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Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis
Tuberculosis xxx (2015) 1e6
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Tuberculosis journal homepage: http://intl.elsevierhealth.com/journals/tube
MOLECULAR ASPECTS
Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis Alberto Rodríguez-Castillo a, 1, Alma Y. Arce-Mendoza a, Armando Quintanilla-Siller a, Jose Adrian Rendon b, Mario C. Salinas-Carmona a, Adrian G. Rosas-Taraco a, * n, Monterrey 64460, Mexico Departamento de Inmunología, Facultad de Medicina y Hospital Universitario, Universidad Autonoma de Nuevo Leo n, Prevencio n y Tratamiento de Infecciones Respiratorias) Hospital Universitario, Universidad Autonoma de Nuevo Leon, CIPTIR (Centro de Investigacio Monterrey 64460, Mexico
a
b
a r t i c l e i n f o
s u m m a r y
Article history: Received 25 August 2014 Received in revised form 30 March 2015 Accepted 8 April 2015
Human P-glycoprotein (P-gp) is a membrane transporter encoded by ABCB1 (also known as MDR1) that plays a critical role in pharmacokinetics of many unrelated drugs. Rifampin (RMP) and ethambutol (ETB), two anti-tubercular agents, are substrates of P-gp. Single nucleotide polymorphisms (SNPs) in ABCB1 have been associated with resistance to several drugs; however, their association with RMP and ETB resistance in tuberculosis patients has not yet been studied. Genotype/allele frequencies in C1236T, G2677T/A and C3435T SNPs of ABCB1 were obtained from 99 tuberculosis patients susceptible or resistant to RMP and ETB (NoRER or RER). 2677G>A allele prevalence was found to be significantly higher in the RER group compared to NoRER (5 resistant vs 2 non-resistant patients, P < 0.01; OR, 11.0; 95% CI, 2.00e56.00). No differences were found in genotype/allele frequencies in C1236T and C3435T SNPs of ABCB1 and resistance to RMP and ETB in tuberculosis patients (P > 0.05). The present study suggests the 2677G>A allele of ABCB1 could be associated with simultaneous resistance to RMP and ETB in pulmonary tuberculosis patients. Further studies with larger sample sizes are needed to confirm this association and explore its nature. © 2015 Elsevier Ltd. All rights reserved.
Keywords: P-glycoprotein Polymorphisms Drug-resistant tuberculosis SNPs
1. Introduction Pulmonary tuberculosis (TB) is the most frequent form of infection caused by Mycobacterium tuberculosis (Mtb). Nine million new TB cases are reported worldwide and two million people die each year due to this disease [1]. Combination treatment with rifampin (RMP), isoniazid (INH), ethambutol (ETB) and pyrazinamide (PZA) is the standard and generally effective therapy for new TB cases. Nevertheless, in last decades Mtb strains resistant to anti-tubercular agents have been emerging [2e4]. Multidrug-resistant TB (MDR-TB) is caused by Mtb strains resistant to at least INH and RMP simultaneously [5]. MDRTB represents a world health problem. Its burden differs among
* Corresponding author. Tel.: þ52 81 83294211; fax: þ52 81 833310 58. E-mail addresses: [email protected], [email protected] (A.G. Rosas-Taraco). 1 Alberto Rodríguez Castillo: Dept. IV, Max Planck Institute Present address. Jose for Heart and Lung Research. Parkstrasse 1, 61231 Bad Nauheim, Germany.
countries, however, rates as high as 28.9% for new TB cases and 51.5% for previously treated cases have been reported [6]. Mutations in several genes of Mtb have been documented to be associated to drug-resistant phenotypes in bacterial cultures. Additionally, accumulation of such mutations may lead the bacteria to acquire a multidrug-resistant phenotype [3]. Positive selection of resistant strains is known to occur as consequence of environmental factors, which are related to repeated exposition to antibiotic therapy, and inadequate or insufficient treatment [5]. Although the involvement of bacterial genetics in the genesis of MDR-TB has been relatively well explored, and several authors have investigated the role of host genetics on Mtb infection [7,8], much less is known about host genetic factors on the development of resistant forms of TB. In this regard, Takahashi et al. reported a possible association between a variant of the gen SLC11A1 and MDR-TB [9]. Human P-glycoprotein (P-gp) is an extensively studied ATPdependent pump that actively transports several different molecules out of the cell [10]. Its most evident function is protection of cells from the toxic effect of some endogenous compounds and xenobiotics [11,12]. P-gp is expressed on apical surface of some
http://dx.doi.org/10.1016/j.tube.2015.04.004 1472-9792/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Rodríguez-Castillo JA, et al., Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.04.004
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epithelia, kidney, bloodebrain barrier and phagocytic cells as macrophages (main target of Mtb) [9,13,14]. In addition, P-gp plays a critical role in pharmacokinetics of some drugs [15]. ABCB1/MDR1 polymorphisms affect the expression of P-gp and/or its activity against some cardiovascular, anti-cancer, central nervous system drugs, etc. [16e20]. A study demonstrated that two of the first line drugs for tuberculosis treatment, RMP and ETB, are substrates of Pgp [21], setting up the rationale for considering P-gp as an interesting candidate for human genetics' involvement in the generation of a fraction of the MDR-TB cases. Up to date, there are no studies that explore whether P-gp polymorphisms are associated with drug-resistant TB. In this study, we investigated whether polymorphisms in ABCB1 may be associated with higher risk to develop resistant forms of TB. We explored for associations between alleles, genotypes and haplotypes of C1236T, G2677T/A and G3435T polymorphisms and resistance to any of four first line drugs INH, RMP, ETB and streptomycin (SM), focusing on MDR-TB and simultaneous resistance to RMP and ETB. 2. Materials and methods 2.1. Participants From 2010 to 2013, a total of 122 consecutive patients who had culture proven TB and attended the TB Clinic of the University Hospital of Monterrey (A referral center for MDR-TB cases from north-east Mexico) were evaluated. The ethics committee approved this study and written informed consent was obtained from all participants. Only individuals with positive Mtb cultures and results of drug sensitivity tests (DST) were recruited. DST performed to samples from all patients tested for resistance to INH, RMP, ETB and SM. Subjects with extra-pulmonary infection and those co-infected with HIV were excluded. Demographic data was recorded and a peripheral blood sample was obtained from each patient. Finally, samples that presented genotyping problems were discarded, ending up with 99 participants for analysis (Figure 1). Patients were allocated in 4 non-mutually exclusive study groups according to their DST results: MDR, NoMDR, RMP-ETB resistant (RER) and NoRER. Information about patients' resistance status, risk factors and co-morbidities was obtained from their medical records.
