Analysis of MDR1 Polymorphisms Among HIV-Infected Individuals on ARV Therapy from Western India

Background: MDR1 is involved in the transport of numerous drugs. Polymorphism of MDR1 is linked with the treatment outcome. ARV regimen is being used to manage the progression of HIV infection. Ethnic disparities have been observed in the distribution of MDR1 genotypes. Methods: MDR1 polymorphism (1236 C/T, 3435 C/T) was genotyped in 34 individuals with ARV-associated hepatotoxicity, 131 HIV-infected, and one-fty-ve healthy by utilization of PCR-RFLP. Results: Haplotype TC exposed the greater risk for hepatotoxicity severity when compared between individuals with hepatotoxicity and HIV infected (OR=1.96, P=0.06). While haplotypes TT and CC bared a reduce risk for hepatotoxicity severity (OR= 0.16, P=0.006; OR= 0.46, P=0.06). Haplotype TT and CC displayed a decrease risk of hepatotoxicity severity while compared between individuals with hepatotoxicity and healthy (OR=0.09, P=0.003; OR=0.34, P=0.03). A higher occurrence of MDR1 1236TT genotype was seen among patients with hepatotoxicity who consumed alcohol (28.6% versus 14.8%, OR=1.50). In patients with hepatotoxicity taking nevirapine, there was an increased incidence of MDR1 1236TT genotype in contrast with efavirenz (21.7% versus 9.1%, OR=2.11). In HIV-infected people taking nevirapine, MDR1 1236CT, 1236TT genotypes found to be increased compared with efavirenz (43.7% versus 33.3%, OR=1.66; 12.6% versus 8.3%, OR=1.96). A higher occurrence of MDR1 1236TT genotype has happened in hepatotoxicity cases having both alcohol and nevirapine (40.0% versus 16.67%, OR=2.21). N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes.


Background
Antiretroviral (ARV) regimen is being extensive utilized for the management of HIV patients, the death rate among individuals with HIV is constantly increasing. Long-term e cacy and toxicity are signi cant issues of worry with the ARV regimen. Hepatotoxicity is an adverse outcome of ARV drugs in HIV-infected individuals [1,2]. A higher occurrence of hepatotoxicity was seen with the utilization of nevirapine-based regimen than the efavirenz-based regimen [3]. The shown occurrence of hepatotoxicity because of nevirapine was 3.19% [4]. In another investigation, the stated occurrence of grade 3 or 4 hepatotoxicity had 10.8% in the patients treated with efavirenz and 8.9% in patients treated with nevirapine (5). ABCB1 is one of the universal adenosine triphosphate (ATP)-binding cassette (ABC) genes is responsible for cell homeostasis [6][7][8].
ABCB1 gene is situated on chromosome 7q21. It is a part of the MDR subfamily [8]. ABCB1 protein is expressed in a few tissues including epithelial cells of the blood-brain barrier [9,10] and has appeared to transport numerous drugs [11]. P-glycoprotein (P-GP), a transmembrane transporter protein, is encoded by the MDR1.P-gp system of ATP-subordinate e ux transports outside particles including drugs from intracellular to the extracellular matrix [12][13][14]. The variation in P-gp expression may vary the function, thus affect the transport of nevirapine (NVP) [15]. The absorption and penetration of efavirenz (EFV) and NVP are impacted by the P-gp expression [16]. Chelule et al., (2003) have shown the commonness of MDR1 3435CC genotype had 85.9% in Africans, 41.70% in Indians, and 35.7% in whites of Kwazulu-Natal, South Africa, respectively. In the African population, the predominance of the MDR1 3435CC genotype showed overexpression of P-glycoprotein, while in patients with TT genotype it was demonstrated a lower expression of P-GP [17,18]. Studies have been reported a signi cant increase of CD4 cell counts with MDR1 3435TT genotype in HIV patients [19,20]. Haas et al., (2005) proposed that MDR1 3435C/T polymorphism was not linked to efavirenz exposure [19]. Salem et al., (2014) suggested that MDR1 3435C/T polymorphism had no impact on efavirenz clearance [21]. Zhu et al., (2013) proposed that polymorphisms of MDR1 3435T/C and 2677T/G was linked to the response of nevirapine treatment (P = 0.031, P = 0.001) and could help to predict the drug response in HIV patients [22]. MDR1 3435TT genotype among individuals treated with EFV or nel navir was connected with a more elevated level of CD4 + count than the CT/CC genotypes [22]. Leschziner et al., (2007) recommended that MDR1 3435 TT genotype was linked with higher adverse outcome of 3TC and NVP treatment than EFV treatment [23]. Ritchie et al., (2006) indicated a signi cant reduced risk of hepatotoxicity with NNRTI-containing regimens in the presence of MDR1 3435C/T polymorphism [24]. The link between polymorphism of MDR1 3435C/T and nevirapine induced hepatotoxicity was documented by studies [24,25].
Few studies have shown a link between MDR1 polymorphism and adverse outcome of ARV drug and other studies do not suggest the link. Moreover, the link between MDR1 polymorphism and ARV-related hepatotoxicity has not been examined from India. Thus, we analyzed the relationship between MDR1 (1236C/T and 3435C/T) polymorphism and hepatotoxicity induced by ARV regimen.

