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

Abstract

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.
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.
(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.
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.

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RESEARCHOpen Access
S gene mutations of HBV in children with
HBV-associated glomerulonephritis
Hongzhu Lu1,2, Hui Zhu1and Jianhua Zhou1*
Abstract
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 virus-
associated 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.
Results
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
* Correspondence: jhzhou@tjh.tjmu.edu.cn
1Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong
University of Science and Technology, Wuhan 430030, People’s Republic of
China
Full list of author information is available at the end of the article
Lu et al. Virology Journal 2012, 9:59
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© 2011 Lu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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patients had proteinuria and hematuria. Result of liver
function test was normal in 21 patients. Serology mar-
kers 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. There-
fore, 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 geno-
type-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 com-
pared with AY167097.1, an endemic HBV strain of sero-
type 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 sub-
stitutions at Threonine, Serine or Tyrosine phosphoryla-
tion 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 syn-
drome 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 chil-
dren, 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 detect-
able HBV antigens in the kidney. However, HBsAg
immune complex deposits are detectable in kidney in
some patients with viral hepatitis but without renal dis-
eases [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 geno-
types 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 chil-
dren, we were not sure whether HBV strain other than
the serotype adw and the genotype B might be asso-
ciated with HBV-GN in children. It is interesting that
the serotype ayw and genotype A of HBV are predomi-
nant 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 [12-14].
Kim et al. [15] reported 209 patients with HBV infec-
tion, in which the adr, adw and ayr serotypes were
found in 193, 12 and 1 subjects, respectively. The extra-
ordinary predominance of the genotype C was found in
chronic HBV patients in Korean, suggesting that the
clinical manifestations of chronic HBV patients in Kor-
ean 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 with HBV-
GN (21 of 30, 70%). Moreover, most of them (16 of 21,
76.2%) were involved in point mutations which may
result in amino acid substitutions in HBsAg protein.
Threonine, serine and tyrosine are the most likely
amino acid substitution sites. Eleven of 16 children with
missense mutation in HBV S gene showed lose or gain
Table 1 Serology markers of HBV in children with
HBV-GN
Findings Number of participants
HBsAg positive 30
HBeAg positive19
HBV DNA positive 30
ALT/AST increased14
ALT: alanine aminotransferase; AST: aspartate aminotransferase
Table 2 HBV genotype in HBV-GN and control groups
Group (Number of cases)BCE
HBV-GN (30)2901
Control 1 (5)500
Control 2 (18)1710
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Table 3 HBV S gene mutations and the inferred amino acid substitutions in children with HBV-GN
Case No.amino acid loci in S regionCodon change
TTG®TCG
TTC®TGC
TCG®ATG
TTC®TGC
TAT®TTT
AAA®AAG
ACA®ACG
TCA®TCC
TCT ®TCC
AAA®AAG
GAG®GGG
TAT®TTT
ACA®ACG
TCG®ATG
AGT®AGA
ACA®ACG
GTT®GGT
TAT®TTT
TTC®TGC
TCT ®TCC
AAA®AAG
TCA®TCC
ACA®ACT
CCT®ACT
TCG®ATG
TTC®TGC
AAA®AAG
ACA®ACG
TCA®TCC
TCT ®TCC
CAA®CAC
GGA®GGC
CAA®CAC
GGA®GGC
ACA®ACG
CAA®CAC
ACA®ACG
CAT®CGT
ATG®ACG
Amino acid substitutions
Leu®Ser
Phe®Cys
Ser®Met
Phe®Cys
Tyr®Cys
Lys
Thr
Ser
Ser
1 94
2161
3 143
161
200
122
125
136
155
4122Lys
Glu®Gly
Tyr®Cys
5 164
6 200
7 125Thr
Ser®Met
Ser®Arg
Thr
Val®Gly
Tyr®Cys
Phe®Cys
8143
9 204
125
1096
200
11161
12 155Ser
13122
136
Lys
Ser
1445Thr
Pro®Thr
Ser®Met
Phe®Cys
Lys
Thr
Ser
Ser
Gln®His
Gly
Gln®His
Gly
Thr
Gln®His
Thr
His®Arg
Met®Thr