Figure 1. Flow diagram of patient selection.
was performed under the following conditions: Initial denaturation step for 5 min at 94 C, 40 cycles of denaturation at 94.5 for 45 s, annealing at 63 C for 30 s and extension at 72 C for 30 s. All restriction enzymes were from New England Biolabs (Ipswich, MA). Each PCR product was separately digested with RsaI or BseYI. Fragments were run in 3% agarose gel and stained with ethidiumbromide or RedGel™ (Biotium, Hayward, CA). Products with restriction patterns matching TA or AA genotypes were also doubledigested with NlaIV and RsaI in order to discriminate between them.
2.2. Genotyping of C1236T, C3435T and G2677T/A polymorphisms 2.3. Statistical analysis Genomic DNA isolation was performed from 0.5 ml of EDTAanticoagulated blood using the TSNT technique. DNA samples were stored at 20 C until their use. Real time PCR was performed using Taqman® technology for C1236T [rs1128503] and C3435T [rs1045642] genotyping (Assay IDs: C___7586662_10 and C___7586657_20 respectively). PCR conditions for C1236T were as follows: Initial denaturation step by 10 min at 95 , 40 cycles of 15 s at 95 C and annealing-extension step at 60 C for 60 s. PCR conditions for C3435T were 10 min at 95 , 40 cycles of 30 s at 95 C and annealing-extension step at 60 C for 90 s. Both reactions contained 10 ml of IQ Super Mix (2x, Bio-Rad, Hercules, CA), 8 ml miliQ water, 1 ml of isolated genomic DNA and 1 ml of the corresponding Taqman probes (20x, Applied Biosystems, Carlsbad, CA), real time PCR reactions were performed in a CFX96 thermocycler (Bio-Rad, Hercules, CA). G2677T/A [rs2032582] genotyping was tested using the PCRRFLP method (Table 1). Forward and reverse primers reported previously by Levran et al. (2008) were used (F: 50 -TCT CAT GAA GGT GAG TTT TCA GA-30 , R: 50 -AAA CAC ATT CTT AGA GCA TAG TAA GCA-30 ). Primers were synthesized by Alpha DNA (Montreal, QC). G2677T/A PCR (PTC-200 thermocycler, MJ Research, St.-Bruno, QC)
Power calculations to detect a relative risk of 1.5 assuming a dominant model were between 7% for the A allele of G2677T/A (2677G>A), and 14% for the C allele of C1236T (1236C). Concordance of genotype distribution with the HardyeWeinberg equilibrium was assessed for individual alleles at each locus using Arlequin software v3.5.1.3 (Bern University, Switzerland), which makes use of a test analogous to Fisher exact test [22]. HardyeWeinberg test was performed on the NoMDR patients, as genotype frequencies of this group are expected to be more representative of the general population than those of MDR patients. Polymorphisms that were in equilibrium were further analyzed with a Fisher bilateral test comparing among patients with MDR, NoMDR, RER and NoRER using Prism v5.0 (Graphpad, La Jolla, CA). P-values below 0.05 were considered statistically significant. Haplotype inference was performed from G2677T/A and C3435T genotypes using the ELB algorithm found in Arlequin. The ELB algorithm makes use of a Bayesian method to “reconstruct” the gametic phase of multi-locus genotypes. Frequencies of inferred haplotypes were then analyzed using the software WHAP 2.09,
Please cite this article in press as: Rodríguez-Castillo JA, et al., Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.04.004
J.A. Rodríguez-Castillo et al. / Tuberculosis xxx (2015) 1e6
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Table 1 Restriction pattern of each possible genotype of G2677T/A. Allele/Genotype
Restriction enzymes RsaI
G T A GG GT TT GA TA AA
200 200 200 200 200 200 200 200 200
NlaIV þ RsaI
BseYI þ þ þ þ þ þ þ þ þ
194 194 112 194 194 194 194 194 112
þ 82
þ 112 þ 82 þ 112 þ 82 þ 82
282 394 394 282 394 394 394 394 394
þ 112 194 þ 104 þ 96 112 þ 104 þ 96 þ 82 þ 112 þ 282 þ 112 þ 282 þ 112 194 þ 112 þ 104 þ 96 þ 82 112 þ 104 þ 96 þ 82
Product size: 394 bp. Double digestions with NlaIV and RsaI were only performed in samples matching patterns of TA/AA genotypes after simple digestions.
which utilizes a logistic regression model for analysis of binary traits. WHAP was chosen for haplotype based association tests because it supports analysis of multiallelic polymorphisms [23]. Haplotype-specific tests with 1 degree of freedom were conducted for each haplotype versus all the other haplotypes. Additionally, omnibus analyses were performed to test for all haplotype effects. P-values from the omnibus tests were obtained from 10,000 permutations. 3. Results 3.1. Characteristics of participants 99 individuals were analyzed for this study. Among them, 38 individuals were MDR and 61 were NoMDR (53 sensible to all tested drugs and 9 mono or multi-resistant); 22 subjects were RER and 77 were NoRER (Table 2, data not shown and Supplementary Table 1). Clinical characteristics of MDR and NoMDR patients are shown in Table 2. Among MDR group, 26 individuals were male and 12 were female, while 37 NoMDR were male and 24 were female. MDR-TB was more common among relapses (18 MDR vs 11 NoMDR patients) and this difference probed to be statistically significant (P < 0.01) (Table 2). 3.2. Alleles and genotypes in TB patients For C1236T, the CT genotype was more frequent in the studied population (61.6%), followed by CC (19.2%) and TT (19.2%). Overall genotype frequencies of C3435T were found as follows: CT, 45.4%;