Subjects
A case-control design was undertaken between August 2014 to September 2017. The number of individuals with HIV infection experienced a liver function test (LFT) was one hundred sixty-ve. Out of which, thirty-four individuals of hepatotoxicity and one thirty-one individual with no hepatotoxicity were a rmed and one hundred fty-ve healthy people age-matched were recruited. The patients were enlisted from the clinic of the National Institute of AIDS Research, Pune. Tuberculosis, Hepatitis B and C, immune reconstitution syndrome, untreated opportunistic infections, and other known hepatotoxic medications have been excluded from the hepatotoxicity case group. At the same time, one hundred fty-ve people (those from a similar family have been excluded) lacking diseases for example, TB, Hepatitis B, C, and HIV, have been recruited as controls. Clinical information was noted through the reviews of case records, questionnaires, and personal interviews. The status of the liver enzymes was assessed by the utilization of LFT. In patients with hepatotoxicity, males with SGOT > 93.8 U/ml, Alkaline phosphatase > 550.8 U/ml, total bilirubin > 3. 22 mg /ml, SGPT > 229.5 U/ml and females with SGOT > 163.2 U/ml, Alkaline phosphatase > 550.8 U/ml, total bilirubin > 3. 22 mg /ml, SGPT > 173.4 U/ml were considered. In HIV-infected male and female controls, SGOT < 32 U/ml, Alkaline phosphatase < 108 U/ml, total bilirubin < 1.
24 mg /ml, and SGPT < 34 U/ml were considered. FACS analysis was utilized to estimate the CD4 + count. The stages of patients were characterized based on current CD4 status. CD4 + cell count ranges from < 200 cells/mm3, 201-350 cells/mm3, and > 350cells /mm3 were considered as advanced, intermediate, and early stage of HIV infection. HBsAg and hepatitis C testing was completed by ELISA with the Ortho HCV ELISA test system. A questionnaire was utilized to record the usage of tobacco and alcohol. The ethical endorsement was taken from the Ethics Committee, National AIDS Research Institute, Pune, India (Reference number: August 28, 2013, EC/NARI/Genetic Susceptibility/13-14/146) and written consents has been taken from every single quali ed subject.

Extraction of DNA
The collection of 2 ml peripheral blood was done from all subjects and put away at − 70 0 C until DNA extraction. The DNA extraction has been completed from blood leukocytes pellets utilizing the QIAamp DNA Mini Kit according to the guidance of the company.

Genotyping
Restriction fragment length polymorphism (RFLP) analysis was utilized to genotype the MDR1 (1236 C/T and 3435 C/T) polymorphism. Primers to amplify the MDR1 (C1236T and C3435T) polymorphism were utilized as designated by the previous report [26]. PCR was performed in a volume of 20 µl. The PCR conditions for ampli cation of MDR1 C1236T and C3435T polymorphisms were used as described previously [27]. A thermocycler was utilized to complete all the reactions. PCR products were visualized by ethidium bromide staining. The PCR products of MDR1 C1236T and C3435T polymorphism was digested by utilization of restriction enzymes HaeIII and MboI at 37 °C for 16 hours separately. 10% polyacrylamide gel along with molecular weight markers was utilized to the genotype of MDR1 C1236T and C3435T polymorphism. The sequences and location of SNP were employed for genotyping of MDR1 C1236T and C3535T polymorphisms. Genotypes were: for MDR1 C1236T: 93 and 87 bp for CC, 87, 58, and 35 bp for TT, and 93, 87, 58, and 35 bp for CT, for MDR1 C3435T: 130 and 76 bp for CC, 206 bp for TT and 206, 130, and 76 bp for CT. Additional staff of the laboratory was done re-genotyping in 20% samples to check the disparities in genotyping. The genotyping mistake was cross-veri ed by DNA sequencing in 10% of samples.

