Rapid Communication Open Access
Copyright ©2006 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. May 14, 2006; 12(18): 2928-2931
Published online May 14, 2006. doi: 10.3748/wjg.v12.i18.2928
Relationship between IFN-γ gene polymorphism and susceptibility to intrauterine HBV infection
Hui Yu, Qi-Rong Zhu, Shao-Qing Gu, Lin-E Fei
Hui Yu, Qi-Rong Zhu, Shao-Qing Gu, Lin-E Fei, Department of Infectious Disease, Children’s Hospital of Fudan University, Shanghai 200032, China
Supported by the National Natural Science Foundation of China, No.30271365
Correspondence to: Dr. Yu Hui, Department of Infectious Disease, Children’s Hospital of Fudan University, Shanghai 200032, China. yuhui4756@sina.com.cn
Telephone: +86-21-54524666-3003 Fax: +86-21-64038992
Received: November 14, 2005
Revised: November 14, 2005
Accepted: November 14, 2005
Published online: May 14, 2006

Abstract

AIM: To explore the susceptibility of children to intrauterine HBV infection by studying the relationship between IFN-γ gene polymorphism, including IFN-γ+874A/T single nucleotide polymorphism(SNP) and CA repeat microsatellite polymorphism and intrauterine HBV infection.

METHODS: A TaqMan fluorescence polymerase chain reaction in the IFN-γ+874A/T single nucleotide polymorphism was tested in the intrauterine HBV infection group(group I) and the normal immune children group(group II). Capillary electrophoresis was performed in the above two groups to assay the IFN-γ CA repeat microsatellite polymorphism.

RESULTS: Frequencies of AA, AT and TT genotypes were 67.4%, 19.6% and 13.0% in the intrauterine HBV infection group, and 45.2%, 30.1% and 24.7% in the normal immune children group, respectively. A significant difference was found in the frequency distribution of IFN-γ+874 genotype between the two groups (χ2 = 5.102, P = 0.02389). In the intrauterine HBV infection group the AA genotype was more common than in the normal immune group. Frequency of IFN-γ+874A allele was 77.17% in the intrauterine HBV infection group, and 60.27% in the normal immune children group. In the intrauterine HBV infection group the IFN-γ+874A allele was more common than in normal immune group. A significant difference was found in the frequency distribution between the two groups (χ2 = 7.238, P = 0.02389, OR = 2.228, 95% CI = 1.244-3.992). (CA12)+/(CA12)+ of IFN-γ CA microsatellite polymorphism was 11.90% in the intrauterine HBV infection group and 26.47% in the normal immune children group. A significant difference was found in the frequency distribution between the two groups (χ2 = 5.64, P = 0.0176). Frequency of IFN-γ CA repeat was 25% in the intrauterine HBV infection group and 43.38% in the normal immune children group. The frequency of IFN-γ CA repeat was less in the intrauterine HBV infection group than in normal immune group. A significant difference was found in the frequency distribution between the two groups (χ2 = 7.548, P = 0.0060).

CONCLUSION: There is a relationship between IFN-γ+874A/T SNP and intrauterine HBV infection as well as between IFN-γ CA microsatellite polymorphism and intrauterine HBV infection. IFN-γ gene polymorphism might be important in determining individual’s susceptibility to intrauterine HBV infection.

Key Words: Interferon-γ, Gene polymorphism, Hepatitis B virus, Intrauterine



INTRODUCTION

IFN-γ+874A/T SNP within the first intron can increase or decrease the binding of a particular transcription factor and ultimately the export of cytokine product[1-2].The registered DNA sequences of the human IFN-γ gene deposited in Genbank show the presence of a CA repeat element in the first intron, 875 bp downstream from the start of the first exon. There are seven CA repeats of variable lengths In a consecutive study, 12 CA repeats are confirmed its role as a marker of high IFN-γ production[2]. We have reported in a previous study that higher IFN-γ secretion than spontaneous secretion could be found in the intrauterine HBV infection group and the normal immune children group with HBsAg stimulation, but the increase in the intrauterine HBV infection group is strikingly lower than that in the normal immune children group[3]. IFN-γ production is positively associated with IFN-γmRNA expression[3]. In the present study, we studied the IFN-γ+874 SNP and CA repeat microsatellite polymorphism in the intrauterine HBV infection group and the normal immune children group in order to explore the association between IFN-γ gene polymorphisms and susceptibility to intrauterine HBV infection.

