Estimating High Mobility Group Box Protein 1 (HMGB1) Single Nucleotide Polymorphisms Among Hepatitis B Virus Infected Patients of Pakistan Origin

HMGB1 is nuclear non-histone protein and unique member of cytokines. In viral hepatitis infection HMGB1 serum level increases and translocates towards cytoplasm and extracellular spaces where it activates single stimulating hepatic stellate cell proliferation which induces brogenic protein expression and causes hepatocellular carcinoma. In this study, total 150 subjects were recruited to assess the association between HMGB1 SNPs and HBV. Three types of genotypes were found visible in rs3742305 of HMGB1; wild type homozygous GG with 65%, homozygous minor type CC with 6% and heterozygous minor type GC with 26% frequency distribution. High prevalence of GG genotype in the selected population presenting that GG genotype may have higher risk for susceptibility to HBV infection. Our results showed signicant correlation of HMGB1 polymorphism with HBV infection in the selected Pakistani population.


Introduction
Hepatitis B virus may cause either acute or chronic liver infection. It may also involve in liver cirrhosis and hepatic-cellular carcinoma. Chronic infection with HBV is a major worldwide health problem despite the availability of vaccine (1). The HBV infection prevalence is highly changeable as estimated more than 10% in some Western Paci c countries and Asian's to under 0.5% in the northern European countries and United States. Globally the estimated HBV infected individuals are 350 million (2)(3)(4). Approximately 7-9 million carriers of HBV are found in Pakistan with an increase in carrier rate of 3-5 (5). The lifecycle of HBV has been studies exclusively however, the host factors involved in HBV replication and HBV associated HCC mechanisms are not fully understood.
The high mobility group (HMG) proteins binds to nucleosomes in non-sequence speci c manner and have the ability to bring structural changes in chromatin (6). HMG proteins belonged to three families: HMGA, HMGB, and HMGN. These members are structurally divergent but share functional similarity (6). The most abundant protein of this family is HMGB (high-mobility group box) which is further divided into four groups (HMGB1, HMGB2, HMGB3 and HMGB4) (6). HMG-box family showcases diversity of characters including chromatin remodeling, transcription, replication, repair, recombination and genomic stability, moreover extracellular HMGB1 involves in cell growth and mitotic activity, receptor signaling (6). This emphasis that HMGB1 plays a critical role in the pathogenesis and treatment of liver diseases.
A single nucleotide polymorphism (SNP) is the stable replacement of single base in human gene. It is considered the most common genetic mutation in humans appearing in more than 1% of population.
SNP is not identi ed as a disease-causing variant, but genetic variation may impact suggestively on the disease susceptibility (7). Common and rare SNPs and structural genomic changes may in uence or restrict HCC development. Early identi cation of moderate to high genetic variants associated with HBVrelated HCC will help to determine susceptibility of infection better outcomes (8)(9)(10). For instance, mutational screening for BRCA1 and BRCA2 is used to identify women at risk for breast and ovarian cancer and is routinely used in accuracy of treatment protocols (9-10). It is already a known fact that HBV infection is closely related with cytokines and polymorphisms of cytokine gene (ESR1) is reported to be associated with HBV infection (11)(12). One of the SNP rs3742305 was identi ed in HMGB1 in HBV infected Chinese patients (14). Since there is no evidence of reported SNP in Pakistani population, identifying the association of HMGB1 and HBV. We rst time reported the study based on polymorphism of HMGB1 gene in HBV patients identi ed in population of Pakistan origin.

Materials And Methods
Patients Data In the current study, total 150 peoples were selected in which 100 patients with HBV infection (Acute, Chronic) were randomly selected from the Nuclear Medicine, Oncology and Radiotherapy Institute, Islamabad. Control sample size was 50 from normal population. Informed consent was obtained from all patients to participate in this study. All those patients were excluded from the study that was co-infected with HBV, HIV or patients infected with the alcoholic liver disease. 5 ml blood samples from patients were collected along with singed consent forms. The diagnostic criteria for HBV infected patient were positive patients of HBsAg, HBeAg and anti-HBc and the patients have following symptoms of jaundice, fatty liver, abdominal pain and dark urine. Acute hepatitis B (AHB), chronic hepatitis B (CHB) and severe hepatitis B (SHB) were selected. Patients having HBV were inducted with micro complications along with age, sex-matched, jaundice, fatty liver, abdominal pain, dark urine. HBV patients were assessed for AHB, CHB and SHB. The features/parameters recorded for the HBV patients are summarized in Table 1 and 2.