Statistical examination
The age variable was communicated as mean ± standard deviation (SD). Hardy-Weinberg equilibrium was examined by the utilization of Chi-Square Goodness -of -t test in healthy people. Fisher's exact test was utilized to analyze the genotype frequencies between groups. Logistic regression was utilized to compute the odds ratios (ORs) and 95% con dence interval (CI). SPSS (SPSS Inc) programming form 17.0 was utilized for statistical examination and the two-sided value was taken as a test of statistical signi cance. A p-value under ≤ 0.05 was considered for signi cance. SNPStats online software was utilized to compare the frequency of haplotypes among groups (25). Linkage disequilibrium (LD) was analyzed between both the loci by computing the relative LD value (D') as D' = Dij/ Dmax (28). The Dij value was compared between various groups by the comparison of con dence intervals. 3.0

Results
An aggregate of 165 individuals of HIV infection incorporating thirty-four individuals with hepatotoxicity, one-thirty-one HIV-infected and one-fty-ve healthy were used to analysis the polymorphism of MDR1. The mean age ± SD of individuals with hepatotoxicity, HIV infected, and healthy have been 37.24 ± 3.29, 40.27 ± 2.45, and 37.25 ± 6.30 years. The demographic pro les of the participants are outlined in Table 1.

MDR1 polymorphism in HIV-infected people
The occurrence of polymorphisms of MDR1 (1236C/T, 3435C/T) in people with HIV infection and healthy is introduced in Table 3. The deviation from Hardy-Weinberg equilibrium in healthy people was investigated for MDR1 polymorphism, we found that it was followed (P = 0.36, 0.13). The distribution of MDR1 polymorphism was alike between HIV-infected and healthy people. HIV-infected people had more occurrence of MDR1 3435TT genotype than healthy people (43.5% versus 34.83%, OR = 1.24, 95%CI: 0.59-2.61, P = 0.57). The dispersion of other genotypes and alleles of MDR1 polymorphisms were comparable between both groups.

Haplotypes distribution
We have likewise investigated the occurrence of MDR1 haplotypes among people of hepatotoxicity, HIVinfected, and healthy people. Haplotype CT (1236*C/3435*T) was considered as a reference. Haplotype occurrence has appeared in Table 4.

Gene-environment interaction
The dispersion of MDR1 polymorphism between individuals with hepatotoxicity and HIV infected utilizing tobacco, alcohol, and NNRTI regimen and nonusers appeared in Tables 6-9. The occurrence of polymorphisms of MDR1 (1236C/T and 3435C/T) was not varied between tobacco utilizing people with hepatotoxicity, HIV infected versus nonusers. MDR1 1236TT genotype was overrepresented in tobacco utilizing hepatotoxicity patients than nonusers (28.6% versus 14.8%) (    NS, not signi cant. N = number of subjects, (%) = frequency of subjects, OR and 95% CIs were derived from logistic regression model comparing the homozygous wild-type genotype/allele (CC genotype and C allele for MDR1 1236 C/T and 3435 C/T) with other genotypes.