15127
16 143
161
122
125
136
155
17 129
119
18129
119
125
19 129
125
20175
21198
othersNo mutation
Table 4 Gene mutations in HBV S region in control groups
Control groups Case No. Amino acid lociGene mutations
ACA®ACT
ACC®ACT
AAA®AAG
CAA®CAG
ACA®ACG
GTC®GTT
Amino acid
Nephrosis with HBV carrier1
2
45
118
122
Thr
Thr
Lys
HBV carrier1
2
54
115
190
Gln
Thr
Val
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of these three amino acids. Kim et al. [16] also found
similar amino acid substitutions in HBsAg in 6 of 7 chil-
dren with HBV associated membranous nephropathy
(HBV-MN). Furthermore, one of the substitutions in
each subject was involved in serine gaining. It is well
known that serine and threonine could be phosphory-
lated by MAPK and tyrosine could be phosphorylated
by PTK. The phosphorylation is critical to the intracel-
lular function of S proteins, large proteins and middle
proteins. The important amino acid substitutions
?
?
?
Figure 1 Point mutation-caused amino acid substitutions in HBV surface antigen (HBsAg) were shown in children with HBV-GN. Panel
A, Pro 127 Thr (CCT®ACT); Panel B, Gln 129 His (CAA®CAC); Panel C, Thr 131 Asn (ACC®AAC); Panel D, Thr 143 Met (ACG®ATG).
Lu et al. Virology Journal 2012, 9:59
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secondary to their gene mutations may result in differ-
ent biological behavior in HBV-infected cells. The signif-
icance 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
“a” 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 “a” determinant
region, were frequently discovered in renal tissue of
children with HBV-MN. Further, changes in HBV sur-
face 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 muta-
tions 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 pre-
sent study.
Methods
Procedures to detect point mutations of HBV S gene by
PCR and direct sequencing analysis
Sera samples from child participants ® DNA purifica-
tion ® HBV S gene amplification ® 1.5% Agarose gel
electrophoresis ® PCR-DNA purification ® Automatic
sequencing ® Comparing with AY167097.1
Participants and samples
Thirty children with HBV-GN diagnosed by renal biopsy
from Department of Pediatrics, Tongji Hospital, Tongji
Medical College, Huazhong University of Science and
Technology, from February 1992 to October 2003, were
included in this study. Additional 5 nephrotic children
with HBV carrier but not HBV-GN served as control
group 1, and 18 HBV carriers without nephrosis as con-
trol group 2. The blood was collected from these chil-
dren 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, accord-
ing to the ethical principles of international ethical
guidelines for biomedical research involving human
subjects.
Extraction of HBV DNA
HBV DNA was extracted from serum samples. Briefly,
100 μl serum was added to 400 μl buffer containing pro-
teinase K (10 mM Tris-Cl pH 8.0, 10 mM EDTA pH8.0,
0.5% proteinase K) (Jingmei Biotech Co. Ltd.), water
bath at 55°C for 3 hrs, and then DNA was extracted
with chloroform and phenol.
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 Bio-
tech 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.
Abbreviations
HBV: Hepatitis B virus; HBV-GN: HBV-associated glomerulonephritis; MAPK:
Mitogen-activated protein kinase; PTK: Protein tyrosine kinase; HBV-MN: HBV
associated membranous nephropathy.
Acknowledgements
This study was supported in part by funds from the National Natural Science
Foundation of China (NO:30772360, 81100514). The authors thank Prof.
Hengsheng Wu and Feng Fang for collecting blood samples and for data
statistical analysis.
Author details
1Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong
University of Science and Technology, Wuhan 430030, People’s Republic of
China.2Department of Pediatrics, the First Affiliated Hospital, Clinical Medical
College, Yangtze University, Jingzhou, Hubei, People’s Republic of China.
Authors’ contributions
LH and ZJ designed and carried out the study and wrote the manuscript.
ZH carried out the serological and molecular biology assays. All authors read
and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 11 March 2011 Accepted: 5 March 2012
Published: 5 March 2012
Lu et al. Virology Journal 2012, 9:59
http://www.virologyj.com/content/9/1/59
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