High Frequency of Variant Alleles of the Mannose-Binding Lectin 2 ( MBL2) Gene Are Associated with Patients Infected by Hepatitis B Virus

Abstract

Patients with hepatitis B virus (HBV) infection may develop severe chronic liver disease. Carriers of HBV have an increased risk of developing cirrhosis, hepatic decompensation, and hepatocellular carcinoma. Worldwide an estimated 350 million people are infected with HBV, and 15–40% will develop serious sequelae in their lifetime. In our study we investigated the association of single nucleotide polymorphisms (SNPs) in the first exon and promoter region of the mannose-binding lectin gene 2 (MBL2) situated on chromosome 10, with susceptibility to HBV infection. One-hundred and two patients infected with HBV were included in this study, and 232 uninfected individuals were used as healthy controls. Genotyping of the first exon (alleles A/O) was performed using a melting temperature assay. Genotyping of the promoter region (−550 H/L; −221 Y/X) was performed using the Taqman PCR technique. In the HBV-infected group we found a significantly increased frequency of haplotypes associated with low serum MBL. Our findings may indicate that MBL has a protective role against HBV infection in the studied population.

Introduction

H epatitis B virus (HBV) is highly infectious and may lead to chronic severe liver disease. HBV carriers have an increased risk of developing liver cirrhosis, hepatic decompensation, and hepatocellular carcinoma (3). Worldwide an estimated 350 million individuals are chronically infected with HBV (13), and 15–40% of them will develop serious sequelae during their lifetime (5). Genetic factors are likely to be involved in HBV infection and disease progression (6).

Mannose-binding lectin (MBL) is a protein of the innate immune system that plays an important role in the defense mechanism against infections, due to its role in activation of the complement system and induction of phagocytosis (20). It is also believed that MBL acts as a regulatory factor of the inflammatory pathway (12). Low serum MBL levels are associated with an increased risk of chronic HBV infection (8), and it has been suggested that high serum MBL levels protect against chronic HBV infection (18). MBL binds through multiple lectin domains to carbohydrate arrays of the HBsAg-activating complement system in in vitro assays (8).

The MBL gene (MBL2) is located on chromosome 10 and contains four exons. In exon 1 three single nucleotide polymorphisms (SNPs) are present at codons 52, 54, and 57, which are grouped together as allele O, and the wild-type is described as allele A (9). The effect of polymorphisms in exon 1 are still being studied, but it is believed that allele O causes low serum MBL level and impairs its biological activity (17). SNPs have also been found in the promoter region of MBL2 at positions −550 H/L, −221 X/Y, and +4 P/Q, which can also influence MBL serum levels (14). Allele X on position −221 has been shown to have an important influence on low serum MBL expression (21).

Low serum MBL levels due to SNPs in MBL2 are also associated with increased susceptibility to and severity of a variety of infectious illnesses, particularly when immunity is compromised, specifically in young children, patients with cystic fibrosis, and after chemotherapy or transplantation (21).

Previous studies have focused on the polymorphisms of MBL in HBV-infected patients. In Vietnamese persons with a higher frequency of polymorphisms in MBL2 in patients infected with HBV had worse outcomes than a healthy control group (16). One study among patients of Caucasian and Asian extraction found an association between mutations in codon 52 of MBL2 and persistent HBV infection in Caucasian, but not in Asian, patients (19). In a group of German patients, the frequency of codon 52 and 54 mutations with chronic HBV infection were shown to be no different from those of controls or patients with acute infection (11).

Most of the studies of MBL polymorphism and HBV infection have focused on the polymorphism of the first exon, or to the −221 (X/Y) allele variants. However, the −550 (H/L) are also variants that determine serum levels of MBL, and thus the investigation of this region is an important area of research (14).

The aim of our study was to investigate the frequency of variant alleles of the first exon and promoter region of MBL2 in individuals who had been exposed to HBV infection, and to compare them with those of non-infected individuals, to gain insights into the influence of this polymorphism on HBV susceptibility.

Materials and Methods

Study subjects

In this study 102 patients with HBV infection from northeastern Brazil were included. The study group consisted of 33 females and 69 males with a mean age of 45 y (SD ± 13.2 y), and the individuals in this group were patients with hepatitis B surface antigen (HBsAg) positivity for more than 6 mo as tested by electrochemiluminescence immunoassay (ELECSYS HBsAg; Roche, Mannheim, Germany); and individuals who had recovered from HBV infection and were simultaneously positive for anti-HBsAg and anti-HBc antibodies as assessed by serological analysis (ELECSYS anti-HBs; Roche).

All patients were negative upon anti-hepatitis C virus and anti-human immunodeficiency viral serological testing. The control group consisted of 97 females and 135 males, with a mean age of 33 y (SD ± 8.3 y), without HBV infection, all of whom were blood donors, and were matched for place of origin.