CC, 34.3% and TT; 20.2%. The most prevalent genotype of G2677T/A was the GT heterozygous (51.5%), followed by GG, TT, GA, TA and AA (24.24%, 17.17%, 5.05%, 1.01% and 1.01% respectively). Allele and genotype frequencies of each polymorphism of MDR and NoMDR are shown in Table 3. Allele frequencies of C3435T and G2677T/A showed to be consistent with HardyeWeinberg equilibrium (P > 0.05, data not shown). Allele distribution of C1236T was not in equilibrium (P < 0.05, data not shown), so further statistical analysis could not be performed for this polymorphism. Analysis of genotype and allele frequencies of C3435T and G2677T/A showed no significant differences between MDR and NoMDR groups (P > 0.05). Due to RMP and ETB are both the only first-line drugs used against TB that have shown to be substrates of P-gp, we additionally formed groups of RER and NoRER individuals. Same features showed in Table 2 were compared between RER and NoRER patients and no significant differences were found (Supplementary table 1). Analysis of differences of genotype frequencies among RER versus NoRER subjects showed no statistical differences for any of the studied polymorphisms (Table 4). Even so, we observed a significantly higher prevalence of the rare allele A of G2677T/A (2677G>A) in the RER group compared to NoRER (5 and 2 individuals respectively, P < 0.01; OR, 11.0; 95% CI, 2.00e56.00). Details about characteristics and genotypes of 2677G>A carriers are given in Supplementary table 2. No significant differences of allele frequencies between RER and NoRER were found for C3435T (Table 4). In a post-hoc analysis, genotype and allele frequencies of subjects mono-resistant to each drug (INH-resistant, ETB-resistant,
Table 2 Demographical and clinical characteristics of MDR and NoMDR individuals. Characteristics
MDR (%) [n ¼ 38]
NoMDR (%) [n ¼ 61]
P-value
Gender (Male:Female) Mean age Reactivation cases Household contacts with TB Alcoholism Smoking habit Drug abuse Comorbidities Diabetes mellitus Hypertension Asthma COPD Malnutrition Renal disorders Cardiac disorders Arthropaties Others
26:12 (68.4e31.6) 39.9 18 (47.3) 9 (23.7) 3 (7.9) 3 (7.9) 3 (7.9)
37:24 (60.0e40.0) 47.0 11 (18.0) 9 (14.8) 5 (8.2) 3 (4.9) 1 (1.4)
0.52 0.14 <0.01 0.29 0.52 0.68 0.30
5 2 2 1 0 2 0 1 3
15 (24.6) 8 (13.1) 3 (4.9) 1 (1.4) 1 (1.4) 1 (1.4) 2 (3.3) 1 (1.4) 4 (6.6)
(13.2) (5.3) (5.3) (2.6) (0.0) (5.3) (0.0) (2.3) (7.9)
0.20 0.12 0.70 1.00 0.56 1.00
Statistical comparisons were performed using Fisher bilateral test. Statistically significant data (P < 0.05) is written in bold. MDR, patients with multidrug-resistant tuberculosis; NoMDR, patients without multidrug-resistant tuberculosis; COPD, chronic obstructive pulmonary disease; others: neurological disorders, depressive disorder, migraine, hypertiroidism, hypoparatiroidism, mammary fibroadenoma and benign prostatic hyperplasia.
Please cite this article in press as: Rodríguez-Castillo JA, et al., Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.04.004
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Table 3 Genotype and allele frequencies of ABCB1 polymorphisms of MDR and NoMDR patients. Genotype C1236T CC CT TT Allele frequencies C T C3435T CC CT TT Allele frequencies C T G2677T/A GG GT GA TT TA AA Allele frequencies G T A
MDR [n ¼ 38] (%)
NoMDR [n ¼ 61] (%)
11 (28.9) 21 (55.3) 6 (15.8)
8 (13.1) 40 (65.6) 13 (21.3)
43 (56.6) 33 (43.4)
56 (45.9) 66 (54.1)
14 (36.8) 16 (42.1) 8 (21.1)
OR
95% CI
P-value
20 (32.8) 29 (47.5) 12 (29.7)
0.79 0.95
0.32e2.00 0.31e2.90
0.65 1.00
44 (57.9) 32 (42.1)
69 (56.6) 53 (43.4)
0.95
0.53e1.70
0.88
10 17 3 6 1 1
14 34 2 11 0 0
0.70 2.10 0.76
0.26e1.90 0.29e15.00 0.21e2.80
0.61 0.63 0.75
0.86 4.8
0.47e1.60 0.92e25.00
0.65 0.06
(26.3) (44.7) (7.9) (15.8) (2.6) (2.6)
40 (52.6) 30 (39.5) 6 (7.9)
(23.0) (55.7) (3.3) (18.0) (0.0) (0.0)
64 (52.2) 56 (45.9) 2 (1.6)
Statistical comparisons were performed using Fisher bilateral test. Statistically significant data (P < 0.05) is written in bold. MDR, patients with multidrug-resistant tuberculosis; NoMDR, patients without multidrug-resistant tuberculosis; OR, odds ratio; CI, confidence interval.
RMP-resistant and SM-resistant) and their corresponding drugsensitive group were compared. Significant difference was only found between the 2677G>A frequencies of ETB-resistant and ETBsensitive individuals (Data not shown, P ¼ 0.01; OR, 8.20; 95% CI, 1.50e44.00).
of the A-C haplotype in RER versus NoRER (4 versus 1 haplotypes, P < 0.01). However, no significant differences were found in the omnibus test (P > 0.05). On the other hand, no associations were found either in the global test or in the haplotype-specific tests between MDR and NoMDR individuals (P > 0.05).
3.3. Haplotype analysis in TB patients
4. Discussion
Inferred haplotype frequencies from unphased genotypes and results from haplotype analyses are shown in Table 5. Haplotypespecific tests showed a significant difference between frequencies
Nowadays, several genetic factors of the host have been linked to susceptibility to mycobacterial infections [7,24]. Additionally, polymorphisms of several genes, such as SLC11A1, some HLA genes,
Table 4 Genotype and allele frequencies of ABCB1 polymorphisms of RER and NoRER patients. Genotype C1236T CC CT TT Allele frequencies C T C3435T CC CT TT Allele frequencies C T G2677T/A GG GT GA TT TA AA Allele frequencies G T A
RER [n ¼ 22] (%)
NoRER [n ¼ 77] (%)
6 (27.3) 14 (63.6) 2 (9.1)
13 (16.9) 47 (61.0) 17 (22.1)
26 (59.1) 18 (40.9)
73 (47.4) 81 (52.6)
9 (40.9) 10 (45.5) 3 (13.6)
25 (32.5) 35 (45.4) 17 (22.1)
28 (63.6) 16 (36.4)
85 (55.2) 69 (44.8)
6 8 3 3 1 1
(27.3) (36.4) (13.6) (13.6) (4.5) (4.5)
23 (52.3) 15 (34.1) 6 (13.6)
18 43 2 14 0 0
(23.4) (55.8) (2.6) (18.2) (0.0) (0.0)
81 (52.6) 71 (46.1) 2 (1.3)
OR
95% CI
P-value
0.79 0.49
0.28e2.20 0.12e2.10
0.79 0.50
0.70
0.35e1.40
0.39
0.56 4.5 0.64
0.17e1.80 0.60e34.00 0.14e3.00
0.36 0.29 1.00
0.74 11
0.36e1.50 2.00e56.00
1.0 0.004
Statistical comparisons were performed using Fisher bilateral test. Statistically significant data (P < 0.05) is written in bold. RER, patients with tuberculosis simultaneously resistant to ethambutol and rifampin; NoRER, individuals without simultaneous resistance to ethambutol and rifampin; OR, odds ratio; CI, confidence interval.
