Association of Polymorphism in MicroRNA 219-1

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Journal of Medical Virology 85:808814 (2013)

Association of Polymorphism in MicroRNA 219-1With Clearance of Hepatitis B Virus Infection

Jae Youn Cheong,1 Hyoung Doo Shin,2 Yoon Jun Kim,3* and Sung Won Cho1

1Department of Gastroenterology, Ajou University School of Medicine, Suwon, Republic of Korea2Department of Life Science, Sogang University, Seoul, Republic of Korea3Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine,Seoul, Republic of Korea

Polymorphisms in the primary microRNA re-gion may be associated with natural course ofhepatitis B virus (HBV) infection. This studyevaluated if the mircoRNA 219-1 (miR-219-1)polymorphism can influence the susceptibilitytowards persistence of HBV infection and theprogression to hepatocellular carcinoma(HCC) in patients with chronic HBV infection.A total of 1,439 individuals having either pastor present evidence of HBV infection were en-rolled for the study. The subjects were divid-ed into four groups; (1) spontaneous recovery(n 404), (2) chronic HBV carrier (n 313),(3) chronic HBV carrier with cirrhosis(n 305), and (4) hepatocellular carcinoma

(n 417). Genotyping was performed at threepolymorphic variants (rs421446, rs107822, andrs213210) in the pri-miRNA region of miR-219-1.The rs421446 T allele was found to be stronglyassociated with HBV clearance (OR 0.73,P 0.0005 in a codominant model andOR 0.67, P 0.0009 in a dominant model,OR 0.69, P 0.04 in a recessive model, re-spectively). The rs107822 G allele was alsofound to be associated with HBV clearance(OR 0.79, P 0.008 in a codominant modeland OR 0.72, P 0.01 in a dominant model,respectively). In haplotype analysis, ht2 (T-G-T)and ht1 (C-A-C) were found to be in significantassociation with the clearance of HBV. However,no significant association was observed be-tween miR-219-1 polymorphism and the risk ofHCC occurrence. This result suggests that poly-morphisms in the pri-miRNA region of miR-219-1 might be a genetic factor for HBV clearanceafter infection. J. Med. Virol. 85:808814,2013. 2013 Wiley Periodicals, Inc.

KEY WORDS: polymorphism; microRNA-219; hepatitis B virus; hepato-cellular carcinoma

INTRODUCTION

Hepatitis B virus (HBV) infection is a major globalhealth problem for more than 350 million HBV car-riers around the world [Iino, 2002; Lok and McMahon,2007; Suk et al., 2012]. The clinical outcome of theinfection varies widely and may include spontaneousrecovery, the presence of inactive hepatitis B surfaceantigen (HBsAg) carriers, chronic hepatitis, liver cir-rhosis, or hepatocellular carcinoma (HCC). Perinataltransmission of HBV is the major mode of infection inHBV-endemic areas, including Korea. The most sig-nificant factor affecting the chronicity of HBV infec-tion is the age at time of infection; the chronicity inKorean patients who are vertically infected seems to

be determined by host factors or genetic differencesrather than viral factors such as variations in viru-lence of the viral strains [Stevens et al., 1975; Coursa-get et al., 1987; Tassopoulos et al., 1987]. Geneticfactors are also likely to modify the risk of HCC devel-opment among patients with HBV infection in caseswhere family history of HCC is a well-known risk fac-tor [Yu et al., 2000]. Recent studies by the authorsreported that genetic variations such as single nucleo-tide polymorphism (SNP) of transforming growth fac-tor beta receptor III gene and tumor necrosis factoralpha gene are significantly associated with the risk

Grant sponsor: SNUH Research Fund; Grant number:03-2012-021; Grant sponsor: Basic Science Research Program;Grant number: 2010-0009943; Grant sponsor: Basic ResearchLaboratory Program, National Research Foundation of Korea;Grant sponsor: Ministry of Education, Science and Technology;Grant number: 2010-0001200.