DNA extraction method
The leukocytes genomic DNA from 5 mL whole blood was isolated using organic phenol chloroform extraction method (15). DNA samples were diluted to 10 ng/µL. Genetic Analysis SNP rs3742305 of HMGB1 was selected on the basis of previous ndings (14), showing evidence of strong association of this SNP with HMGB1. Genotyping of allelic variant (G>C) for rs3742305 was done by PCR-RFLP method. All samples were ampli ed for target DNA fragment spanning rs3742305 region.

PCR ampli cation and Gel Electrophoresis
PCR ampli cation was carried out with 26 µl of master mix containing 8 µl of Go Taq, 6 µl of water, 1µl of each forward and reverse primer and template with 10µl DNA was added. The reaction conditions were as follows 95 •C for 5 minutes, 94 •C for 1 minute 57 •C for 1 minute and fteen second and 72•C for 1 minute and thirty seconds with 40 cycles and 72•C for 7 minutes in elongation. Ampli ed product of 676bp was analyzed by electrophoresis on 2% agarose gel. 2X TBE buffer was prepared from 10X TBE buffer solution and 2 grams of agarose was added to 100 ml of 1X TBE buffer solution. For visualization of DNA, 30 µl of Ethidium Bromide was added to the gel solution. For comparing the fragment size, a 50 bp molecular marker was used (MBI Fermentas, Catalogue no SM1153). The gel was observed in gel DOC system. The PCR ampli ed product was digested with BClI (recognition site T/GATCA) restriction enzyme at 56 •C for 15 hours. The digested product was then analyzed by agarose gel and the required pattern of bands was obtained. 2 % agarose gel solution was used for the separation of DNA fragments. A 50bp ladder was used to compare the band sizes (MBI Fermentas, Catalogue no SM1153).

Statistical Analysis
The mean, frequency and standard deviation was calculated by SPSS. 16.0 statistical software (SPSS Inc. Chicago, IL, United States). Genotype, allelic frequency, P-value were calculated by Hardy -Weinberg frequency calculator. P < 0.01 considered in Hardy-Weinberg equilibrium was considered signi cant.

