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IJE Advance Access first published online on May 11, 2007
This version published online on May 24, 2007
International Journal of Epidemiology, doi:10.1093/ije/dym093
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Published by Oxford University Press on behalf of the International Epidemiological Association © The Author 2007; all rights reserved.

SPP1 polymorphisms associated with HBV clearance and HCC occurrence

Hyoung Doo Shin1, Byung Lae Park1, Hyun Sub Cheong1, Jung-Hwan Yoon2, Yoon Jun Kim2 and Hyo-Suk Lee2,*

1 Department of Genetic Epidemiology, SNP Genetics, Inc., Rm 1407, 14th floor, Complex B, WooLim Lion's Valley, 371-28, Gasan-Dong, Geumcheon-Gu, Seoul, 153-803, Republic of Korea.
2 Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 28 Yungun-dong, Chongro-Gu, Seoul, 110-744, Republic of Korea.

* Corresponding author. Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 28 Yungun-Dong, Chongno-Gu, Seoul, 110-744, Republic of Korea. E-mail: hsleemd{at}snu.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Appendix
 Acknowledgements
 References
 
Background Secreted phosphoprotein-1 (SPP1) is a secreted arginine-glycine-aspartate (RGD)–containing phosphoprotein. SPP1 is overexpressed in metastatic hepatocellular carcinoma (HCC), and therefore could act as both a diagnostic marker and a potential therapeutic target for metastatic HCC. We investigated the genetic polymorphisms in SPP1 to determine whether it is a potential candidate gene for a host genetic study of hepatitis B virus (HBV) clearance and HCC occurrence.

Methods Five genetic variants in SPP1 were genotyped by TaqMan assay and the genetic association with HBV clearance and HCC occurrence was analysed.

Results Genetic association analysis of SPP1 polymorphisms with an HBV cohort (n = 1069) from the Korean population revealed that the most common haplotype (SPP1-ht2 [T-T-C-T-A]) was associated with HBV clearance. The frequency of the SPP1-ht2–bearing genotype in the chronic carrier (CC) group was higher than in the spontaneously recovered (SR) group (OR = 1.44 [95% CI 1.11–1.87], P = 0.006, Pcorr = 0.02). By Cox relative hazard analysis, both SPP1-ht2 and –1800G > T were associated with age of HCC occurrence among chronic hepatitis patients, e.g. ht2/h2- and 1800T/T-bearing patients showed earlier progression to HCC than did others (RH = 1.85, P = 0.004, Pcorr = 0.01 and RH = 1.85, P = 0.003, Pcorr = 0.01, respectively).

Conclusion Our findings suggest that SPP1 polymorphisms might be among the genetic factors for HBV clearance and/or HCC occurrence.

Keywords SPP1, HBV, HCC, single-nucleotide polymorphism, haplotype

Accepted 3 April 2007


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Appendix
 Acknowledgements
 References
 
Hepatitis B virus (HBV) infection is a global public health problem, and it is also a major risk factor for chronic hepatitis (CH), liver cirrhosis (LC), and hepatocellular carcinoma (HCC).1,2 The clinical course of HBV infection varies from spontaneous recovery after acute hepatitis to a chronic persistent infection. Chronic carriers of HBV have a much higher risk of developing LC and HCC compared with uninfected individuals.3–5 Although the mechanisms underlying resolution and different outcomes of HBV infection still remain undetermined, it is apparent that host genetic factors are likely to influence outcome of HBV infection based on twin and family studies.6,7

Age at infection has the most significant impact on the clinical outcome of HBV infection because chronic infection occurs in approximately 90% of infants infected at birth, in 25–50% of children infected between the ages of 1 and 5 years, and in <5% of those infected during adult life.8–10 It is well-known that the major mode of infection in Korea, one of the HBV endemic areas, is perinatal transmission.11,9 When determining the chronicity of HBV infection within a group of Korean patients who are presumed to have been infected at the same age, i.e. perinatally, the outcome of the infection does not appear to be determined by variations in virulence of the viral strains,12,13 but rather, host factors are likely to influence disease outcome.14,6 Thus, it is conceivable that genetic differences play an additional role.