MATERIALS AND METHODS
Subjects

The subjects were selected from outpatients who were in our hepatitis B (HB) vaccine follow up clinics. These subjects whose mothers were HBV carriers were inoculated with HB vaccine or HB vaccine plus hepatitis B immunoglobulin (HBIg) and followed up for serum alanine transaminase (ALT) and HBV marker. Intrauterine HBV-infected children were defined based on their positive HBsAg and/or HBV-DNA at birth and lasting for six months (group I). Normal immune children were defined based on their negative HBV marker after birth and high HBsAb titers (group II). ALT of all subjects was normal.

TaqMan fluorescence polymerase chain reaction

Genomic DNA was extracted from peripheral blood using the kits supplied by Sangon Bioengineering Company (Shanghai, China). Polymorphisms at + 874 were identified using the ABI Prism 7700 fluorescent automatic sequencer. PCR products were obtained after amplification in a 10 μL volume containing 1 × Taqman buffer A, 200 μmmol/L(each) dATP, dCTP, dGTP, 400 μmmol/LdUTP, 3.5 mmol/L MgCl2, 300 μmmol/Lβ-actin forward primer, 300 μmmol/L β-actin reverse primer, 200 μmmol/Lβ-actin probe, AmpliTaq Gold, 0.025 U/μL DNA polymerase, 0.01 U/μL AmpErase UNG, 900 nmmol/L(each) forward & reverse primers, 250 nmol/L TaqMan MGB probe, 20ng DNA template. The forward primer sequence is 5’-ACA TTC CAC AAT TGA TTT TAT TCT TAC AAC A-3’. The reverse primer sequence is 5’-ACG AGC TTT AAA AGA TAG TTC CAA ACA-3’. The TaqMan MGB probe sequences are as follows: Fam: AAA TCA AAT CtC ACA CAC ACA C; Vic: AAA TCA AAT CaC ACA CAC ACA C. Forty-five cycles of PCR amplification were performed at 50 °C for 2 min, at 95 °C for 10 min, at 95 °C for 30 s and at 60 °C for 30 s.

Capillary electrophoresis

DNA was amplified in a 7.5 μL reaction volume containing 0.75 μL of 10 × buffer, 0.75 μL 25 mmol/L MgCl2, 0.75 μL 2.5 μmmol/LdNTP, 2.5 U/μL Taq Gold, 0.06 μL DNA polymerase, 1 μL (each) forward & reverse primers, 2 μL DNA template, 1.19 μL ddH2O. The forward primer sequence is CTT CGT TGC TCA CTG GGA TT-6-FAM. The reverse primer sequence is GCA AAG CCA CCC CAC TAT AA. The microsatellite region in the first intron of the IFN-γ gene was amplified on a PTC-100 thermal cycler. Following an initial denaturation (12 min at 95 °C), samples were subjected to 10 cycles of PCR amplification, for each cycle, the sample was amplified at 94 °C for 30 s, at 53 °C for 30 s, at 75 °C for 45 s, followed by 30 cycles at 89 °C for 30 s, at 53 °C for 30 s, at 75 °C for 45 s, and a final extension at 4 °C. The IFN-γ CA repeat microsatellite polymorphisms were monitored by ABI PRISM 3700 capillary electrophoresis automatic analysis.

Statistical analysis

IFN-γ+874A/T allele frequencies and IFN-γ CA microsatellite frequencies were evaluated by gene count. Allelic frequency was calculated with the following equation: allelic frequency = n/2N (n represents the number of alleles, and N represents the total number of subjects). Allelic frequencies were compared using the chi-square test. A P value less than 0.05 was considered statistically significant. The strength of an association was expressed as odds ratio (OR) with 95% confidence interval. STATA 6.0 software was used to determine the statistical significance.