*Correspondence to: Yoon Jun Kim, M.D, Department ofInternal Medicine, Seoul National University College of Medi-cine, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Republic ofKorea. E-mail: [email protected]

Accepted 14 January 2013

DOI 10.1002/jmv.23551Published online in Wiley Online Library(wileyonlinelibrary.com).

2013 WILEY PERIODICALS, INC.


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of HCC occurrence or the clearance of HBV [Kimet al., 2003, 2011].

MicroRNAs (miRNAs) are small, single-stranded,2123 nucleotide-long non-coding RNA that functionsto modulate protein expression [Lagos-Quintanaet al., 2001]. Recent estimates indicate that miRNAs

regulate at least 30% of all protein-coding genes,building complex regulatory networks that controlalmost every cellular process [Filipowicz et al., 2008].Association studies using SNPs in miRNA genomicregions might be of help to evaluate the involvementof miRNAs in disease. Sequence variations in miRNAgenes including pri-miRNAs, pre-miRNAs, couldpotentially influence the processing and/or targetselection of miRNAs [Duan et al., 2007]. In addition,SNPs located within miRNA target sites have beenshown to affect the expression of the target gene andcontribute to susceptibility to human disease [Boreland Antonarakis, 2008]. Xu et al. [2008] havereported that polymorphisms in miRNA and theirtargets are useful in predicting the risk of HCC occur-rence. A G > C polymorphism in miR-146a precursor(rs2910164) has been reported to predict HCCdevelopment.

The function of mircoRNA 219-1 (miR-219-1) hasnot been studied extensively. Recent report showedthat miR-219 modulates N-methyl-D-aspartate recep-tor mediated neurobehavioral dysfunction [Kocerhaet al., 2009]. Another investigation indicates thatmiR-219 plays an important role in oligodendrocytedifferentiation [Dugas et al., 2010]. However, the bio-logical roles of miR-219 in disease progression inpatients with HBV infection are not elucidated.

This study evaluated if the miR-219-1 polymor-phisms can influence the susceptibility towards per-sistence of HBV infection and the progression to HCCin patients with HBV infection.

MATERIALS AND METHODS

Study Patients

To serve as primary patients for the casecontrolstudy, a total of 1,439 individuals having either pastor present evidence of HBV infection were recruitedfor the present study. The selected subjects were theones, who had enrolled in the outpatient clinic of theliver unit and the Center for Health Promotion Centerat Seoul National University Hospital and Ajou Uni-versity Hospital. Diagnoses of chronic carriers andspontaneously recovered individuals were establishedby repeated seropositivity for the hepatitis B surfaceantigen (HBsAg; Enzygnost1 HBsAg 5.0; DadeBehring, Marburg, Germany) over a 6-month period,and for both anti-HBs (antibody to hepatitis B surfaceantigen; Enzygnost Anti-HBs II; Dade Behring) andanti-HBc (antibody to hepatitis B core antigen; AB-Corek; DiaSorin s.r.l., Saluggia, Italy) of the IgG typewithout HBsAg, respectively. The chronic carriergroup was assessed further for disease progression tocirrhosis or HCC. All of the patients in the chronic

carrier group had undergone regular medical follow-ups and had been evaluated with serum alpha-fetoprotein level, abdominal ultrasonography, and/ora four-phase spiral liver CT scan more than twice ayear to detect early stages of HCC. Based on the clini-cal decision, dynamic contrast enhanced abdominal

MRI, bone scans, and chest CT, brain MRI, brain CT,hepatic angiography, or PET scan was also carriedout in some of the patients.

Diagnosis of HCC was based on imaging findings ofnodules larger than 1 cm showing intense arterial up-take followed by washout of contrast in the venous-delayed phases in four-phase multi-detector CT scanor dynamic contrast enhanced MRI and/or biopsy[Bruix and Sherman, 2011]. Cirrhosis of the liver, onthe other hand, was diagnosed pathologically or thediagnosis was based on the clinical evidence of portalhypertension such as the presence of visible collateralvessels on the abdominal wall, esophageal varies onesophagogastroscopy, palpable splenomegaly, and sono-graphically definite findings of cirrhotic liver or ascites.