Secreted phosphoprotein-1 (SPP1, located on 4q21 [PDB] –q25; alternatively Osteopontin) is a secreted arginine-glycine-aspartate (RGD)–containing phosphoprotein with cell-adhesive and chemotactic properties both in vitro and in vivo.15 SPP1 mainly contributes to host defence, bone formation, and wound healing by stimulating macrophage migration as well as protecting against viral and bacterial infections through its pro-Th1 effect.16–18 It has been shown that SPP1 is up-regulated in many kinds of cancer, including HCC,19 breast cancer,20,21 prostate cancer,22 ovarian cancer,23 brain cancer24,25 and lung cancer.26 The SPP1-specific antibody effectively blocks HCC cell invasion in vitro and has been shown to inhibit pulmonary metastasis of HCC cells in nude mice.27 These results indicate that SPP1 could act as both a diagnostic marker and a potential therapeutic target for metastatic HCC.

Despite the importance of SPP1 to HBV infection and/or HCC progression, evidence of the genetic contribution of SPP1 polymorphisms is lacking. We hypothesized that polymorphisms in the SPP1 gene could affect the clearance of HBV as well as HCC progression among HBV-infected patients.28


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Appendix
 Acknowledgements
 References
 
Subjects
A total of 1069 Korean subjects having either present or past evidence of HBV infection were prospectively enrolled from the outpatient clinic of the liver unit or from the Center for Health Promotion of Seoul National University Hospital between January 2001 and August 2003. All the study subjects were of Korean ethnicity. Subjects were placed in two different groups, chronic carrier (CC) and spontaneously recovered (SR), according to serological markers. The CC and SR cohorts consisted of 641 and 428 subjects, respectively, and the CC cohort was composed of 331 CH/LC and 310 HCC patients, respectively. Clinical profiles are described in Table 1.


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  		Table 1 Clinical profiles of study subjects

 
The diagnoses of the CC and SR subjects were established by repeated seropositivity for the hepatitis B surface antigen (HBsAg) (Enzygnost® HBsAg 5.0; Dade Behring, Marburg, Germany) over a 6-month period, and for both anti-HBs (Enzygnost® Anti-HBs II; Dade Behring, Marburg, Germany) and anti-HBc (AB-Corek; DiaSorin s.r.l., Saluggia, Italy) of the IgG type without HBsAg. We excluded subjects who were positive only for anti-HBs and not for anti-HBc, and those positive for anti-HCV or anti-HIV (GENEDIA®; Greencross Life Science Corp., Yongin-shi, Korea, HCV®3.2; Dong-A Pharmaceutical Co., Seoul, Korea). Subjects whose average alcohol consumption assessed by interview was ≥10 g/day or whose average cigarette smoking was > 1 pack/day were excluded. Patients who had any other types of liver disease such as autoimmune hepatitis, toxic hepatitis, primary biliary cirrhosis or Budd-Chiari syndrome were also excluded. No patients in our study had a previous history of immunosuppression or anti-viral treatment.

All the patients in the CC group had been on regular medical follow-up and had been evaluated with serum alpha-fetoprotein level assessment, abdominal ultrasonography, and/or 2-phase spiral liver CT scan more than twice a year to detect early stages of HCC. We also performed abdominal MRI, bone scan, chest CT, brain MRI, brain CT, hepatic angiography or PET scan in some patients according to the clinical decisions. Liver cirrhosis was diagnosed pathologically or by the clinical evidences of portal hypertension such as visible collateral vessels on the abdominal wall, esophageal varices on esophagogastroscopy, palpable splenomegaly and sonographically definite findings of cirrhotic liver or ascites. HCC was diagnosed as described previously (the age of onset was determined by the date of the diagnosis.).29

Genotyping with fluorescence polarization detection
For genotyping of polymorphic sites in our HBV study, probes were designed for TaqMan.30 Information regarding the primers used is available on our website (Appendix Table A2 and 3; http://www.snp-genetics.com/reference/SupplementaryInformationToSPP1.doc).

Primer Express (Applied Biosystems, Foster City, CA) was used to design the MGB TaqMan probes. One allelic probe was labelled with the fluorescent FAM dye and the other with the VIC dye. PCRs were run in a TaqMan Universal Master mix without UNG (Applied Biosystems), with PCR primer concentrations of 900 nM and TaqMan MGB-probe concentrations of 200 nM. Reactions were performed in a 384-well format in a total reaction volume of 5 µl using 20 ng of genomic DNA. The plates were then placed in a thermal cycler (PE 9700, Applied Biosystems) and heated at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min with a final soak at 25°C. The TaqMan assay plates were transferred to the Prism 7900HT instrument (Applied Biosystems) where the fluorescence intensity in each well of the plate was read. Fluorescence data files from each plate were analysed by automated allele-calling software (SDS 2.1).

