ApaI VDR Polymorphism as a Risk Factor of Treatment Failure in Chronic Hepatitis C Patients

Noora Hassan Hezam Al-Aqmer1*, Zain Aamir2, Muhammad Farooq Hanif3, Soumble Zulfiqar4, Sibgha Zulfiqar1, Mateen Izhar5 and Abdul Rauf Shakoori4* 1Department of Physiology, Shaikh Zayed Postgraduate Medical Institute, Shaikh Zayed Medical Complex, Lahore, Pakistan 2Primary and Secondary Healthcare Department, Lahore, Pakistan 3Gastroenterology and Hepatology, Pakistan Kidney and Liver Institute and Research Center, Lahore, Pakistan 4School of Biological Sciences, University of the Punjab, Lahore, Pakistan 5Department of Microbiology, Shaikh Zayed Postgraduate Medical Institute, Shaikh Zayed Medical Complex, Lahore, Pakistan Article Information Received 12 June 2021 Revised 19 October 2021 Accepted 27 October 2021 Available online 16 November 2021

INTRODUCTION H epatitis C virus is an important world-wide health problem (Stanaway et al., 2016). It is a main cause of cirrhosis, hepatocellular carcinoma, and mortality (Perz et al., 2006). Hepatitis C virus is also shown to cause complications beyond the liver such as lymphoma, diabetes, and chronic renal disease (Younossi et al., 2016). It is estimated that approximately 71 million individuals are infected with hepatitis C virus worldwide (Polaris Observatory HCV Collaborators, 2017). More than 50% of HCV infections are in China, Pakistan, Egypt, Nigeria, Russia, and India (Gower et al., 2014).

*
Corresponding author: arshakoori.sbs@pu.edu.pk, drnooraalaqmer@gmail.com 0030-9923/2021/0001-0001 $ 9.00/0 Copyright 2021 Zoological Society of Pakistan In Pakistan, the prevalence of hepatitis C virus is the 2 nd highest in the world (Hill et al., 2017;Abbas and Abbas, 2020) and is about 5% nationwide (Al Kanaani et al., 2018) which is persistently high without evidence of a decline since three decades (Mahmud et al., 2019) and the prevalence in rural areas and peri-urban areas is up to 25% (Umer and Iqbal, 2016). In Pakistan, genotype 3a is common (Haqqi et al., 2019) and to achieve WHO target of elimination of hepatitis C by 2030, treatment has to be provided to a million hepatitis C infected patients yearly (Altaf and Pasha, 2020).
Vitamin D receptors are hormonal receptors in the nucleus of the cell. They are ligand-activated regulatory proteins that direct the transcription machine to specific genomic sites to influence RNA production and therefore encoding proteins which are important for specific biological functions (Pike and Meyer, 2012). Vitamin D receptor is involved in different physiological processes O n l i n e

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and that include cell differentiation and proliferation and immune modulation (Adams and Hewison, 2008). Several vitamin D receptor single nucleotide polymorphisms were found to be associated with the risk of hepatitis C infection , progression of the disease (Baur et al., 2012) as well as with the response to treatment (Garcia-Martin et al., 2013;Al-Aqmer et al., 2021). Baur et al. (2012) found ApaI vitamin D receptor polymorphism to be inversely associated with the response to pegylated interferon with ribavirin and considered it as risk factor for failure of treatment. However, there were studies which reported no association between ApaI vitamin D receptor polymorphism and the response to treatment (Arai et al., 2015;Abdelsalam et al., 2016;Wang et al., 2016;Thanapirom et al., 2019).
As no study was conducted specifically on hepatitis C genotype 3 patients receiving directly acting antiviral treatment, this study aimed to find out the association of ApaI vitamin D receptor polymorphism with the response to directly acting antiviral treatment in hepatitis C genotype 3 Pakistani patients.

MATERIALS AND METHODS
This case control study was conducted after approval from the Ethical Review Board of Pakistan Kidney and Liver Institute, Lahore and included 66 responders who received hepatitis C antiviral treatment, daclatasvir and sofosbuvir (with ribavirin in case of cirrhotic patients), and achieved a sustained virologic response (HCV-RNA negative) three months after completing the treatment, males and females ≥ 18 years in age, and were matched in age and gender with 66 non-responders who received the same treatment and did not achieve a sustained virologic response (HCV-RNA positive) three months after completing the treatment. Patients with advanced liver disease, renal disease, hepatitis B, and HIV were excluded. After written consent was taken from the participants, demographic data and reports of hemoglobin, liver function tests, platelet count, prothrombin time, and INR were recorded. About 5 mL of blood was drawn for DNA extraction followed by polymerase chain reaction (PCR) of the DNA sequence containing the ApaI restriction sites (rs7975232), restriction fragment length polymorphism analysis and gel electrophoresis.
Using the kit (Thermo Scientifc #K0781), DNA extraction was done followed by PCR using the primers.
Restriction fragment length polymorphism (RFLP) analysis for ApaI was done using the enzyme Thermo Scientific ApaI (#ER1411). The 30 µL mixture contained 10 µL PCR product (0.1-0.5 µg), 2 µL 10X buffer B, 2 µL ApaI enzyme, and 16 µL nuclease free water. The mixture was incubated at 37°C for 8-16 h. Samples were run on 2% agarose gel and visualization was done under ultraviolet light and stored in the documentation system.