RESULTS
General clinical characteristics

No significant difference was found in sex, HBeAg positivity of mother and HBIg injection to pregnant women between the two groups (Table 1).

Table 1 Clinical characteristics of patients and controls.
SubjectsnSex
HBV marker of mother
HBIg injection in pregnancy
MaleFemaleHBsAg+HBsAg+ and HBeAg+YesNo
Group I46262015311030
Group II73413225481647
χ20.00150.03390.0020
P0.96950.85390.9640
IFN-γ+874A/T SNP

The distribution of IFN-γ+874A/T allele frequencies in two groups was in accordance with Hardy-Weinberg hereditary equilibrium law. Frequencies of AA, AT and TT genotypes were 67.4%, 19.6% and 13.0% in the intrauterine HBV infection group and 45.2%, 30.1% and 24.7% in the normal immune children group, respectively. A significant difference was found in the frequency distribution of IFN-γ+874 genotype between the two groups (χ2 = 5.102, P = 0.02389). In the intrauterine HBV infection group, the AA genotype was more common than in normal immune group (Table 2). Frequency of IFN-γ+874A allele was 77.17% in the intrauterine HBV infection group and 60.27% in the normal immune children group. In the intrauterine HBV infection group, the IFN-γ+874A allele was more common than in normal immune group. A significant difference was found in the frequency distribution between the two groups (χ2 = 7.238, P = 0.02389, OR = 2.228, 95%CI1.244-3.992)(Table 3).

Table 2 Frequencies of IFN-γ+874 genotype in patients and controls.
SubjectsnNumber of subjects and percentage ofIFN-γ+874 genotype (%)
AAATTT
Group I4631 (67.4)9 (19.6)6 (13.0)
Group II7333 (45.2)22 (30.1)18 (24.7)
χ25.102
P0.02389
Table 3 +874T and +874A IFN-γ allele frequencies in patients and controls.
Allele(n)IFN-γ+874A/T allelefrequencies (%)
OR(95% confidence interval)χ2P
+874A+874T
3.228
Group I46 (92)71 (77.17)21 (22.83)(1.244-3.992)7.2380.0071
Group II73 (146)88 (60.27)58 (39.73)
IFN-γ CA microsatellite polymorphism

(CA12)+/(CA12)+ of IFN-γ CA microsatellite polymorphism was 11.90% in the intrauterine HBV infection group and 26.47% in the normal immune children group. A significant difference was found in the frequency distribution between the two groups (χ2 = 5.64, P = 0.0176)(Table 4). Frequency of IFN-γ CA repeat was 25% in the intrauterine HBV infection group and 43.38% in the normal immune children group. In the intrauterine HBV infection group, the frequency of IFN-γ CA repeat was less than in normal immune group. A significant difference was found in the frequency distribution between the two groups (χ2 = 7.548, P = 0.0060)(Table 5, Figure 1).

Figure 1
Figure 1 IFN-γ CA microsatellite polymorphisms in patients and controls. Group I: the intrauterine HBV infection group; Group II: the normal immune children group.
Table 4 IFN-γCA microsatellite morphisms in patients and controls.
nNumber and percentage of genotype of IFN-γCA (%)
(CA12)+/(CA12)+(CA12)+/(CA12)-(CA12)-/(CA12)-
Group I425 (11.90)11 (26.19)26 (61.91)
Group II6818 (26.47)23 (33.82)27 (39.71)
χ25.640
P0.0176
Table 5 IFN-γ CA repeat times in patients and control.
IFN-γ CArepeatGroup 1Group IIχ2P
(84)
(136)
% (n)% (n)
111.19 (1)0.74 (1)0.1190.7302
1225 (21)43.38 (59)7.5480.00601
1313.10 (11)8.82 (12)1.0080.3155
1440.48 (34)33.82 (46)0.9890.3201
1513.10 (11)7.35 (10)1.9740.1600
164.76 (4)3.68 (5)0.1550.6936
172.38 (2)2.21 (3)0.0070.9327
DISCUSSION