Statistics
Linkage disequilibrium (LD) was inferred using the algorithm (Haploview) developed by the Broad Institute, which searches for a spine of strong |D'| and LD coefficient r2 running from one marker to another.31 Haplotypes of each individual were inferred using the algorithm (PHASE, version 2.0) developed by Stephens et al.32 Subjects harbouring missing genotypes were omitted in the analysis of individual single-nucleotide polymorphisms (SNPs) and haplotypes. The genotyping success rate was >99%, so it is unlikely that omitting a small number of individuals introduced any bias in the analysis.

For analysis of viral clearance as an outcome, logistic regression models were used for calculating odds ratios (95% confidential interval) and corresponding P-values controlling for age (continuous value) and sex (male = 0, female = 1) as covariates. To analyse HCC occurrence as an endpoint from chronic HBV infection, Cox models were used for calculating relative hazards and P-values controlling for sex, adjusted age (0, <40; 1, 40–60; and 2, >60), LC (LC = 0, no LC = 1), and HBeAg (negative = 0, positive = 1). The effective number of independent marker loci in SPP1 was calculated to correct for multiple testing, using the software SNPSpD (http://genepi.qimr.edu.au/general/daleN/SNPSpD/), which is based on the spectral decomposition (SpD) of matrices of pair-wise LD between SNPs.33 The number of independent marker loci in SPP1 was calculated as 3.7895, and this was applied to correct for multiple testing (P-value x 3.7895).

HBV genotypes, HBV DNA and ALT levels have been regarded as important factors influencing HBV clearance and the development of HCC. However, HBV genotype C is predominant among CCs of the virus in Korea,34–38 and the HBV DNA and ALT levels were found to be fluctuating during the follow-up for the majority of our HBV cohort. Therefore, logistic models for HBV clearance were adjusted only for age and sex, and Cox models were controlled for HBeAg status, which can represent HBV DNA levels and ALT levels, 39–41 in addition to categorized age, sex and LC status.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Appendix
 Acknowledgements
 References
 
In this study, we examined the association of SPP1 polymorphisms with persistent HBV infection and HCC occurrence. We analysed two SNPs in the 5' flanking region (–1800G > T and –1627T > C, which correspond to –616G > T and –443T > C, respectively, in the previous study42) and three SNPs in the coding region (+4645C > T (D80D)) in exon 6, +5806T > C (A236A) in exon 7 and +6139A > G in 3' UTR, which correspond to +8090C > T, +9250T > C, and +9583A > G, respectively, in the previous study28] (Figure 1a). The minor allele frequencies were 0.296 (–1800G > T), 0.384 (–1627T > C), 0.278 (+4645C > T), 0.278 (+5806T > C) and 0.324 (+6139A > G) in the Korean population (n = 1069) (Figure 1a). No significant deviations from Hardy–Weinberg equilibrium were observed in all polymorphisms (P > 0.05, Appendix Table A1; http://www.snp-genetics.com/referrence/SupplementaryInformationToSPP1.doc). Five SNPs showed tight LDs (Figure 1c), and four major haplotypes accounted for over 91.4% of the distribution (Figure 1b). +5806T > C was excluded in statistical analysis because of almost absolute LD with +4645C > T (|D'| = 1 and r2 = 0.998; Figure 1b).


Figure 1
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  		Figure 1 Gene maps and haplotypes of SPP1. Coding exons are marked by black blocks, and 5' and 3' UTRs by white blocks. The first base of the translational start site is denoted as nucleotide +1. (a) Polymorphisms in SPP1 on chromosome 4q21-q25 (Ref. Genome Seq. NT_016354.17). (b) Haplotypes of SPP1. Four major haplotypes accounted for over 91.4% of distribution. The others are GCTCA, GCCTA, TTCTG, GTCTG, TTTCA and GCCCG. (c) Linkage disequilibrium coefficients (|D'| and r2) among SPP1 gene polymorphisms

 
The genotype frequencies among CC and SR groups for each polymorphism were analysed using logistic regression models (Table 2), controlling for age and sex as covariates (gender and age were significantly associated with HBV clearance; P < 0.05, data not shown.) in our Korean HBV study (648 CC and 428 SR individuals). SPP1-ht2 [T-T-C-T-A] was found to be associated with HBV clearance in that the frequency of individuals bearing the SPP1-ht2 allele among the CC group was significantly higher than among the SR group (OR = 1.44 [95% CI: 1.11–1.87], P = 0.006, Pcorr = 0.02 in the dominant model). A similar association was also observed in –1800G > T, although the significance was not retained after multiple test correction (Table 2). The LC occurrence among CC and HCC occurrence among patients with LC were also analysed. None of the polymorphisms in SPP1 were found to be significantly associated (please see Tables A4 and A5).