Discussion
We analyzed the relationship between MDR1 polymorphism and ARV-related hepatotoxicity from Western India. MDR1 encodes for the ATP-dependent membrane e ux transporter (14). The genetic variants that impact patients drugs are substrates of P-glycoproteins. The occurrence of MDR1 polymorphism changes from the population to the population [17]. We analyzed the MDR1 genotypes and haplotypes between individuals infected with HIV and hepatotoxicity.
The occurrence of MDR1 3435C/T polymorphism in our healthy people was identical to the investigations done in European, North India, Turkish, and Asian populations [29][30][31][32][33][34] and contrasted with the examinations did in Chinese, Iranian and Thailand populations [26,32,35]. The genotypic dispersal of MDR1 1236C/T polymorphism in our healthy people was almost alike to the investigation announced from North India [34], however, contrasted with the examinations shown from Indians, Mexicans, Chinese and South Africans populations [18,30,37,38]. We have done the genotype-phenotype analysis and found that the MDR11236TT genotype was displayed a hazard for hepatotoxicity severity (OR = 1.37, P = 0.57). However, due to the small sample size in the hepatotoxicity group, the risk could not reach statistical signi cance. The low phenotypic expression is dictated by common polymorphisms inside Pgp. People with 3435TT genotype were connected with lower levels of P-gp than CC and CT genotype. MDR1 3435C/T polymorphism was related with the reduced risk of NNRTI-induced liver toxicity [24].
We have examined the relationship of gene-gene interaction to understand the additive impact of MDR1 polymorphism on ARV-related hepatotoxicity. The gene-gene collaboration had a greater effect on gene expression than a single gene [39]. In our investigation, haplotype TC was shown with a greater risk for the severity of hepatotoxicity (OR = 1.96, P = 0.06). While, haplotypes TT and CC were linked with reduced risk of severity of hepatotoxicity (OR = 0.16, P = 0.006; OR = 0.46, P = 0.06; OR = 0.09, P = 0.003; OR = 0.34, P = 0.03). It is likely that individuals with haplotype TC may have prone to the severity of hepatotoxicity, whereas haplotypes TT and CC may have reduced risk for hepatotoxicity severity.
Likewise, we analyzed the relationship between the MDR1 genotype and the stage of HIV infection. In our investigation, the prevalence of MDR1 genotypes did not signi cantly vary between people of various stages of HIV and healthy. MDR1 1236CT, 1236TT, and 3435CT genotypes were correlated with the HIV disease progression, however, these polymorphisms did not regulate the susceptibility of HIV-1 [40].
Following the treatment of a half year, patients with 3435 TT genotype had raised the CD4 + count [22].
Analysis of the relationship of gene environment was done to take a look at the impact on the etiology of the disease [41,42]. We had selected a case-only analysis. We did not examine the case-control in light of the fact that in the case-control investigation, cases must be coordinated with the controls in the population [43]. Hence, we utilized the case-only analysis. HIV patient's naïve ART utilizing alcohol has demonstrated a reduction in CD4 + cell count [44]. In women with HIV disease and tobacco utilization, ART response was seen to be diminished [45]. In our examination, the patients of hepatotoxicity utilizing alcohol with MDR11236TT genotype exposed a hazard for severity of hepatotoxicity (OR = 1.50, P = 0.88), while individuals of HIV contamination using alcohol with 3435CT genotype were at higher danger of HIV disease progression (OR = 2.47, P = 0.12). In hepatotoxic patients utilizing nevirapine and MDR1, 1236TT genotype demonstrated a higher threat for the hepatotoxicity severity (OR = 2.11, P = 0.55).
Individuals with HIV infection and utilizing nevirapine with MDR1 1236CT, 1236TT genotypes exposed a risk for the progression of HIV infection (OR = 1.66, P = 0.45; OR = 1.96, P = 0.55). In people with hepatotoxicity who took both alcohol and nevirapine, MDR1 1236TT genotype exposed the higher vulnerability for hepatotoxicity severity (OR = 2.21, P = 0.55). In people having an infection of HIV with MDR1 3435CT genotype and taking both nevirapine and alcohol showed a vulnerability for the progression of HIV infection (OR = 2.04, P = 0.23). This suggests that individuals with MDR1 1236TT and 3435CT genotypes having HIV infection or ARV-related hepatotoxicity have an additive effect on vulnerability of hepatotoxicity severity and progression of HIV disease. Individuals with 3435 T allele having infection of HIV taking nevirapine were connected with the reduced risk of hepatotoxicity [25].
People with MDR1 1236T and 1235T alleles were related to diminished plasma NNRTI concentration in uencing the virological response to HAART [21]. Haas et al., (2005) recommended no signi cant relationship between ABCB1 variations and plasma EFV concentrations [19]. This work has a few limit points, it can just assess association and not indicate causation. In the beginning, the present investigation was planned for a 1:4 proportion of case controls. However, we were not able to complete to select a similar proportion of controls. Though, we recruited a 1:3 proportion which may be su cient.

Conclusions
MDR1 haplotypes may have impact on hepatotoxicity severity. Individual with MDR1 1236TT and 3435CT genotypes in presence of alcohol and nevirapine had an additive effect for vulnerability of severity of hepatotoxicity and progression of HIV disease.
MDR1 is associated with drug clearance. MDR1 expression differed in response to NVP and EFV administration. Hence, further examination of the relationship between MDR1 polymorphism and plasma drug concentration would be done with a bigger sample size in different populations. In addition, the correlation of polymorphisms of other drug transporter genes with plasma drug levels is required to comprehend the effect of genetic variants on treatment effect.