Please cite this article in press as: Rodríguez-Castillo JA, et al., Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.04.004
J.A. Rodríguez-Castillo et al. / Tuberculosis xxx (2015) 1e6
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Table 5 Frequencies of ABCB1 inferred haplotypes (G2677T/A-C3435T) of MDR, NoMDR, RER and NoRER patients. Haplotype Omnibus G-C TeT T-C G-T A-C A-T
MDR (%) [n ¼ 76] 35 (46.1%) 26 (34.2%) 5 (6.6%) 5 (6.6%) 4 (5.3%) 1 (1.3%)
NoMDR (%) [n ¼ 122]
P-value
RER (%) [n ¼ 44]
NoRER (%) [n ¼ 154]
P-value
57 (46.7%) 45 (36.9%) 11 (09%) 7 (5.7%) 1 (0.8%) 1 (0.8%)
0.60 0.97 0.70 0.51 0.89 0.07 0.50
21 (47.7%) 13 (29.5%) 3 (6.8%) 2 (4.5%) 4 (9.1%) 1 (2.3%)
71 (46.1%) 58 (37.7%) 13 (8.4%) 10 (6.5%) 1 (0.6%) 1 (0.6%)
0.18 0.81 0.30 0.71 0.55 0.006 0.22
Empirical P-values for global (Omnibus) tests with 4 degrees of freedom were obtained by permutation. Haplotypes A-C and A-T were clustered in a single group for this test. Statistical comparisons for haplotype-specific tests are based on chi-square tests of 1 degree of freedom. Statistically significant data (P < 0.05) is written in bold. MDR, patients with multidrug-resistant tuberculosis; NoMDR, patients without multidrug-resistant tuberculosis; RER, patients with tuberculosis simultaneously resistant to ethambutol and rifampin; NoRER, individuals without simultaneous resistance to ethambutol and rifampin.
TLR7, TLR8 and INFG, have been associated with increased susceptibility to pulmonary tuberculosis [25]. Nevertheless, only a few reports exist regarding the role of host genetics in factors that may lead to the development of resistant forms of TB. In this regard, Takahashi et al. found a possible association between two linked polymorphisms of the gene SLC11A1 and both, MDR-TB and cavity formation in the lung [9]. In the present study, a novel association has been proposed between the 2677G>A allele of human ABCB1 and combined resistance to RMP and ETB. P-gp is an efflux pump involved in uptake, distribution and excretion of many drugs. Polymorphisms of this protein have shown to differently affect its function against different compounds. In addition, some of these polymorphisms have been associated to pharmacoresistance to several anti-cancer and antiepileptic drugs, and to higher risk of interactions or toxicity by cardiovascular drugs [18e20]. In 2007, it was demonstrated that both RMP and ETB are substrates of P-gp but not INH [21]. It is currently unknown whether SM and PZA are substrates for P-gp. In this study, prevalence of MDR-TB in the 99 patients with pulmonary TB was 38.4%. This rate is by far higher than the estimated in Mexico, which is 2.4% for new cases and 6.5% for previously treated patients [6]. This difference could be explained by the fact that patients were recruited from a TB clinic that is also a referral center for MDR-TB cases, leading to selection of higher proportion of MDR individuals. As expected, MDR-TB cases were more common among relapse cases, which include true relapses and reinfections [26e28]. Nevertheless, none of the studied polymorphisms showed significant association to MDR-TB in our sample. This finding is consistent with results of a previous study conducted by Lian, Wu and Zhou [29]. They reported no differences among P-gp mRNA levels among peripheral blood mononuclear cells from patients with MDR-TB, Non-MDR and healthy subjects. Even though these results suggest no relationship between P-gp and MDR-TB, data is lacking about differences in its expression on cell's membrane surface or functionality of P-gp in relation to the resistance status of TB patients. Although the investigation of factors that may contribute to generation of MDR-TB cases is of particular interest due to its clinical relevance [2], we also wanted to evaluate if ABCB1 polymorphisms are associated specifically to simultaneous resistance to RMP and ETB since pharmacokinetics of these drugs is more likely to be affected by P-gp. Interestingly, we observed for the first time an association between the A allele of G2677T/A and the presence of RER. G2677T/A is a triallelic nonsynonymous polymorphism whose 2677G>T and 2677G>A alleles respectively cause 893Ala/ Ser and 893Ala/Thr substitutions in P-gp [30]. In a previous study, Sakurai et al. expressed variant forms of human P-gp polymorphisms in insect Sf9 cells. Among the studied polymorphisms, G2677T/A had the greatest impact on both the activity and substrate specificity of P-gp. Enhanced ATP-ase activity of P-gp and
increased efflux of several drugs was observed in cells transfected with 2677G>A [16]. Nonetheless, whether G2677T/A or other polymorphisms of ABCB1 have any effect on P-gp activity against RMP or ETB remains to be elucidated. In a study conducted in African subjects, no difference was found in RMP pharmacokinetic profiles of 57 individuals with different genotypes of C1236T, C3435T and G2677T [31]. Unfortunately, only 2677G and 2677G>T alleles but not 2677G>A were assessed and such data cannot be contrasted properly with our results. Another similar study with 16 healthy controls and 72 TB patients evaluated the C3435T polymorphism in ABCB1 gene, and it was not associated to the area under the curve of RMP [32]. Notably, in our study C3435T was neither associated to resistance status in TB patients. Haplotype-specific tests in our sample showed a possible association between the A-C haplotype (Formed by the 2677G>A and 3435C alleles) and RER. Nonetheless, the omnibus test failed to show a significant association, suggesting that the single effect of the 2677G>A allele may account for the association found at the haplotype level. No additional significant effects of the haplotypes were detected on the RER or MDR status. Although the present work provides the first insight into an association between the 2677G>A allele and resistant-TB, limitations of this study should be noted. 1) The high rate of MDR-TB patients attending the clinic led to an increased proportion of drug-resistance in our sample compared to the general population with TB, potentially introducing bias. 2) Due to its small sample size, the study had low power to resolve additional associations. 3) The absolute number of drug-resistant TB patients might have been too small to effectively dissect the effect of the allele involved. Given the association found between 2677G>A and ETBmonoresistance in the post-hoc analysis, whether the association found for RER was caused secondary to single association with ETBmonoresistance remains a possibility. Further studies with higher power might show a stronger association of G2677T/A with either RMP or ETB-monoresistance or even with MDR-TB. Therefore, if this association is confirmed in future studies, use of alternative regimens could be a reasonable approach for the treatment of these cases. 5. Conclusion This study showed a possible association between RER-TB and the 2677G>A allele of human ABCB1. This finding suggests that 893Ala/Thr substitution in P-gp, could be a facilitator for the development of drug resistant TB. Further research is needed to better understand that relationship. Acknowledgment We thank Susana Covarrubias for technical support.