The patients in the study and the controls were all from the same metropolitan area of the city of Recife in northeastern Brazil, and are descendents of a mixed African, Caucasian, and Native American population. The control group was recruited from the Hematological Institute of Northeastern Brazil. The HBV-infected group was recruited from the Gastroenterology Service of the Oswaldo Cruz University Hospital of the University of Pernambuco, and the Liver Institute of Pernambuco. This study was approved by the Ethical Committee in Research of the University of Pernambuco under protocol no. CEP/UPE/058/07. Informed written consent was obtained from all participants.

DNA extraction and MBL2 genotyping

DNA extraction from whole blood was done using the GenomicPrep Blood DNA Isolation Kit (Amersham Biosciences, Buckinghamshire, U.K.) according to the manufacturer's protocol. MBL2 genotyping was done with real-time PCR using two methods. The first method was the Sybr Green I chemistry kit (Applied Biosystems, Foster City, CA), which uses a melting temperature assay following the protocol of Hladnik et al. (10). On the basis of distinct melting profiles there is simultaneous detection of the three SNPs in exon 1 (at codons 52, 54, and 57) of the MBL2 gene. All three variant alleles of MBL2 were grouped as allele O, while the wild-type allele was called allele A (9). The haplotypes of the MBL2 gene were grouped into two groups according to MBL production levels: high (HYA and LYA) and intermediate/low levels (LXA, HYO, and LYO), as previously described (9).

The second method, the Taqman PCR technique, was used to detect SNPs in the promoter of MBL2 at positions −550 and −221. A variant allele at position −550 was called H, while the wild-type allele was called L. A variant allele at position −221 was called X, while the wild-type was called Y. The Taqman PCR technique (Applied Biosystems) is able to detect one variant allele of multiple DNA samples in every PCR run.

Statistical analysis

Allele and haplotype frequencies were calculated by Arlequin Software (Excoffier, LG, Laval, LG, and Schneider S, 2005). Statistical significance was set at p < 0.05, odds ratios (OR) were used with 95% confidence intervals (95% CI), and were calculated by Epinfo V.6 (Centers for Disease Control and Prevention, Atlanta, GA), using 2 × 2 and 3 × 2 contingency tables.

Results

There was no difference in gender numbers between HBV patients and controls. With regard to age, the patients were older than the controls (p < 0.001; data not shown).

In Table 1 the allelic frequencies and genotype distributions of the polymorphisms in the first exon and the promoter region of the MBL2 gene are shown. The genotype distributions were in agreement with Hardy-Weinberg equilibrium. There was no significant association found among either the separate alleles or the genotypes. Our results suggest that in most cases, genotypes that cause low MBL are more common in HBV-infected patients than in controls.

Table 1.

Frequency of Alleles and Genotypes in HBV-Infected Patients Compared to Controls

	HBV patients (n = 102)	Controls (n = 232)	p Value
Promoter (−550)
Allele
L	143/204 (70.0%)	310/464 (66.8%)	0.454
H	61/204 (30.0%)	154/464 (33.2%)
Genotype
L/L	51/102 (50.0%)	106/232 (45.7%)	L/L versus
H/L	41/102 (40.2%)	98/232 (42.2%)	H/L+H/H
H/H	10/102 (9.8%)	28/232 (12.1%)	0.543
Promoter (−221)
Allele
Y	163/204 (79.9%)	388/464 (83.6%)	0.291
X	41/204 (20.1%)	76/464 (16.4%)
Genotype
Y/Y	64/102 (62.7%)	165/232 (71.1%)	Y/Y versus
Y/X	35/102 (34.3%)	58/232 (25.0%)	Y/X+X/X
X/X	3/102 (3.0%)	9/232 (3.9%)	0.164
Exon 1
Allele
A	154/204 (75.4%)	372/464 (80.2%)	0.207
O	50/204 (24.6%)	92/464 (19.8%)
Genotype
A/A	57/102 (55.8%)	147/232 (63.3%)	A/A versus
A/O	40/102 (39.2%)	78/232 (33.6%)	A/O+O/O
O/O	5/102 (5.0%)	7/232 (3.0%)	0.242

Abbreviation: HBV, hepatitis B virus.

The frequencies of haplotypes in HBV-infected patients and controls are shown in Table 2. The results show that haplotypes that are associated with a high level of MBL (HYA, and LYA) (4) were found more frequently in the control group than in the HBV-infected group (63.7% versus 55.3%). Haplotypes associated with low MBL levels (LXA, HYO, and LYO) (4) were found significantly more frequently in the HBV-infected group than in the control group (44.7% versus 36.3%). These results show that haplotypes inducing low MBL levels are common in HBV-infected patients from northeastern Brazil (p = 0.04; OR 1.42; 95% CI = 1.00,2.01).

Table 2.