Statistical analysis
Data was analyzed using SPSS 24. T-test (for normally distributed data) and Mann-Whitney test (for not-normally distributed data) were used to compare age, BMI, hemoglobin, liver function tests, platelet count, prothrombin time and INR in cirrhotic and non-cirrhotic patients. Frequencies of ApaI polymorphisms were studied in accord with the Hardy-Weinberg equilibrium. Chisquare test was used to assess the association of vitamin D polymorphisms with the response to treatment and with cirrhosis. Logistic regression was used to find out the association of ApaI and other independent variables with the response to treatment. A p-value of ˂ 0.05 was considered significant.
The restriction fragment length polymorphism analysis showed a single band of 744 base pairs in the AA wild homozygous genotype, three bands of 744, 527, and 217 base pairs in the Aa heterozygous genotype, and two bands of 527 and 217 base pairs in the aa homozygous mutant genotype (Fig. 1).
The frequencies of the AA, Aa, and aa ApaI genotypes were 28 (42.4%), 27 (40.9%), and 11 (16.7%) in responders and 22 (33.3%), 26 (39.4%), and 18 (27.3%) in non-responders. The allelic distribution for the "A" and "a" alleles was 83 (62.9%) and 49 (37.1%) in responders and 70 (53%) and 62 (47%) in non-responders. Logistic regression showed ApaI genotype ''aa'' as a significant predictor of treatment failure (p-value= .024, OR= 3.589, 95% CI= 1.181-10.911) ( Table I).  There was no association of ApaI VDR polymorphism with cirrhosis in responders and non-responders (Table  II). The frequencies of ApaI VDR genotypes in males and females are shown in Table III and no significant difference was seen in the frequencies of ApaI VDR genotypes between males and females in both responder and non-responders  Significant differences were found in the levels of total bilirubin, direct bilirubin, ALP, serum albumin, PT, and INR between cirrhotic and non-cirrhotic patients in both the groups, responders and non-responders, and in the AST levels between cirrhotic and non-cirrhotic patients in the non-responder group (p-value ˂ .05) (Tables IV and  V).

DISCUSSION
Our study found the mutant homogenous ApaI genotype aa to be a risk factor and a predictor of treatment failure (OR= 3.589, 95% CI= 1.181-10.911). The mutant homogenous ApaI aa genotype was also found to be a risk factor for treatment failure by Baur et al. (2012) (OR=2.67, 95% CI= 1.24-5.70). They found ApaI polymorphism to be associated with failure to pegylated interferon and ribavirin treatment.
Contrary to our findings, Thanapirom et al. (2019) found no association between ApaI polymorphism and the response to treatment; however, this could have been due to the presence of HCV genotypes 1, 2, 3, and 4 patients O n l i n e

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ApaI VDR Polymorphism as a Risk Factor of Treatment Failure in Chronic Hepatitis C Patients in their study whereas our study included only genotype 3 hepatitis C patients and the hepatitis C genotype could influence the response to treatment and might interact with the pharmacokinetics of the different drugs. Wang et al. (2016) found no association between ApaI VDR polymorphism and the response to treatment in HCV Chinese patients and similar results were reported by Abdelsalam et al. (2016) in genotype 4 Egyptian patients. In all these studies, the used antiviral treatment was interferon and ribavirin whereas in our study the treatment was directly acting antiviral treatment. The difference in the results could be due to pharmacogenetics, ethnicity, or the different HCV genotypes as the three factors do affect the response to treatment.
The association of ApaI polymorphism with failure to treatment (daclatasvir and sofosbuvir with ribavirin in cirrhotic patients) could be explained in view of the effect of ApaI polymorphism in forming a VDR protein that is less active and might cause a disturbance in the balance of T helper cell type 1/ T helper cell type 2, thereby causing diminished activity of the signaling pathways of vitamin D (Triantos et al., 2018).
This study is the first on the association of ApaI polymorphisms with the response to directly acting antiviral treatment i.e., daclatasvir and sofosbuvir (with ribavirin in cirrhotic patient) in chronic hepatitis C genotype 3 patients. The previous studies were conducted on patients who received pegylated interferon and ribavirin and none was specific to hepatitis C genotype 3 patients. ApaI VDR polymorphism could be considered as a new marker to predict the failure of treatment in chronic hepatitis C patients.

CONCLUSION
ApaI VDR genotype aa is a predictor of failure to treatment in chronic hepatitis C genotype 3 patients and can be considered as a risk factor for treatment failure.