed whole genome amplification
for a total of 486 samples using GenomiPhi v2 DNA Amplification
kit (GE Healthcare Life Sciences, UK), in accordance with the
A total of 2,895 samples were successfully genotyped and
characteristics of these samples are summarized in Table S1.
Fishers exact test in two-by-two cross tables was used to
examine the associations between HLA-DP allele and chronic
HBV infection or disease progression in chronic HBV patients,
using statistical software R2.9. To avoid false-positive results due
to multiple testing, significance levels were adjusted based on the
number of observed alleles at each locus in each population. For
HLA-DPA1 alleles, the number of observed alleles was 3 in
Japanese, 4 in Korean, 5 in Hong Kong, and 5 in Thai subjects.
Therefore, the significant levels for a were set at a = 0.05/3 in
Japanese, a = 0.05/4 in Korean, a = 0.05/5 in Hong Kong, and
a = 0.05/5 in Thai subjects. In the same way, significant levels for
HLA-DPB1 alleles were a = 0.05/10, 0.05/11, 0.05/12, and 0.05/
16, respectively. Multivariate logistic regression analysis adjusted
for age and sex (used as independent variables) was applied to
assess associations between the number of DPB1*02:01 alleles (i.e.,
0, 1, or 2) and disease progression in CHB patients. To examine
the effect of DPB1*02:01 allele on disease progression in all
populations, population was further adjusted by using three
dummy variables (i.e., (c1, c2, c3) = (0, 0, 0) for Japanese, (1, 0, 0)
for Korean, (0, 1, 0) for Hong Kong, and (0, 0, 1) for Thai) in a
multivariate logistic regression analysis. We obtained the following
regression equation: logit(p) =
Significance levels in the analysis of disease progression in CHB
patients were set as a = 0.05/10 in Japanese, a = 0.05/11 in
Korean, a = 0.05/15 in Hong Kong, and a = 0.05/15 in Thai
subjects. The phase of each individual (i.e., a combination of two
DPA1-DPB1 haplotypes) was estimated using PHASE software
, assuming samples are selected randomly from a general
population. In comparison of the estimated DPA1-DPB1 haplotype
frequencies, significant levels were set as a = 0.05/14 in Japanese,
a = 0.05/17 in Korean, a = 0.05/17 in Hong Kong, and a = 0.05/
18 in Thai subjects. Meta-analysis was performed using the
DerSimonian-Laird method (random-effects model) in order to
calculate pooled OR and its 95% confidence interval (95% CI).
We applied meta-analysis for alleles with frequency.1% in all
four Asian populations. The significance levels in meta-analysis
were adjusted by the total number of statistical tests; a = 0.05/20
for DPA1 alleles, a = 0.05/57 for DPB1 alleles, and a = 0.05/74 for
Figure S1 Comparison of odds ratios in association
analyses for HLA-DP with chronic HBV infection among
four Asian populations: (A) HLA-DPA1 alleles; (B)
HLADPB1 alleles; and (C) HLA DPA1-DPB1 haplotypes.
Metaanalysis was performed using the DerSimonian-Laird
method (random-effects model) to calculate pooled OR
and its 95% confidence interval (95% CI). Bold depicts a
statistically significant association after correction of significance
Table S2 Frequencies of HLA-DP alleles in HBV
patients and healthy controls among Asian populations.
Table S4 Associations of HLA-DPB1 alleles with disease
progression in CHB patients among Asian populations.
We would like to thank all the patients and families who contributed to the
study. We are also grateful to Ms. Mayumi Ishii (National Center for
Global Health and Medicine), Ms. Megumi Sageshima, Yuko Hirano,
Natsumi Baba, Rieko Shirahashi, Ayumi Nakayama (University of Tokyo),
and Yuko Ohara (Japanese Red Cross Kanto-Koshinetsu Block Blood
Center) for technical assistance.
Conceived and designed the experiments: NN HS MS KT M. Mizokami.
Performed the experiments: NN HS KK Y. Mawatari M. Kawashima M.
Minami. Analyzed the data: NN HS M. Kawashima JO. Contributed
reagents/materials/analysis tools: W-KS M-FY NP YP SHA K-HH K.
Matsuura YT M. Kurosaki YA NI J-HK SH TI KY IS Y. Murawaki YI
AT EO YH MH SK EM KS KH ET SM MW YE NM K. Murata M.
Korenaga KT M. Mizokami. Wrote the paper: NN HS JO KT M.
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