Haplotypes in HBV-Infected Patients and Controls

	HBV patients ( ^a n = 204)		Controls ( ^a n = 464)
MBL2 haplotype	n	%	n	%
High levels
HYA	56	27.4	142	30.6
LYA	57	27.9	154	33.1
Total	113	55.3	296	63.7
Intermediate to deficient levels
LXA	41	20.1	76	16.3
HYO	5	2.4	12	2.5
LYO	45	22.2	80	17.5
Total	91	44.7	168	36.3

n = number of chromosomes.

High versus intermediate to deficient levels: p = 0.04; OR 1.42, 95% CI (1.00,2.01).

Abbreviations: OR, odds ratio; CI, confidence interval.

Discussion

Vaccination against HBV in Brazil has been guaranteed by the government since 1988. However, despite the fact that seroprevalence of hepatitis B surface antigen in endemic areas has decreased from 20% to 3.5%, viral hepatitis remains a significant health problem in Brazil, with an estimated prevalence of 7.9%. Variables such as geographic region, socioeconomic status, and racial and cultural differences may contribute to the significant differences in its prevalence seen in different parts of the Brazilian population (15).

In previous studies, both negative (7,11) and positive (8,16,18) associations have been reported between low serum MBL haplotypes and persistence of HBV infection or liver disease progression.

Höhler et al. (1998) analyzed 149 Caucasians, but they found no significant correlation for the first exon polymorphism of the MBL2 gene between a control group and patients with acute or chronic HBV infection (11). A large population study of Korean patients infected with HBV did not show any association between the codon 54 polymorphism of MBL2 and recovery from hepatitis B infection or with disease progression in chronic patients (7).

Conversely, increased allele B (codon 54) frequency was described in Vietnamese patients with acute HBV infection compared to controls (16). In another study, 498 North American persons divided into two groups (recovered and chronic patients) were studied in a nested case-control study (18). The group with chronic infection was positively associated with allele X at the −221 position and with allele B in the first exon of the MBL2 gene. The haplotype YA/YA, which is associated with high MBL levels, was associated with recovery (18).

Chong et al. (2005) analyzed 1090 Chinese persons classified as non-progressed carriers, progressed carriers, healthy recovered persons, and controls. MBL levels were significantly lower in progressed carriers, and patients with low MBL genotypes more often had cirrhosis and hepatocellular carcinoma (8).

These different results may be due to the fact that the populations were from different genetic backgrounds. Individuals enrolled in the present study came from a northeastern Brazilian region, and were of mixed African, Caucasian, and Native American extraction (44%, 34%, and 22%, respectively) (1). Our findings support the concept that one's MBL genetic background may play a role in susceptibility to HBV infection.

The age differed between the HBV patients and controls, with the mean age being higher in the patient group. It is well known that age is an important factor in the acquisition of HBV infection. However, it is difficult to determine the age at which infection occurred. In Brazil, specifically in the northeastern population, HBV acquisition is associated with the initiation of sexual activity (15), indicating that the age difference should not influence the results.

The data showed an increased frequency of haplotypes associated with low serum MBL levels in patients infected with HBV, compared to non-infected individuals (p = 0.04). Our report included for the first time analysis of the MBL2 allelic variants in exon 1 (A/O), simultaneously with analysis of the promoter region of MBL2 at positions −550 (H/L) and −221(X/Y), in patients with HBV infection. The results suggest that individuals carrying haplotypes for low expression of MBL have an increased risk of infection with HBV. Variant alleles or genotypes related to low levels of serum MBL were no more common in the HBV patients than in the healthy controls. The lack of association of HBV infection with genotypes related to low levels of MBL may reflect the small number of individuals studied, while the haplotype analyses utilized chromosomes, which allowed more accurate sampling.

Although the determination of MBL levels is an important issue, the present study aimed only to verify the association of the frequency of MBL polymorphisms with HBV infection. Also, the association of serum levels of MBL with genotype has been well-documented by many authors (9,21). On the other hand, MBL is an acute-phase protein, and its levels may be influenced by HBV infection.

The preS2 hepatitis B surface antigen has a mannose-rich oligosaccharide region, which could be a potential binding site for MBL (8). In fact, there is evidence that MBL binds to HBsAg via its multiple carbohydrate recognition domains, suggesting that this lectin may play a role in HBV opsonization. Also, HBsAg-MBL complexes activate C4 in vitro (8). Accordingly, low levels of serum MBL may lead to susceptibility to HBV infection.

One potential mechanism for MBL's protection against HBV infection may involve the ability of MBL to target the virion or subviral particles of HBV for antigen-presenting cells, either by opsonization or by deposition of complement system components C4b and C3b.

Our intent is to expand this study to gain more insight about the role of polymorphisms in HBV-related liver disease progression. MBL infusion therapy has been suggested to treat MBL-deficient individuals that are undergoing chemotherapy or have immune deficiency (2). Thus studies with larger number of patients are needed to establish the importance of MBL polymorphisms in the predisposition toward HBV infection or progression.