Please cite this article in press as: Rodríguez-Castillo JA, et al., Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.04.004
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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tube.2015.04.004 Funding: Programa de Apoyo a la Investigacion Cientifica y Tecnologica (PAICYT)-UANL. Conflicts of interest: All authors: No reported conflicts. The funders had no role in the study design, data collection, data analysis, decision to publish, or preparation of the manuscript. Ethical approval: The Bioethics and Research Committee from the Facultad de Medicina at the Universidad Autonoma de Nuevo Leon approved the project, ensuring that it was in accordance with Institutional Review Board criteria. Participation was voluntary and participants gave informed, written consent. References [1] World Health Organization. The global plan to stop TB 2011-2015: transforming the fight towards elimination of tuberculosis. Geneva: Stop TB Partnership; 2010. [2] Gandhi NR, Nunn P, Dheda K, Schaaf HS, Zignol M, van Soolingen D, Jensen P, Bayona J. Multidrug-resistant and extensively drug-resistant tuberculosis a threat to global control of tuberculosis. Lancet 2010;375:1830e43. http:// dx.doi.org/10.1016/S01406736(10)60410-2. [3] Almeida Da Silva PE, Palomino JC. Molecular basis and mechanisms of drug resistance in Mycobacterium tuberculosis: classical and new drugs. J Antimicrob Chemother 2011;66:1417e30. http://dx.doi.org/10.1093/jac/ dkr173. [4] Jain A, Dixit P. Multidrug-resistant to extensively drug resistant tuberculosis: what is next? J Biosci 2008;33:605e16. [5] WHO Stop TB Initiative. . Standard regimen treatments. Treatment of tuberculosis: guidelines. Geneva: World Health Organization; 2010. p. 29e52. [6] Zignol M, van Gemert W, Falzon D, Sismanidis C, Glaziou P, Floyd K, Raviglione M. Surveillance of anti-tuberculosis drug resistance in the world: an updated analysis, 2007-2010. Bull World Health Organ 2012;90: 111e119D. http://dx.doi.org/10.2471/BLT.11.092585. [7] Yim JJ, Selvaraj P. Genetic susceptibility in tuberculosis. Respirology 2010;15: 241e56. http://dx.doi.org/10.1111/j.1440-1843.2009.01690.x. [8] Gao L, Tao Y, Zhang L, Jin Q. Vitamin D receptor genetic polymorphisms and tuberculosis: updated systematic review and meta-analysis. Int J Tuberc Lung Dis 2010;14:15e23. [9] Takahashi K, Hasegawa Y, Abe T, Yamamoto T, Nakashima K, Imaizumi K, Shimokata K. SLC11A1 (formerly NRAMP1) polymorphisms associated with multidrug-resistant tuberculosis. Tuberculosis 2008;88:52e7. http:// dx.doi.org/10.1016/j.tube.2007.08.008. [10] Sharom FJ. The P-glycoprotein multidrug transporter. Essays Biochem 2011;50:161e78. http://dx.doi.org/10.1042/bse0500161. [11] Huls M, Russel FG, Masereeuw R. The role of ATP binding cassette transporters in tissue defense and organ regeneration. J Pharmacol Exp Ther 2009;328: 3e9. http://dx.doi.org/10.1124/jpet.107.132225. [12] Mruk DD, Su L, Cheng CY. Emerging role for drug transporters at the bloodtestis barrier. Trends Pharmacol Sci 2011;32:99e106. http://dx.doi.org/ 10.1016/j.tips.2010.11.007. [13] O'Brien JP, Cordon-Cardo C. Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J Histochem Cytochem 1990;38:1277e87. http://dx.doi.org/10.1177/38.9.1974900. [14] Puddu P, Fais S, Luciani F, Gherardi G, Dupuis ML, Romagnoli G, Ramoni C, Cianfriglia M, Gessani S. Interferon-ɣ up-regulates expression and activity of P-glycoprotein in human peripheral blood monocyte-derived macrophages. Lab Invest 1999;79:1299e309. [15] Zhou SF. Structure, function and regulation of P-glycoprotein and its clinical relevance in drug disposition. Xenobiotica Fate Foreign Compd Biol Syst 2008;38:802e32. http://dx.doi.org/10.1080/00498250701867889.
[16] Sakurai A, Onishi Y, Hirano H, Seigneuret M, Obanayama K, Kim G, Liew EL, Sakaeda T, Yoshiura K, Niikawa N, Sakurai M, Ishikawa T. Quantitative structure-activity relationship analysis and molecular dynamics simulation to functionally validate nonsynonymous polymorphisms of human ABC transporter ABCB1 (P-Glycoprotein/MDR1). Biochemistry 2007;46:7678e93. http://dx.doi.org/10.1021/bi700330b. [17] Hoffmeyer S, Burk O, von Richter O, Arnold HP, Brockmoller J, Johne A, Cascorbi I, Gerloff T, Roots I, Eichelbaum M, Brinkmann U. Functional polymorphisms of the human multidrug-resistance gene: multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc Natl Acad Sci U S A 2000;97:3473e8. http://dx.doi.org/ 10.1073/pnas.050585397. [18] Horinouchi M, Sakaeda T, Nakamura T, Morita Y, Tamura T, Aoyama N, Kasuga M, Okumura K. Significant genetic linkage of MDR1 polymorphisms at positions 3435 and 2677: functional relevance to pharmacokinetics of digoxin. Pharm Res 2002;19:1581e5. http://dx.doi.org/10.1023/A: 1020433422259. [19] Lazarowski A, Czornyj L. Potential role of multidrug resistant proteins in refractory epilepsy and antiepileptic drugs interactions. Drug Metabolism Drug Interact 2011;26:21e6. http://dx.doi.org/10.1515/DMDI.2011.006. [20] Wessler JD, Grip LT, Mendell J, Giugliano RP. The P-glycoprotein transport system and cardiovascular drugs. J Am Coll Cardiol 2013;61:2495e502. http:// dx.doi.org/10.1016/j.jacc.2013.02.058. [21] Hartkoorn RC, Chandler B, Owen A, Ward SA, Bertel Squire S, Back DJ, Khoo SH. Differential drug susceptibility of intracellular and extracellular tuberculosis, and the impact of P-glycoprotein. Tuberculosis 2007;87:248e55. http://dx.doi.org/10.1016/j.tube.2006.12.001. [22] Excoffier L, Lischer HE. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 2010;10:564e70. http://dx.doi.org/10.1111/j.17550998.2010.02847.x. [23] Purcell S, Daly MJ, Sham PC. WHAP: haplotype-based association analysis. Bioinformatics 2007;23:255e6. http://dx.doi.org/10.1093/bioinformatics/ btl580. [24] Cottle LE. Mendelian susceptibility to mycobacterial disease. Clin Genet 2011;79:17e22. http://dx.doi.org/10.1111/j.1399-0004.2010.01510.x. [25] Qu HQ, Fisher-Hoch SP, McCormick JB. Knowledge gaining by human genetic studies on tuberculosis susceptibility. J Hum Genet 2011;56:177e82. http:// dx.doi.org/10.1038/jhg.2010.164. [26] Espinal MA, Laserson K, Camacho M, Fusheng Z, Kim SJ, Tlali E, Smith I, Suarez P, Antunes ML, George AG, Martin-Casabona N, Simelane P, Weyer K, Binkin N, Raviglione MC. Determinants of drug-resistant tuberculosis: analysis of 11 countries. Int J Tuberc Lung Dis 2001;5:887e93. [27] Moonan PK, Teeter LD, Salcedo K, Ghosh S, Ahuja SD, Flood J, Graviss EA. Transmission of multidrug-resistant tuberculosis in the USA: a cross-sectional study. Lancet Infect Dis 2013;13:777e84. http://dx.doi.org/10.1016/s14733099(13)70128-2. [28] Chen S, Huai P, Wang X, Zhong J, Wang X, Wang K, Wang L, Jiang S, Li J, Peng Y, Ma W. Risk factors for multidrug resistance among previously treated patients with tuberculosis in eastern China: a case-control study. Int J Infect Dis 2013;17:e1116e1120. http://dx.doi.org/10.1016/j.ijid.2013.06.006. [29] Lian Z, Wu P, Zhou J. Expression of P-glycoprotein and multidrug resistanceassociated protein in peripheral blood mononuclear cells from multidrug resistant tuberculosis patients. Zhonghua Jie He He Hu Xi Za Zhi 2011;34: 520e32. [30] Kroetz DL, Pauli-Magnus C, Hodges LM, Huang CC, Kawamoto M, Johns SJ, Stryke D, Ferrin TE, DeYoung J, Taylor T, Carlson EJ, Herskowitz I, Giacomini KM, Clark AG. Sequence diversity and haplotype structure in the human ABCB1 (MDR1, multidrug resistance transporter) gene. Pharmacogenetics 2003;13:481e94. http://dx.doi.org/10.1097/ 01.fpc.0000054113.14659.b9. [31] Chigutsa E, Visser ME, Swart EC, Denti P, Pushpakom S, Egan D, Holford NH, Smith PJ, Maartens G, Owen A, McIlleron H. The SLCO1B1 rs4149032 polymorphism is highly prevalent in South Africans and is associated with reduced rifampin concentrations: dosing implications. Antimicrob Agents Chemother 2011;55:4122e7. http://dx.doi.org/10.1128/AAC.01833-10. [32] Weiner M, Peloquin C, Burman W, Luo CC, Engle M, Prihoda TJ, Mac Kenzie WR, Bliven-Sizemore E, Johnson JL, Vernon A. Effects of tuberculosis, race, and human gene SLCO1B1 polymorphisms on rifampin concentrations. Antimicrob Agents Chemother 2010;54:4192e200. http://dx.doi.org/10.1128/ AAC.00353-10.
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MOLECULAR ASPECTS
Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis Alberto Rodríguez-Castillo a, 1, Alma Y. Arce-Mendoza a, Armando Quintanilla-Siller a, Jose Adrian Rendon b, Mario C. Salinas-Carmona a, Adrian G. Rosas-Taraco a, * n, Monterrey 64460, Mexico Departamento de Inmunología, Facultad de Medicina y Hospital Universitario, Universidad Autonoma de Nuevo Leo n, Prevencio n y Tratamiento de Infecciones Respiratorias) Hospital Universitario, Universidad Autonoma de Nuevo Leon, CIPTIR (Centro de Investigacio Monterrey 64460, Mexico
a
b
a r t i c l e i n f o
s u m m a r y
Article history: Received 25 August 2014 Received in revised form 30 March 2015 Accepted 8 April 2015
Human P-glycoprotein (P-gp) is a membrane transporter encoded by ABCB1 (also known as MDR1) that plays a critical role in pharmacokinetics of many unrelated drugs. Rifampin (RMP) and ethambutol (ETB), two anti-tubercular agents, are substrates of P-gp. Single nucleotide polymorphisms (SNPs) in ABCB1 have been associated with resistance to several drugs; however, their association with RMP and ETB resistance in tuberculosis patients has not yet been studied. Genotype/allele frequencies in C1236T, G2677T/A and C3435T SNPs of ABCB1 were obtained from 99 tuberculosis patients susceptible or resistant to RMP and ETB (NoRER or RER). 2677G>A allele prevalence was found to be significantly higher in the RER group compared to NoRER (5 resistant vs 2 non-resistant patients, P < 0.01; OR, 11.0; 95% CI, 2.00e56.00). No differences were found in genotype/allele frequencies in C1236T and C3435T SNPs of ABCB1 and resistance to RMP and ETB in tuberculosis patients (P > 0.05). The present study suggests the 2677G>A allele of ABCB1 could be associated with simultaneous resistance to RMP and ETB in pulmonary tuberculosis patients. Further studies with larger sample sizes are needed to confirm this association and explore its nature. © 2015 Elsevier Ltd. All rights reserved.
Keywords: P-glycoprotein Polymorphisms Drug-resistant tuberculosis SNPs
1. Introduction Pulmonary tuberculosis (TB) is the most frequent form of infection caused by Mycobacterium tuberculosis (Mtb). Nine million new TB cases are reported worldwide and two million people die each year due to this disease [1]. Combination treatment with rifampin (RMP), isoniazid (INH), ethambutol (ETB) and pyrazinamide (PZA) is the standard and generally effective therapy for new TB cases. Nevertheless, in last decades Mtb strains resistant to anti-tubercular agents have been emerging [2e4]. Multidrug-resistant TB (MDR-TB) is caused by Mtb strains resistant to at least INH and RMP simultaneously [5]. MDRTB represents a world health problem. Its burden differs among
* Corresponding author. Tel.: þ52 81 83294211; fax: þ52 81 833310 58. E-mail addresses: [email protected], [email protected] (A.G. Rosas-Taraco). 1 Alberto Rodríguez Castillo: Dept. IV, Max Planck Institute Present address. Jose for Heart and Lung Research. Parkstrasse 1, 61231 Bad Nauheim, Germany.