Exclusion criteria for the study patients includedthe following: (i) tested positive for anti-HBs but notfor anti-HBc; (ii) tested positive for anti-HCV or anti-HIV (Genedia1; Greencross Life Science, Yongin-shi,Korea, HCV13.2; Dong-A Pharmaceutical, Seoul,Korea); (iii) average alcohol consumption of10 g/dayor an average number of 1 cigarette pack/s smokeddaily assessed through individual interviews; and (iv)incurrence of other types of liver diseases such asautoimmune hepatitis, toxic hepatitis, hemochromato-sis, Wilsons disease, non-alcoholic steatohepatitis,primary biliary cirrhosis, or BuddChiari syndrome.

None of the patients had a previous history of immu-nosuppressant or anti-viral treatment. Finally, writ-ten consent was secured from the patients prior toconducting the study and ethical approval wasobtained from the Institutional Review Board for Hu-man Research at Seoul National University Hospitaland Ajou University Hospital.

Genotyping of miR-219-1 Genome RegionPolymorphisms

Using the Wizard genomic DNA purification kit(Promega, Madison, WI), genomic DNA was extractedfrom patients peripheral blood samples. The SNP

genotyping was performed using the TaqMan1

[Livak,1999] assay in the ABI prism 7900HT sequence detec-tion system (Applied Biosystems, Foster City, CA).Genotyping quality control was performed in 10% ofthe samples by duplicate checking (rate of concor-dance in duplicates 100%). Assay IDs of rs421446,rs107822, and rs213210 were C__27015692_10,C___2215075_20 and C___2215074_10, respectively(Applied Biosystems).

Statistical Analyses

To determine the association of rs421446, rs107822,and rs213210 with HBV clearance and HCC

MicroRNA 219-1 and Hepatitis B Virus Infection 809

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occurrence, the odds ratio (95% Confidence Interval)was calculated using logistic analysis adjusted for age(continuous value) and sex (male 0, female 1) ascovariates to eliminate or reduce any confounds thatmight influence the findings. Data was managed andanalyzed using the Statistical Analysis System (SAS)

version 9.1 (SAS, Cary, NC). Using the PHASEalgorithm, version 2.0 [Stephens et al., 2001], haplo-types were then inferred from the genotyped SNPs.Lewontins D0 (jD0j) and the linkage disequilibrium(LD) coefficient r2 were then examined using theHaploview algorithm [Barrett et al., 2005] to measurethe LD between all pairs ofBiallelic loci.

RESULTS

Clinical Profiles of the Study Patients

The clinical profiles of the subjects enrolled for thestudy are listed in Table I. A total of 1,439 Koreansubjects, having either past or present evidence ofHBV infection, were enrolled in this study and classi-fied into two groups: spontaneously recoveredsubjects (n 404) and chronic HBV infected patients(n 1,035). Chronic patients infected with HBVcomprised of 313 chronic hepatitis B patients, 305liver cirrhosis patients, and 417 HCC patients(Table I). In chronic HBV infection, subjects withmore progressive diseases tended to be older in ageand had higher male to female ratio, lower positiverate of hepatitis B e antigen (HBeAg), and higher pos-itive rate of anti-HBe.

Genotype and Minor Allele Frequency of

pri-miRNA Region of miR-219-1 SNPs in KoreanPopulation

By direct sequencing, three SNPs were identified inthe pri-miRNA region of miR-219-1. Figure 1A depictsschematic gene map of rs421446, rs107822, andrs213210 in pri-miRNA region of miR-219-1 on chro-mosome 6p21.32. Black blocks represent miR-219-1,HSD17B8, and RING1 genes. The frequency in paren-theses was based on the genotyping data (n 1,439).The minor allele frequencies of the three genotypedpolymorphisms were 0.345 (rs421446 C > T), 0.387(rs107822 A>G) and 0.470 (rs213210 C >T),

respectively (Table II). No significant deviation fromthe HardyWeinberg equilibrium was observed in allthe polymorphisms (P >0.05, Table II).