This is the first study of Brazilian individuals that shows preliminary evidence supporting the hypothesis that there is a protective role of MBL against HBV infection, as shown by the higher frequency of haplotypes associated with high MBL in the control group than in HBV patients.

Footnotes

Acknowledgments

We would like to thank Drs. Gustavo Guerra and Antônio Guelfler Campos for their help with patient follow-up, the Pernambuco State Diagnosis Center for the serological analysis, Ms. Coriene Catsman-Berrevoets for her help in preparing the manuscript, and the National Council for Research and Development (CNPq).

Author Disclosure Statement

No competing financial interests exist.

References

Alves-Silva

, Da Silva Santos

, Guimaraes

, Ferreira

, Bandelt

, Pena

, Prado

. The ancestry of Brazilian MT DNA lineages. Am J Hum Genet, 2000; 67:444–461.

Bang

, Laursen

, Thornberg

et al. The pharmacokinetic profile of plasma-derived mannan-binding lectin in healthy adult volunteers and patients with Staphylococcus aureus septicaemia. Scand J Infect Dis, 2008; 40:44–48.

Beasley

. Hepatitis B virus, the major etiology of hepatocellular carcinoma. Cancer, 1988; 61:1942–1956.

Boldt

, Petzl-Erler

. A new strategy for mannose-binding lectin gene haplotyping. Hum Mutat, 2002; 19:296–306.

Bosch

, Ribes

, Cleries

, Diaz

. Epidemiology of hepatocellular carcinoma. Clin Liver Dis, 2005; 9:191–211.

Brown

, Ryder

, Irving

, Sim

, Hickling

. Mannan binding lectin and viral hepatitis. Immunol Lett, 2007; 108:34–44.

Cheong

, Cho

, Lim

et al. Lack of association between hepatitis B virus infection and polymorphism of mannose-binding lectin gene in Korean population. J Korean Med Sci, 2005; 20:65–69.

Chong

, To

, Ip

et al. Mannose-binding lectin in chronic hepatitis B virus infection. Hepatology, 2005; 42:1037–1045.

Garred

, Larsen

, Madsen

, Koch

. Mannose-binding lectin deficiency-revisited. Mol Immunol, 2003; 40:73–84.

10.

Hladnik

, Braida

, Boniotto

, Pirulli

, Gerin

, Amoroso

, Crovella

. Single-tube genotyping of MBL-2 polymorphisms using melting temperature analysis. Clin Exp Med, 2002; 2:105–108.

11.

Höhler

, Wünschel

, Gerken

, Schneider

, Meyer zum Büschenfelde

, Rittner

. No association between mannose binding lectin alleles and the susceptibility to chronic hepatitis B infection in German patients. Exp Clin Immunogenet, 1998; 15:130–133.

12.

Jack

, Read

, Tenner

, Frosch

, Turner

, Klein

. Mannose-binding lectin regulates the inflammatory response of human professional phagocytes to Neisseria meningitidis serogroup B. J Infect Dis, 2001; 184:1152–1162.

13.

Lavanchy

. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat, 2004; 11:97–107.

14.

Madsen

, Garred

, Thiel

, Kurtzhals

, Lamm

, Ryder

, Svejgaard

. Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein. J Immunol, 1995; 155:3013–3020.

15.

Pereira

, Martelli

, Merchán-Hamann

et al. Population-based multicentric survey of hepatitis B infection and risk factor differences among three regions in Brazil. Am J Trop Med Hyg, 2009; 81:240–247.

16.

Song Le

, Binh

, Duya

et al. Binding lectin gene polymorphisms and hepatitis B virus infection in Vietnamese patients. Mutat Res, 2003; 522:119–125.

17.

Sumiya

, Super

, Tabona

, Levinsky

, Arai

, Turner

, Summerfield

. Molecular basis of opsonic defect in immunodeficient children. Lancet, 1991; 337:1569–1570.

18.

Thio

, Mosburger

, Astemborski

, Greer

, Kirk

, O'Brien

, Thomas

. Mannose binding lectin genotypes influence recovery from hepatitis B virus. Infect Virol, 2005; 79:9192–9196.

19.

Thomas

, Foster

, Sumiya

, Mclntosh

, Jack

, Turner

, Summerfield

. Mutation of gene for mannose binding protein associated with chronic hepatitis B viral infection. Lancet, 1996; 348:1417–1419.

20.

Turner

. Mannose-binding lectin: the pluripotent molecule of the innate immune system. Immunol Today, 1996; 17:532–540.

21.

Worthley

, Bardy

, Mullighan

. Mannose-binding lectin: biology and clinical implications. Int Med J, 2005; 35:548–555.