S gene mutations of HBV in children with HBV-associated glomerulonephritis

Background Hepatitis B virus-associated glomerulonephritis (HBV-GN) is a kind of immune complex-induced glomerulonephritis. The present study was designed to determine whether mutation of Hepatitis B virus (HBV) S gene is associated with glomerulonephritis in Chinese children. Methods Total 53 subjects, including 30 HBV-GN, 5 nephrosis with HBV carriers (control group 1), and 18 HBV carriers (control group 2) were included in this study. Polymerase chain reaction (PCR) was used to detect the HBV-GN S gene mutation. Results (1) The serotype of HBV was adw in the majority (52/53) of subjects, and was adr in only 1 subject in the control group 2; (2) the genotype of HBV was the type B in 51 subjects, the type E in 1 HBV-GN child, and the type C in 1 HBV carrier; (3) Seventeen point mutations in the S gene of HBV were identified in 21 of 30 (70%) HBV-GN patients. Among them, 16 of 21 (76.2%) mutations may cause amino acid substitutions of the HBV proteins, which occur predominantly (11/16 mutations) at threonine, serine or tyrosine phosphorylation sites of mitogen-activated protein kinase (MAPK) or protein tyrosine kinase (PTK). (4) In addition, single nucleotide mutations without amino acid substitutions (same sense mutation) were found in 2 subjects in each control group and 5 subjects in HBV-GN group. Conclusions HBV S gene mutations and the subsequent amino acid substitutions in HBV proteins were found in most children with HBV-GN, suggesting that these mutations may play an important role in the pathogenesis of HBV-GN.


Background
Hepatitis B viruses (HBV) are well-recognized as the causes of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. In addition, HBV infection is also associated with a spectrum of extrahepatic manifestations [1]. Hepatitis B is prevalent in China and hepatitis B virusassociated glomerulonephritis (HBV-GN) is one of the common renal damages secondary to HBV infection in Chinese children [2]. The mechanism of HBV-GN is generally believed to be related to HBV-related immune reactions [3]. This is based on the findings that HBV viral antigens were detected in kidney tissue [4,5], and further HBV surface antigen (HBsAg) and HBV nucleus antigen were detected in the glomerular deposits [6].
However, the precise pathogenesis of HBV-GN is not fully understood. The HBsAg was present in all children with HBV-GN [6] and also many HBV carriers. Thus, we hypothesized that HBV S gene mutations and their subsequent amino acid substitutions in HBsAg may alter antigenity of HBsAg in HBV-GN, causing immuno-reactive complex deposited in subepithelial areas in glomeruli. Therefore, in the present study, we used the PCR technique to examine the S gene sequence of HBV in order to determine whether characteristic variants of HBV S gene are associated with HBV-GN in Chinese children.

Clinical characteristics of the participants
Thirty patients (29 male, 1 female), at the age of 7.7 ± 2.8, were diagnosed as HBV-GN. Percutaneous renal biopsy was performed in these patients. All patients had proteinuria, 21 children had nephrotic syndrome, and 9 patients had proteinuria and hematuria. Result of liver function test was normal in 21 patients. Serology markers of HBV of the participants were shown in Table 1.

HBV serotype
HBV S gene contains 678 bp, which encode 226 amino acids of HBsAg major protein. There are 2 subtype determinants (d/y and w/r) in the major protein. Therefore, HBV has 4 serotypes (adw, adr, ayw and ayr) based upon the 122nd amino acid (Lys/Arg, d/y) and the 160th amino acid (Lys/Arg, w/r) in S protein [7]. We detected 53 subjects, in whom 52 were adw and 1 was adr in the control group 2.

HBV genotype
HBV was classified into 8 genotypes according to genotype-specific restriction enzyme sites (A, B, C, D, E, F, G, and H).
The HBV genotype distributions in the different groups were shown in Table 2. There was no difference between HBV-GN and control groups. Most of them were the genotype B, only 1 HBV-GN was the genotype E, and 1 HBV carrier was the genotype C ( Table 2).

Mutations in HBV S gene
The HBV S gene sequences in 53 subjects were compared with AY167097.1, an endemic HBV strain of serotype adw and genotype B in China. The results were shown in Tables 3 and 4. The chromatograph sequences of mutations in some HBV-GN were shown in Figure 1.
Seventeen point mutations were identified in 21 of 30 (70%) HBV-GN patients. Among them, 16 of 21(76.2%) mutations were involved in amino acid substitutions. Moreover, 11/16 (68.8%) were involved amino acid substitutions at Threonine, Serine or Tyrosine phosphorylation sites of mitogen-activated protein kinase (MAPK) and protein tyrosine kinase (PTK) in HBV proteins. In addition, single nucleotide mutations without amino acid substitution (same sense mutation) were found in 2 subjects in each control group and 5 subjects in HBV-GN group.