Results
It has been reported that HMGB1 genotypes for four SNPs, 2262G/A (rs1045411), 1177G/C (rs3742305), 3814C/G (rs2249825), and rs4540927 (16). In our study, we analyzed genotype distribution of GG, GC and CC by entering observed genotype values in the internet calculator of hardy Weinberg equilibrium. The analysis revealed that in Pakistani population we found polymorphism of rs3742305 in HMGB1 gene as represented in Table 3 and 4. Among 100 HBV patients GG and CC was homozygous genotype and GC was heterozygous genotype. The GG genotype was found in 65(60.84%) of total HBV patients, GC genotypes in 26(34.32%) while CC frequency was lower than GG and GC and detected in 9(4.84%) as shown in Table 3. PCR-ampli ed product of rs3742305 having size length of 676bp is shown in gure 1.
Allelic frequency of G was shown to be 156(78%) and for C, 44(22%) as shown in Table 4. In control individuals GG and CC was homozygous genotype and GC was heterozygous genotype. The GG genotype was found in 20(39.69%), GC in 23(46.62%) while CC frequency was lower than GG and GC and detected in 7(13.69%). Allelic frequency of G is 63% and C is 37%.Chi square and P-value calculated using Chi square and P-value calculator is 0.01 and degree of freedom is 2. Three types of rs3742305 genotypes in HBV infected patients, wild type homozygous GG, minor homozygous CC and minor heterozygous GC. Genotypic distribution of GG 65(60.84%), GC 26(34.32%) and CC 9(4.84%). Allelic frequency of G is 156(78%) and C is 44(22%) and in control individuals 20(39.69%), 23(46.62%) and 7(13.69%) for GG, GC and CC respectively. Hardy-Weinberg equilibrium by chi square test showed that Pvalue is 0.01, which con rmed that there is signi cant association between polymorphism of HMGB1 and HBV patients and studied group was in hardy-Weinberg equilibrium. Figure 2 shows the PCR RFLP assay, where the GC genotype shows band length of 676bp, 496bp and 180bp and GG shows band length of 676bp only. Frequency of three types of rs3742305 genotypes were compared in AHB, CHB and SHB patients ( gure 3) and it was shown that GG genotype was most highest in all forms of hepatitis infection and highest in CHB patients as compared to GC and CC genotypes. Next we have compared the three types of rs3742305 genotypes in control and CHB patients ( gure 4). It has been shown that only GG genotype is showing the signi cantly higher occurrence as compared to control but GC and CC genotypes show non-signi cant difference between control and CHB groups. Our data shows that HMGB1 1176 G/G genotype has progression towards HBV infection. Discussion HMGB1 is highly conserved non-histone nuclear DNA binding protein that contribute to architecture of chromatin DNA (6). It also acts as in ammatory cytokine in response to immune mediated diseases (17). In this study it is evident that most of the individuals of GG genotype having chronic hepatitis B infection (CHB) were male. It means CHB infection has association with GG genotype and CC genotype is less prevalent in population. The most of polymorphism occurs in CHB patients while in SHB infected patients, very little polymorphism occur although SHB infected patients have acute-on-chronic liver failure with reduction in liver size. The fractions calculated reveal that in HMGB1 gene 1176 G/G genotype is more susceptible to CHB with more progressive status towards HBV infection and allele G is clearly related with infection production. The more persistence of 1176 G/G genotypes in HBV patients may leads to liver carcinoma results into liver failure and CHB progress towards SHB. The homozygous 1176 C/C is less progressive then 1176 G/G while 1176 G/C have very little involvement in disease progression (14).
The uniqueness of this study is homozygous genotype which is involved in progression of disease. The homozygous GG genotype is associated with disease progression in HBV patients. In immune responses liver plays an important role. Parenchyma and non-parenchyma cells activate toll like receptors and Receptor for advanced glycation end-products. These receptors identify the HMGB1 in pathogenesis of liver disease (18)(19) Extracellular HMGB1 have critical role in activation of hepatic injury depending upon receptors and post translational modi cation (14). It implicates in several immune-mediated diseases. About six polymorphism and four mutations were rstly identi ed and HMGB1 genotypes effect on the risks of SIRS and sepsis. Many researches reveal that polymorphism in HMGB1 leads to early and late death in individuals. Association between genetic variations and disease severity are also identi ed like polymorphism in HMGB1 leads to systemic in ammatory response syndrome (SIRS) (20). The tag SNPs in the entire gene involves in dysfunction syndrome (21). The 1176G/G genotype of HMGB1 gene associates with HBV infection leads towards disease elevation and current study con rmed this association and progress status similar to work of study.

Conclusion
In conclusion, our result shows that HMGB1 1176 G/G genotype has progression towards HBV infection and have high risks towards liver cirrhosis and hepatocellular carcinoma as compare to others as 1176 C/C and 1176 G/C and it also represent that there is association of HMGB1 which is a proin ammatory mediator between 1176 G/C polymorphism of HMGB1 and hepatitis B virus infection. It shows the role of HMGB1 in pathophysiology of diseases related to HBV and in progression of HBV infection towards severity in the form of hepatocellular carcinoma. The future prospects of this study are that it provides a clue for researchers in nding the pathogenicity of HBV infection and nding of other restriction sites involve in causing of polymorphism association with HBV disease pathogenesis. It will help in risk assessment and prediction of clinical outcomes and provides a clue for diagnostics of acute and chronic hepatitis B. It will offer the counseling for precautions in life style changes to high-risk individual. The study has been approved by ethical review board of Islamic International University Islamabad.

Consent to publication:
All authors approved the submission of the manuscript for publication Availability of data and material: The data is available and can be used for the academic or research purposes.

Competing interests:
The authors have no con ict of interest.    Comparison of three types of rs3742305 genotypes in control samples and CHB patients: black bar shows GG genotype, gray bar shows GC genotype and white bar shows CC genotype.