countries, however, rates as high as 28.9% for new TB cases and 51.5% for previously treated cases have been reported [6]. Mutations in several genes of Mtb have been documented to be associated to drug-resistant phenotypes in bacterial cultures. Additionally, accumulation of such mutations may lead the bacteria to acquire a multidrug-resistant phenotype [3]. Positive selection of resistant strains is known to occur as consequence of environmental factors, which are related to repeated exposition to antibiotic therapy, and inadequate or insufficient treatment [5]. Although the involvement of bacterial genetics in the genesis of MDR-TB has been relatively well explored, and several authors have investigated the role of host genetics on Mtb infection [7,8], much less is known about host genetic factors on the development of resistant forms of TB. In this regard, Takahashi et al. reported a possible association between a variant of the gen SLC11A1 and MDR-TB [9]. Human P-glycoprotein (P-gp) is an extensively studied ATPdependent pump that actively transports several different molecules out of the cell [10]. Its most evident function is protection of cells from the toxic effect of some endogenous compounds and xenobiotics [11,12]. P-gp is expressed on apical surface of some
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Please cite this article in press as: Rodríguez-Castillo JA, et al., Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.04.004
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epithelia, kidney, bloodebrain barrier and phagocytic cells as macrophages (main target of Mtb) [9,13,14]. In addition, P-gp plays a critical role in pharmacokinetics of some drugs [15]. ABCB1/MDR1 polymorphisms affect the expression of P-gp and/or its activity against some cardiovascular, anti-cancer, central nervous system drugs, etc. [16e20]. A study demonstrated that two of the first line drugs for tuberculosis treatment, RMP and ETB, are substrates of Pgp [21], setting up the rationale for considering P-gp as an interesting candidate for human genetics' involvement in the generation of a fraction of the MDR-TB cases. Up to date, there are no studies that explore whether P-gp polymorphisms are associated with drug-resistant TB. In this study, we investigated whether polymorphisms in ABCB1 may be associated with higher risk to develop resistant forms of TB. We explored for associations between alleles, genotypes and haplotypes of C1236T, G2677T/A and G3435T polymorphisms and resistance to any of four first line drugs INH, RMP, ETB and streptomycin (SM), focusing on MDR-TB and simultaneous resistance to RMP and ETB. 2. Materials and methods 2.1. Participants From 2010 to 2013, a total of 122 consecutive patients who had culture proven TB and attended the TB Clinic of the University Hospital of Monterrey (A referral center for MDR-TB cases from north-east Mexico) were evaluated. The ethics committee approved this study and written informed consent was obtained from all participants. Only individuals with positive Mtb cultures and results of drug sensitivity tests (DST) were recruited. DST performed to samples from all patients tested for resistance to INH, RMP, ETB and SM. Subjects with extra-pulmonary infection and those co-infected with HIV were excluded. Demographic data was recorded and a peripheral blood sample was obtained from each patient. Finally, samples that presented genotyping problems were discarded, ending up with 99 participants for analysis (Figure 1). Patients were allocated in 4 non-mutually exclusive study groups according to their DST results: MDR, NoMDR, RMP-ETB resistant (RER) and NoRER. Information about patients' resistance status, risk factors and co-morbidities was obtained from their medical records.
Figure 1. Flow diagram of patient selection.
was performed under the following conditions: Initial denaturation step for 5 min at 94 C, 40 cycles of denaturation at 94.5 for 45 s, annealing at 63 C for 30 s and extension at 72 C for 30 s. All restriction enzymes were from New England Biolabs (Ipswich, MA). Each PCR product was separately digested with RsaI or BseYI. Fragments were run in 3% agarose gel and stained with ethidiumbromide or RedGel™ (Biotium, Hayward, CA). Products with restriction patterns matching TA or AA genotypes were also doubledigested with NlaIV and RsaI in order to discriminate between them.
2.2. Genotyping of C1236T, C3435T and G2677T/A polymorphisms 2.3. Statistical analysis Genomic DNA isolation was performed from 0.5 ml of EDTAanticoagulated blood using the TSNT technique. DNA samples were stored at 20 C until their use. Real time PCR was performed using Taqman® technology for C1236T [rs1128503] and C3435T [rs1045642] genotyping (Assay IDs: C___7586662_10 and C___7586657_20 respectively). PCR conditions for C1236T were as follows: Initial denaturation step by 10 min at 95 , 40 cycles of 15 s at 95 C and annealing-extension step at 60 C for 60 s. PCR conditions for C3435T were 10 min at 95 , 40 cycles of 30 s at 95 C and annealing-extension step at 60 C for 90 s. Both reactions contained 10 ml of IQ Super Mix (2x, Bio-Rad, Hercules, CA), 8 ml miliQ water, 1 ml of isolated genomic DNA and 1 ml of the corresponding Taqman probes (20x, Applied Biosystems, Carlsbad, CA), real time PCR reactions were performed in a CFX96 thermocycler (Bio-Rad, Hercules, CA). G2677T/A [rs2032582] genotyping was tested using the PCRRFLP method (Table 1). Forward and reverse primers reported previously by Levran et al. (2008) were used (F: 50 -TCT CAT GAA GGT GAG TTT TCA GA-30 , R: 50 -AAA CAC ATT CTT AGA GCA TAG TAA GCA-30 ). Primers were synthesized by Alpha DNA (Montreal, QC). G2677T/A PCR (PTC-200 thermocycler, MJ Research, St.-Bruno, QC)
Power calculations to detect a relative risk of 1.5 assuming a dominant model were between 7% for the A allele of G2677T/A (2677G>A), and 14% for the C allele of C1236T (1236C). Concordance of genotype distribution with the HardyeWeinberg equilibrium was assessed for individual alleles at each locus using Arlequin software v3.5.1.3 (Bern University, Switzerland), which makes use of a test analogous to Fisher exact test [22]. HardyeWeinberg test was performed on the NoMDR patients, as genotype frequencies of this group are expected to be more representative of the general population than those of MDR patients. Polymorphisms that were in equilibrium were further analyzed with a Fisher bilateral test comparing among patients with MDR, NoMDR, RER and NoRER using Prism v5.0 (Graphpad, La Jolla, CA). P-values below 0.05 were considered statistically significant. Haplotype inference was performed from G2677T/A and C3435T genotypes using the ELB algorithm found in Arlequin. The ELB algorithm makes use of a Bayesian method to “reconstruct” the gametic phase of multi-locus genotypes. Frequencies of inferred haplotypes were then analyzed using the software WHAP 2.09,
Please cite this article in press as: Rodríguez-Castillo JA, et al., Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.04.004
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Table 1 Restriction pattern of each possible genotype of G2677T/A. Allele/Genotype
Restriction enzymes RsaI
G T A GG GT TT GA TA AA
200 200 200 200 200 200 200 200 200
NlaIV þ RsaI
BseYI þ þ þ þ þ þ þ þ þ
194 194 112 194 194 194 194 194 112
þ 82
þ 112 þ 82 þ 112 þ 82 þ 82
282 394 394 282 394 394 394 394 394
þ 112 194 þ 104 þ 96 112 þ 104 þ 96 þ 82 þ 112 þ 282 þ 112 þ 282 þ 112 194 þ 112 þ 104 þ 96 þ 82 112 þ 104 þ 96 þ 82
Product size: 394 bp. Double digestions with NlaIV and RsaI were only performed in samples matching patterns of TA/AA genotypes after simple digestions.