Linkage disequilibrium coefficient (jD0j and r2)among the three SNPs was calculated based on thegenotypes of whole study subjects in this study. Pair-

wise comparisons between these three polymorphismsrevealed complete LD (jD0j 1 and r2 6 1; Fig. 1C).Using the PHASE algorithm, four common haplotypesof rs421446, rs107822 and rs213210 were inferredfrom those SNPs. They were ht1 (C-A-C), ht2 (T-G-T),ht3 (C-A-T), and ht4 (C-G-T) and used for haplotypeassociation analysis.

Association Analysis of pri-miRNA Region ofmiR-219-1 SNPs With Risk of Liver Disease in

Korean Population

The genotype frequencies of both the chronic HBV

carriers and the spontaneous recovered groups foreach polymorphism were analyzed using a logisticregression model, controlling for age and sex as cova-riates. The rs421446 T allele was found to be stronglyassociated with HBV clearance (OR 0.73, P 0.0005 in a codominant model and OR 0.67,

P 0.0009 in a dominant model, OR 0.69, P 0.04in a recessive model, respectively). The rs107822 G al-lele was also found to be associated with HBV clear-ance (OR 0.79, P 0.008 in a codominant modeland OR 0.72,P 0.01 in a dominant model, respec-tively). The rs213210 T allele was found to be in mar-ginal association with HBV clearance (Table III).

In haplotype analysis, ht2 (T-G-T) and ht1 (C-A-C)were found to be significantly associated with theclearance of HBV. The ht2 (T-G-T) showed the stron-gest genetic effects in codominant model (OR 0.73,

P 0.0004) (Table III).Subsequently, the relationship between haplotypes

or SNPs of miR-219-1 and the development of HCCwere investigated in chronic HBV carriers. In theanalysis of the HCC occurrence in patients withchronic hepatitis or liver cirrhosis, no significant asso-ciation was observed between haplotypes or SNPs ofpri-miRNA region of miR-219-1 and HCC occurrence(Table III).

TABLE I. Clinical Profile of the Study Patients

Clinical profileSpontaneously

recovered

Chronic carrier

Hepatocellularcarcinoma Liver cirrhosis Chronichepatitis

No. of patients 404 417 305 313Age (mean (range)) 53.1 (2881) 57.5 (2582) 50.8 (2290) 44.4 (1879)Sex (male/female) 272/132 279/48 231/74 238/75HBeAg (positive rate) (%) 0 25.7 29.7 37.1

Anti-HBe (positive rate) (%) 37.7 65.8 50.2 44.9HBsAg (positive rate) (%) 0 100 100 100

Anti-HBs (positive rate) (%) 100 0 0 0

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DISCUSSION

This study shows that the genetic polymorphisms

and the haplotypes of miR 219-1 are associated withclearance of HBV. To the best of our knowledge, thisstudy is the first report on the associations betweenmiR 219-1 polymorphisms and the outcome of HBVinfection.

It is widely accepted that the most important factoraffecting the chronicity of HBV infection is age attime of infection [Shimbo et al., 1997]. However, thisis not the sole factor determining the persistence ofHBV and the host genetic factors involving geneticpolymorphisms are believed to be responsible for

clinical outcomes of HBV infection, especially in thepopulation whose transmission route is vertical infec-tion, which signifies that almost all individuals had

been exposed to the virus at the same age. Recentstudies have revealed that HLA class I and II [Ahnet al., 2000; Thio et al., 2003], TNF-a[Kim et al., 2003;Cheong et al., 2006], interleukin 10 [Shin et al., 2003]and other various kinds of cytokines contribute to thevariable clinical outcome of HBV infection [Kim et al.,2010; Park et al., 2010]. The present study alsostrengthens the possibility that genetic factor is in-volved in natural course of HBV infection.

