Study design and participant selection
This was a case-control study. Two hundred and ninety-two (292) participants comprising 146 HBV infected persons as cases (excluding active hepatitis B cases) and 146 apparent healthy HBV non-infected persons as controls were recruited from the Greater Accra Region of Ghana All the participants selected were Ghanaians by birth between 18 and 65 years of age.
Sample size justification
The sample size for the study was calculated using the Cochran–Armitage trend tests . By considering the additive genetic model at a case: control ratios of 1:1, the rare allele frequency (0.079) of the most replicated SNP (rs2296651), and a national HBV average prevalence of 12.3% , the sample size needed to achieve the prespecified 0.05 α-level and a power 80 for a two-sided trend test was 348 (174 cases vs 174 controls). The total response rate of the participants was approximately 84.0% (292/348). The final population of the study constituted 292 participants comprising of 146 cases and 146 controls.
Sample collection, preparation, biochemical analysis, and DNA isolation
Nine (9) millilitres of venous blood was drawn from each participant into a 3 ml serum separator gel tube (SST) and two 3 ml EDTA tubes; thus, one for haematology and the other for DNA extraction. This was done to avoid contamination. The SST sample was allowed to clot and centrifuged at 2500 g for 7mins to obtain serum and preserved between − 16 to − 20 °C till assayed. Mindray 5-parts hematology analyzer (Mindray, Shenzhen-China, 2013) was used to quantify blood hemoglobin, blood cells and other cellular indices one EDTA sample, using the impedance, flow cytometry, and vis -spectrophotometry methods depending on the analyte or index (Mindray, 2013, 2018). HumaStar 200 Clinical chemistry analyzer (Human Diagnostics worldwide, Germany, 2014) was used to quantify the concentrations of total and direct bilirubin, total protein, albumin; and the activity of the enzymes: alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (ϒGT) and alkaline phosphatase (ALP) (Human Diagnostics worldwide, Germany, 2014, 2018). Genomic (g) DNA was isolated from the other EDTA anticoagulated sample as described by Suguna, et al. , and preserved below − 80 °C until genotyped.
Profiling of HBV antigens and HBV -host antibodies
The commercial Clinogen HBV-5 card was used to screen for the presence of two (2) HBV viral antigens and three (3) host antibodies using colloidal gold and membrane chromatography technology (Clinogen Diagnostics, Japan) after ‘running’ HBV profile on ELISA-confirmed controls previously obtained from the Noguchi Memorial Institute for Medical Research (NMIMR), Legon, Accra. HBsAg, HBeAg, and HBsAb were screened for with the Immunochromatographic (dual-antibody sandwich) principle, and the HBeAb and HBcAb were screened for by the neutralization competitive inhibition principle (Clinogen Diagnostics, Japan; Clinogen Diagnostics, UK).
Primers spanning each of the SNP variants (rs2296651, rs61745930, and rs4646287) were designed using the NCBI Primer-BLAST software and the results shown in Table S1 (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi). Table S1 shows PCR primers and their characteristics restriction enzymes.
To detect the presence of the rs2296651 (about 200 bp, 80), rs61745930 (about 400 bp, 250 bp), and rs4646287 (about 140 bp, 250) fragments, a 25 μL PCR reaction volume consisting of 8.5 μL nuclease-free water, 1 μL (10 μM) each of forward and reverse primers, 12.5 μL of One Taq 2x PCR Master mix (New England BioLabs (NEB), USA) and 2 μL of the isolated genomic DNA was prepared. Nuclease free water was used in place of the gDNA in the negative control. PCR amplification was done using the Applied Biosystems thermal cycler (Fisher Scientific, USA). Denaturation was completed at 940 C for 3 mins (initial) and 940 C for 30 s (on rest). Annealing conditions for rs2296651, rs61745930 and rs4646287 were 580 C (30 s), 59.50 C (35 s) and 60.00 C (30 s), respectively. Initial and final extension were completed at 720 C (30 s) and 720 C (10 mins), respectively. The denaturation, annealing and extension steps were done for 35 cycles, and the hold after the final extension was at 4 °C.
The optimal enzyme concentration was prepared with the appropriate assay buffer containing 1 μg of substrate DNA according to Manufacturer’s instructions. Controls were included to ensure there was no contamination or unspecific product obtained. To complete the genotyping of rs2296651, 15 μL of the PCR product was added to 0.2 μL of HphI enzyme (NEB, USA) and dissolved in 1.5 μL of NEB buffer and 3.3 μL of nuclease-free water, and incubated at 37 0 C for 11 min for the enzyme digestion of the PCR product. Similarly, 15 μL of the PCR product was either added to 0.2 μL of BsaBI or 0.2 μL of TaqἀI enzymes (NEB, USA) and dissolved in 1.5 μL of NEB buffer and 3.3 μL of nuclease-free water and incubated at 60 0 C for 60 min (1 h) for the enzyme digestion of either rs61745930 or rs4646287.
The digested fragments were separated with a 2.5% EtBr- incorporated agarose gel at 100 V, 2A for 90 min, and using the Quick-Load purple 100 bp DNA Ladder (NEB, USA) as the molecular marker (MM) and visualized under UV trans-illuminator. The genotypes were determined according to the band patterns/sizes and in comparison, to the molecular marker (MM) as shown in the supplementary figures. Figure S1, Figure S2 and Figure S3 show the gel bands for the SNP rs2296651, rs61745930, and rs4646287, respectively.
Results obtained were entered into the Statistical Package for the Social Sciences (SPSS), coded, and analyzed using this SPSS (version 23.0). Frequencies were used to represent categorical data and compared using Chi-Square test analysis to compare the genotype and allele frequencies between the groups. Skewed data were compared using the Man-Whitney Test. Normally distributed data were represented with mean ± standard deviation and compared between groups using the T-test. To test for associations between HBV and every single SNP, logistic regression models were fitted, in which each SNP was presented as a predictor variable whose values were equal to the number of copies of the minor allele (0, 1, 2) in an additive model, or presence of at least one copy of the minor allele (0, 1) in a dominant model or presence of two copies of the minor allele (0, 1) in a recessive model. Sex, age and family history of HBV status were included as covariates in the fitted model. The structure of the model was represented as: Logit [pr(D = 1)] = α + β 1 G.
Where D denotes HBV phenotype; G denotes SNP coded as an additive, dominant or recessive; β denotes the corresponding coefficient for each variable in the model, and its exponential is the corresponding odds ratio. A P-value of less than 0.05 was considered statistically significant.