Discussion
Recognition of an association between chronic HBV and glomerular disease dates back to the 1970s [8]. This syndrome occurs mainly in children, predominantly in male in HBV endemic areas of the world. The most typical presentations of HBV-GN include nephrotic syndromes and memberanous glomerulonephropathy. In these children, liver function tests are frequently normal. About 60% of patients show spontaneous remission [9]. Although many patients with renal diseases display blood HBsAg-positive, only a few patients have detectable HBV antigens in the kidney. However, HBsAg immune complex deposits are detectable in kidney in some patients with viral hepatitis but without renal diseases [10]. Therefore, the potential role of HBsAg in pathogenesis of HBV-GN remains unclear.
In present study, we attempted to study whether HBV serotype or genotype characteristics are associated with HBV-GN. We found that the HBV serotypes and genotypes were similar in both HBV-GN and control groups. The endemic HBV strain was the serotype adw and the genotype B in south China [11]. Therefore, it was not surprising that all children with HBV-GN and the majority (22 of 23) subjects in control groups were the HBV genotype B. Since the HBV serotype and genotype have not been reported previously in HBV-GN in children, we were not sure whether HBV strain other than the serotype adw and the genotype B might be associated with HBV-GN in children. It is interesting that the serotype ayw and genotype A of HBV are predominant in HBV-GN in South Africa with different endemic HBV strain infection, where the serotype adw and the genotype B were not as epidemic as in China [12][13][14]. Kim et al. [15] reported 209 patients with HBV infection, in which the adr, adw and ayr serotypes were found in 193, 12 and 1 subjects, respectively. The extraordinary predominance of the genotype C was found in chronic HBV patients in Korean, suggesting that the clinical manifestations of chronic HBV patients in Korean are different from those in other Asian countries.
Although no specific HBV genotype and serotype were identified in HBV-GN, it was interesting that HBV S gene mutations were found in most children      MN). Furthermore, one of the substitutions in each subject was involved in serine gaining. It is well known that serine and threonine could be phosphorylated by MAPK and tyrosine could be phosphorylated by PTK. The phosphorylation is critical to the intracellular function of S proteins, large proteins and middle proteins. The important amino acid substitutions secondary to their gene mutations may result in different biological behavior in HBV-infected cells. The significance of the present findings is unclear. Since no similar mutations were found in HBV carriers and those with primary nephrosis, we believe that the S gene mutations observed in the present study may play an important role in the pathogenesis of HBV-GN. The substitution of amino acid in HBV surface protein may change not only the intracellular biological behavior of HBV, but also the immune response of human body. Many amino acid substitutions were identified in the "α" determinant of HBsAg, which was crucial in the binding of neutralizing antibodies. Thus, virus with mutations could escape from clearance by human immune system [17,18]. The persistent infection of HBV is prerequisite for the development of HBV-GN. Kim et al. [16] demonstrated that deletions and point mutations in the HBV pre-S1, pre-S2 region, and point mutations in the HBV S region, especially the "α" determinant region, were frequently discovered in renal tissue of children with HBV-MN. Further, changes in HBV surface protein may facilitate the binding of HBsAg to its receptors in glomerular epithelial cells, a process called "plant of antigen in situ", which may have triggered the development of HBV-MN.
Not all children with HBV-GN showed point mutations in HBV S gene. We did not detect the genome sequence of HBV. It could not be exclude that point mutations existed in other regions of HBV genome. The subepithelial deposition of HBsAg and their antibodies played a key role in the formation of HBV-MN.

Conclusions
The importance of HBsAg encourages us to explore the role of HBV S gene mutations in HBV-GN. The present data suggest that HBV S gene mutations are closely associated with HBV-GN. Further HBV genome analysis is required to confirm the findings observed in the present study. with HBV carrier but not HBV-GN served as control group 1, and 18 HBV carriers without nephrosis as control group 2. The blood was collected from these children and the sera were isolated at 4°C and stored at -20°C until PCR assays. Informed consent was obtained from all of the patients recruited into the study, according to the ethical principles of international ethical guidelines for biomedical research involving human subjects.

PCR amplification of HBV S gene
Modified primers for S gene were designed according to Yan et al. [19] and were synthesized by Beijing Auke Biotech Co. Ltd. Primer YS1, 5' ATGGGAATTCGGGGTT-TTTCTTGTTGA 3'; Primer YS2, 5' CGTAAGCTTGG-GACTCAAGATGTTGTA 3'. The product length is 586 bp. PCR was performed in 50 μl, 94°C denaturation for 4 minutes, and then 94°C for 50 seconds, 47°C for 1 min, 72°C for 1 min, totally 35 cycles were completed. The final extension was performed at 72°C for 5 min. The PCR products were directly sequenced and compared with AY167097.1, an epidemic HBV strain in China, by Fasta software.