The miRNAs are small non-coding transcripts thatcan control expression of protein-coding mRNA at the

Fig. 1. Physical map, haplotypes, and linkage disequilibrium of rs421446, rs107822, and rs213210.A: Schematic gene map and SNPs in the pri-miRNA region of miR-219-1 on chromosome 6p21.32.Black blocks represent miR-219-1, HSD17B8, and RING1 genes, respectively. The frequency in paren-theses was based on the genotyping data (n 1,439). B: Haplotypes of rs421446, rs107822 andrs213210.C: Linkage disequilibrium coefficient (jD0j and r2) among the three SNPs.

TABLE II. Genotype and Minor Allele Frequency of rs421446, rs107822, and rs213210 in Korean Population (n 1,439)

rs# Chromosome Coordinatea Genotype (#samples)Minor allele

frequency Heterozygosity HWEb

rs421446 6 33282761 CC (602) 0.345 0.452 0.138CT (676)TT (157)

rs107822 6 33283553 AA (519) 0.387 0.475 0.078AG (720)GG (196)

rs213210 6 33283802 CC (396) 0.470 0.498 0.319CT (732)TT (310)

aHuman genome build 36.bP-values of deviation from HardyWeinberg Equilibrium among spontaneously recovered group.




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post-transcriptional level. Single miRNA moleculesare predicted to be capable of influencing the expres-sion of hundreds of different targeted genes [Bartel,2009]. In recent years, much effort has been directedtowards understanding the role of SNPs present inpri-, pre-, and mature miRNA and their influences on

susceptibility to various diseases. So far, little isknown about the involvement of miRNAs during HBVinfection. Recently, a differential miRNAs expressionpattern was found in the livers of HBV and HCVinfected individuals with hepatocellular carcinoma[Ura et al., 2009]. Another study has shown that aliver specific micro RNA, miR-122, binds to a highlyconserved HBV pre-genomic RNA sequence and inhib-its HBV gene expression and replication [Chen et al.,2011]. This report suggested that miR-122 mightdown-regulate HBV replication by binding to the viraltarget sequence, thus contributing to the persistentinfection of HBV. In the current study, we identifiedthe association between SNPs of miR-219-1 and HBVclearance. However, the biological role of miR-219-1SNP is not assessed in current study, thus functionalsignificance of miR-219-1 SNP in the natural course ofHBV infection is unclear. It remains to be elucidatedwhether miR-219-1 function to modulate host immu-nity to eradicate virus during the early stage of infec-tion or function through other mechanisms.

The miRNAs have emerged as important genomicregulators with a key role in the development of cancer[Boyd, 2008]. The potential role of miR-219 as an inhib-itor of cellular proliferation has been indicated in previ-ous research [Wong et al., 2008]. Recent investigationshowed miR-219-5p induced inhibition of HCC cell pro-

liferation by targeting glypican-3 [Huang et al., 2012].Other studies identified miR-219 as being downregu-lated in various cancers [Wong et al., 2008; Ferrettiet al., 2009; Izzotti et al., 2009]. However, the associa-tion studies between genetic variation in miR 219-1and various tumors including oral premalignant lesion,renal cell carcinoma and metastatic colon cancershowed negative results. In current study, no signifi-cant association was observed between genetic varia-tions in pri-miRNA region of miR-219-1 and theoccurrence of HCC in patients with chronic HBV infec-tion. However, there still exists the notation that theother miRNA might influence HCC development andfurther studies are warranted on this subject.

In conclusion, it is stated that miR-219-1 SNP isassociated with clearance of HBV infection. Thisresult suggests that miR-219-1 polymorphism mightbe an important genetic factor, which affects variableoutcome of HBV infection.

ACKNOWLEDGMENTS

The biospecimens from Ajou University Hospitalwere provided by the Ajou Human Bio-Resource Bank(AHBB), a member of the National Biobank of Korea,which is supported by the Ministry of Health andWelfare.

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Association of Polymorphism in MicroRNA 219-1