The clinical features of HBV infection depend on the activities of the host immune response and the virus. The cell-mediated immune response to HBV-encoded antigen is responsible both for viral clearance and for disease pathogenesis during this infection. T-helper lymphocyte response mediated through secretion of IFN-γ plays an important role in inducing viral clearance[4]. During the early aspecific phase of host defence, production of IFN-γ by natural killer(NK) cells plays an important role in bringing about acute inflammation. In the subsequent antigen-specific phase of the immune response, IFN-γ acts as a regulator of antigen presentation and proliferation as well as differentiation of lymphocytes. High-level production of IFN-γ during this phase plays an important role in viral clearance[5-6].

Cytokine production is genetically controlled. The cytokine genes SNPs in cis-acting regions can alter transcriptional activity and are associated with the production of cytokine[7]. Genetic susceptibility to diseases is likely influenced by common DNA variants in the form of SNPs[8-9]. Pravica et al[2] described a variable length CA repeat sequence in the first intron of the human IFN-γ gene and showed that 12 CA repeat is associated with high in vitro IFN-γ production. In a further study, a SNP T to A at the end of the CA repeat region in the first intron of the human IFN-γ gene(+874A/T) was described, showing an absolute correlation between the T allele and the high IFN-γ microsatellite polymorphism. The +874T allele is associated with the high production of genotype and the +874A allele is associated with the low production of genotype[10]. In vitro production of IFN-γ has a significant correlation with the CA microsatellite polymorphism. Twelve CA repeats have been shown to have a higher expression of IFN-γ[11-12]. Japanese researchers[13] have reported the frequencies of 13 CA repeats are significantly greater in patients with IgA nephropathy than in the healthy control group (43% versus 23%, P < 0.05). High production of genotype (12 CA repeats) for IFN-γ may have an influence on acute rejection of kidney transplants[14]. The frequency of the IFN-γ12 CA repeats is significantly greater in WHO class V lupus nephritis patients than in WHO class IV patients[15]. Susceptibility to and severity of rheumatoid arthritis are related to a microsatellite polymorphism within the first intron of the IFN-γ gene[16]. There is a highly significant increase in the low-production of IFN-γ genotype(13 CA repeats) in patients with type 1 diabetes compared with normal healthy controls[17]. This result suggests that polymorphisms of the IFN-γ gene may modify the function of this proinflammatory mediator and the response to pancreatic islet βcells. This +874A/T polymorphism coincides with a putative NF-Kappa B binding site, which might have functional consequences for transcription of human IFN-γ gene. Therefore, the T to A polymorphism might directly influence the level of IFN-γ production associated with the CA microsatellite polymorphism[18]. Patients with tuberculosis have a lower frequency of +874TT genotype than the controls, suggesting that genetically determined variability in IFN-γ and expression might be important for the development of tuberculosis[19]. A significant correlation has been detected between the presence of high-expression polymorphisms of the IFN-γ genes and bronchiolitis obliteration syndrome(BOS) after lung transplantation(P = 0.039). +874TT of the IFN-γ gene significantly increases the risk of BOS after lung transplantation[20]. There is no report on the relationship between IFN-γ gene polymorphism and susceptibility to intrauterine HBV infection.

In this study, the frequency of the +874AA genotype of the IFN-γ was greater in the intrauterine HBV infection group than in normal immune group, the (CA12)+/(CA12)+ of IFN-γ CA microsatellite polymorphism was 11.90% in the intrauterine HBV infection group and 26.47% in the normal immune children group. A significant difference was found in the frequency distribution between the two groups(χ2 = 5.640, P = 0.0176). The frequency of IFN-γ CA repeat was less in the intrauterine HBV infection group than in the normal immune group. A significant difference was found in the frequency distribution between the two groups(χ2 = 7.548, P = 0.0060). The findings suggest that the presence of low-expression polymorphism at +874 of the IFN-γ gene significantly increases susceptibility to intrauterine HBV infection. IFN-γ gene polymorphism might be important in determining an individual’s susceptibility to intrauterine HBV infection and might result in viral persistence.