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  		Table 2 Logistic regression analysis of HBV clearance with individual SNPs and haplotypes in SPP1 in Korean subjects (n = 1069)

 
In further evaluation using the Cox relative hazards model among chronic HBV patients, –1800G > T and ht2 were also associated with onset age of HCC, e.g. subjects bearing the homozygote genotype for the minor allele of –1800T/T and ht2/ht2 showed accelerated progression to HCC outcome (RH = 1.85, P = 0.004, Pcorr = 0.01 and RH = 1.85, P = 0.003, Pcorr = 0.01, respectively; Table 3).


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  		Table 3 Cox relative hazards analysis for age of HCC occurrence as a function of individual SNPs and haplotypes of SPP1 among chronic HBV patients

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Appendix
 Acknowledgements
 References
 
SPP1 is a phosphoprotein with cell-adhesive and chemotactic properties. It might be a key target as an important molecule in mediating tumour metastasis through the infiltrating of macrophages in tumours and direct-stimulating macrophage migration.15,18 SPP1 has both inflammatory and anti-inflammatory actions and plays especially critical roles in cell-mediated immunity among its various functions, including macrophage-directed interleukin-10 (IL-10) suppression and anchorage-independent growth of tumour cells, by regulating cell-matrix interactions and cellular signalling.43,44 Based on these roles of SPP1, we scrutinized the genetic effect of SPP1 polymorphisms for various outcomes of HBV infection in a Korean population.

We identified the association of both SPP1 haplotype-2 [T-T-C-T-A] and –1800G > T with the onset age of HCC. The genetic effect of both SPP1–1800G > T and ht2 are supported by previous studies. One recent study reported that SPP1–1800G > T (–616 in that study) was associated with the response to interferon therapies in patients with CH C.42 Another study also reported that expression of SPP1 was likely to be affected by the SPP1 haplotypes constructed with SPP1 + 4645C > T, +5806T > C and +6139A > G, e.g. (1) in the Dianzani autoimmune/lymphoproliferative disease family, higher levels of SPP1 were co-segregated with haplotype B (+4645C:+5806T:+6139A, almost equivalent with ht2 in this study) or haplotype C (+4645C:+5806T:+6139G, equivalent with ht1 in this study) and (2) in healthy controls, haplotype B or haplotype C carriers displayed higher levels of SPP1 than did those with haplotype A (+4645T:+5806C:+6139A, equivalent with ht3 in this study) homozygotes.45

In summary, we demonstrate that genetic polymorphisms in the SPP1 gene are associated with HBV clearance and onset age of HCC in a large Korean HBV study (n = 1069). Our findings might offer an approach to elucidating the molecular mechanisms of HBV clearance and HCC progression.


   Appendix
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Appendix
Acknowledgements
 References
 


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  		Table A1 Genotype and allele frequency of polymorphisms in SPP1

 


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  		Table A2 Primer sequences for SPP1 SNP screening

 


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  		Table A3 Sequences of amplifying and Taqman probe for SPP1 SNP genotyping

 


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  		Table A4 Logistic regression analysis of HCC occurrence in LC with individual SNPs and haplotypes in the SPP1 gene in Korean subjects

 


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  		Table A5 Logistic regression analysis of LC occurrence with individual SNPs and haplotypes in the SPP1 gene in Korean subjects

 

   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Appendix
 Acknowledgements
References
 
We wish to acknowledge and thank the study participants and their families who took part in the HBV cohort study of Seoul National University. This work was supported by grant number FG04-12-02 (03) of the 21C Frontier Functional Human Genome Project from the Ministry of Science and Technology of Korea, and a grant from the National Research Lab Program as part of the National Research and Development Program from the Ministry of Industry and Energy of Korea, contract grant number M1-0302-00-0073.

Conflict of interest: None declared.


   Notes
 
The originally published version of this paper was incorrect. The superscript of the author Yoon Jun Kim was Yoon Jun Kim1 while it should be Yoon Jun Kim2.

Hyoung Doo Shin and Byung Lae Park contributed equally to this work.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Appendix
 Acknowledgements
 References
 
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