Footnotes

S- Editor Guo SY L- Editor Wang XL E- Editor Bi L

References
1.  Billiau A, Heremans H, Vermeire K, Matthys P. Immunomodulatory properties of interferon-gamma. An update. Ann N Y Acad Sci. 1998;856:22-32.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 136]  [Cited by in F6Publishing: 126]  [Article Influence: 5.9]  [Reference Citation Analysis (0)]
2.  Pravica V, Asderakis A, Perrey C, Hajeer A, Sinnott PJ, Hutchinson IV. In vitro production of IFN-gamma correlates with CA repeat polymorphism in the human IFN-gamma gene. Eur J Immunogenet. 1999;26:1-3.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 348]  [Cited by in F6Publishing: 335]  [Article Influence: 15.1]  [Reference Citation Analysis (0)]
3.  Wang JS, Zhu QR. [Interruption of the transmission of hepatitis B virus from mother to babies]. Zhonghua Ganzangbing Zazhi. 2002;10:308-310.  [PubMed]  [DOI]  [Cited in This Article: ]
4.  Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol. 1995;13:29-60.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 1145]  [Cited by in F6Publishing: 1105]  [Article Influence: 42.4]  [Reference Citation Analysis (0)]
5.  Hilleman MR. Overview of the pathogenesis, prophylaxis and therapeusis of viral hepatitis B, with focus on reduction to practical applications. Vaccine. 2001;19:1837-1848.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 59]  [Cited by in F6Publishing: 18]  [Article Influence: 2.8]  [Reference Citation Analysis (0)]
6.  Guidotti LG, Ishikawa T, Hobbs MV, Matzke B, Schreiber R, Chisari FV. Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes. Immunity. 1996;4:25-36.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 793]  [Cited by in F6Publishing: 369]  [Article Influence: 30.5]  [Reference Citation Analysis (1)]
7.  Mullikin JC, Hunt SE, Cole CG, Mortimore BJ, Rice CM, Burton J, Matthews LH, Pavitt R, Plumb RW, Sims SK. An SNP map of human chromosome 22. Nature. 2000;407:516-520.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 95]  [Cited by in F6Publishing: 84]  [Article Influence: 4.3]  [Reference Citation Analysis (0)]
8.  Bidwell J, Keen L, Gallagher G, Kimberly R, Huizinga T, McDermott MF, Oksenberg J, McNicholl J, Pociot F, Hardt C. Cytokine gene polymorphism in human disease: on-line databases. Genes Immun. 1999;1:3-19.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 410]  [Cited by in F6Publishing: 384]  [Article Influence: 19.5]  [Reference Citation Analysis (0)]
9.  Bidwell J, Keen L, Gallagher G, Kimberly R, Huizinga T, McDermott MF, Oksenberg J, McNicholl J, Pociot F, Hardt C. Cytokine gene polymorphism in human disease: on-line databases, supplement 1. Genes Immun. 2001;2:61-70.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 193]  [Cited by in F6Publishing: 172]  [Article Influence: 9.2]  [Reference Citation Analysis (0)]
10.  Pravica V, Perrey C, Stevens A, Lee JH, Hutchinson IV. A single nucleotide polymorphism in the first intron of the human IFN-gamma gene: absolute correlation with a polymorphic CA microsatellite marker of high IFN-gamma production. Hum Immunol. 2000;61:863-866.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 448]  [Cited by in F6Publishing: 139]  [Article Influence: 20.4]  [Reference Citation Analysis (0)]
11.  Pravica VP, Borreiro LF, Hutchinson IV. Genetic regulation of interferon-gamma production. Biochem Soc Trans. 1997;25:176S.  [PubMed]  [DOI]  [Cited in This Article: ]
12.  Sica A, Dorman L, Viggiano V, Cippitelli M, Ghosh P, Rice N, Young HA. Interaction of NF-kappaB and NFAT with the interferon-gamma promoter. J Biol Chem. 1997;272:30412-30420.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 302]  [Cited by in F6Publishing: 297]  [Article Influence: 12.1]  [Reference Citation Analysis (0)]
13.  Masutani K, Miyake K, Nakashima H, Hirano T, Kubo M, Hirakawa M, Tsuruya K, Fukuda K, Kanai H, Otsuka T. Impact of interferon-gamma and interleukin-4 gene polymorphisms on development and progression of IgA nephropathy in Japanese patients. Am J Kidney Dis. 2003;41:371-379.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 41]  [Cited by in F6Publishing: 31]  [Article Influence: 2.2]  [Reference Citation Analysis (0)]
14.  Asderakis A, Sankaran D, Dyer P, Johnson RW, Pravica V, Sinnott PJ, Roberts I, Hutchinson IV. Association of polymorphisms in the human interferon-gamma and interleukin-10 gene with acute and chronic kidney transplant outcome: the cytokine effect on transplantation. Transplantation. 2001;71:674-677.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 95]  [Cited by in F6Publishing: 92]  [Article Influence: 4.5]  [Reference Citation Analysis (0)]
15.  Miyake K, Nakashima H, Akahoshi M, Inoue Y, Nagano S, Tanaka Y, Masutani K, Hirakata H, Gondo H, Otsuka T. Genetically determined interferon-gamma production influences the histological phenotype of lupus nephritis. Rheumatology (Oxford). 2002;41:518-524.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 52]  [Cited by in F6Publishing: 49]  [Article Influence: 2.6]  [Reference Citation Analysis (0)]
16.  Khani-Hanjani A, Lacaille D, Hoar D, Chalmers A, Horsman D, Anderson M, Balshaw R, Keown PA. Association between dinucleotide repeat in non-coding region of interferon-gamma gene and susceptibility to, and severity of, rheumatoid arthritis. Lancet. 2000;356:820-825.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 76]  [Cited by in F6Publishing: 18]  [Article Influence: 3.5]  [Reference Citation Analysis (0)]
17.  Jahromi M, Millward A, Demaine A. A CA repeat polymorphism of the IFN-gamma gene is associated with susceptibility to type 1 diabetes. J Interferon Cytokine Res. 2000;20:187-190.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 39]  [Cited by in F6Publishing: 40]  [Article Influence: 1.8]  [Reference Citation Analysis (0)]
18.  Sica A, Tan TH, Rice N, Kretzschmar M, Ghosh P, Young HA. The c-rel protooncogene product c-Rel but not NF-kappa B binds to the intronic region of the human interferon-gamma gene at a site related to an interferon-stimulable response element. Proc Natl Acad Sci U S A. 1992;89:1740-1744.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 94]  [Cited by in F6Publishing: 98]  [Article Influence: 3.1]  [Reference Citation Analysis (0)]
19.  Lio D, Marino V, Serauto A, Gioia V, Scola L, Crivello A, Forte GI, Colonna-Romano G, Candore G, Caruso C. Genotype frequencies of the +874T-->A single nucleotide polymorphism in the first intron of the interferon-gamma gene in a sample of Sicilian patients affected by tuberculosis. Eur J Immunogenet. 2002;29:371-374.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 114]  [Cited by in F6Publishing: 114]  [Article Influence: 6.0]  [Reference Citation Analysis (0)]
20.  Lu KC, Jaramillo A, Lecha RL, Schuessler RB, Aloush A, Trulock EP, Mendeloff EN, Huddleston CB, Alexander Patterson G, Mohanakumar T. Interleukin-6 and interferon-gamma gene polymorphisms in the development of bronchiolitis obliterans syndrome after lung transplantation. Transplantation. 2002;74:1297-1302.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 54]  [Cited by in F6Publishing: 56]  [Article Influence: 2.7]  [Reference Citation Analysis (0)]