which utilizes a logistic regression model for analysis of binary traits. WHAP was chosen for haplotype based association tests because it supports analysis of multiallelic polymorphisms [23]. Haplotype-specific tests with 1 degree of freedom were conducted for each haplotype versus all the other haplotypes. Additionally, omnibus analyses were performed to test for all haplotype effects. P-values from the omnibus tests were obtained from 10,000 permutations. 3. Results 3.1. Characteristics of participants 99 individuals were analyzed for this study. Among them, 38 individuals were MDR and 61 were NoMDR (53 sensible to all tested drugs and 9 mono or multi-resistant); 22 subjects were RER and 77 were NoRER (Table 2, data not shown and Supplementary Table 1). Clinical characteristics of MDR and NoMDR patients are shown in Table 2. Among MDR group, 26 individuals were male and 12 were female, while 37 NoMDR were male and 24 were female. MDR-TB was more common among relapses (18 MDR vs 11 NoMDR patients) and this difference probed to be statistically significant (P < 0.01) (Table 2). 3.2. Alleles and genotypes in TB patients For C1236T, the CT genotype was more frequent in the studied population (61.6%), followed by CC (19.2%) and TT (19.2%). Overall genotype frequencies of C3435T were found as follows: CT, 45.4%;
CC, 34.3% and TT; 20.2%. The most prevalent genotype of G2677T/A was the GT heterozygous (51.5%), followed by GG, TT, GA, TA and AA (24.24%, 17.17%, 5.05%, 1.01% and 1.01% respectively). Allele and genotype frequencies of each polymorphism of MDR and NoMDR are shown in Table 3. Allele frequencies of C3435T and G2677T/A showed to be consistent with HardyeWeinberg equilibrium (P > 0.05, data not shown). Allele distribution of C1236T was not in equilibrium (P < 0.05, data not shown), so further statistical analysis could not be performed for this polymorphism. Analysis of genotype and allele frequencies of C3435T and G2677T/A showed no significant differences between MDR and NoMDR groups (P > 0.05). Due to RMP and ETB are both the only first-line drugs used against TB that have shown to be substrates of P-gp, we additionally formed groups of RER and NoRER individuals. Same features showed in Table 2 were compared between RER and NoRER patients and no significant differences were found (Supplementary table 1). Analysis of differences of genotype frequencies among RER versus NoRER subjects showed no statistical differences for any of the studied polymorphisms (Table 4). Even so, we observed a significantly higher prevalence of the rare allele A of G2677T/A (2677G>A) in the RER group compared to NoRER (5 and 2 individuals respectively, P < 0.01; OR, 11.0; 95% CI, 2.00e56.00). Details about characteristics and genotypes of 2677G>A carriers are given in Supplementary table 2. No significant differences of allele frequencies between RER and NoRER were found for C3435T (Table 4). In a post-hoc analysis, genotype and allele frequencies of subjects mono-resistant to each drug (INH-resistant, ETB-resistant,
Table 2 Demographical and clinical characteristics of MDR and NoMDR individuals. Characteristics
MDR (%) [n ¼ 38]
NoMDR (%) [n ¼ 61]
P-value
Gender (Male:Female) Mean age Reactivation cases Household contacts with TB Alcoholism Smoking habit Drug abuse Comorbidities Diabetes mellitus Hypertension Asthma COPD Malnutrition Renal disorders Cardiac disorders Arthropaties Others
26:12 (68.4e31.6) 39.9 18 (47.3) 9 (23.7) 3 (7.9) 3 (7.9) 3 (7.9)
37:24 (60.0e40.0) 47.0 11 (18.0) 9 (14.8) 5 (8.2) 3 (4.9) 1 (1.4)
0.52 0.14 <0.01 0.29 0.52 0.68 0.30
5 2 2 1 0 2 0 1 3
15 (24.6) 8 (13.1) 3 (4.9) 1 (1.4) 1 (1.4) 1 (1.4) 2 (3.3) 1 (1.4) 4 (6.6)
(13.2) (5.3) (5.3) (2.6) (0.0) (5.3) (0.0) (2.3) (7.9)
0.20 0.12 0.70 1.00 0.56 1.00
Statistical comparisons were performed using Fisher bilateral test. Statistically significant data (P < 0.05) is written in bold. MDR, patients with multidrug-resistant tuberculosis; NoMDR, patients without multidrug-resistant tuberculosis; COPD, chronic obstructive pulmonary disease; others: neurological disorders, depressive disorder, migraine, hypertiroidism, hypoparatiroidism, mammary fibroadenoma and benign prostatic hyperplasia.
Please cite this article in press as: Rodríguez-Castillo JA, et al., Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.04.004
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Table 3 Genotype and allele frequencies of ABCB1 polymorphisms of MDR and NoMDR patients. Genotype C1236T CC CT TT Allele frequencies C T C3435T CC CT TT Allele frequencies C T G2677T/A GG GT GA TT TA AA Allele frequencies G T A
MDR [n ¼ 38] (%)
NoMDR [n ¼ 61] (%)
11 (28.9) 21 (55.3) 6 (15.8)
8 (13.1) 40 (65.6) 13 (21.3)
43 (56.6) 33 (43.4)
56 (45.9) 66 (54.1)
14 (36.8) 16 (42.1) 8 (21.1)
OR
95% CI
P-value
20 (32.8) 29 (47.5) 12 (29.7)
0.79 0.95
0.32e2.00 0.31e2.90
0.65 1.00
44 (57.9) 32 (42.1)
69 (56.6) 53 (43.4)
0.95
0.53e1.70
0.88
10 17 3 6 1 1
14 34 2 11 0 0
0.70 2.10 0.76
0.26e1.90 0.29e15.00 0.21e2.80
0.61 0.63 0.75
0.86 4.8
0.47e1.60 0.92e25.00
0.65 0.06
(26.3) (44.7) (7.9) (15.8) (2.6) (2.6)
40 (52.6) 30 (39.5) 6 (7.9)
(23.0) (55.7) (3.3) (18.0) (0.0) (0.0)
64 (52.2) 56 (45.9) 2 (1.6)
Statistical comparisons were performed using Fisher bilateral test. Statistically significant data (P < 0.05) is written in bold. MDR, patients with multidrug-resistant tuberculosis; NoMDR, patients without multidrug-resistant tuberculosis; OR, odds ratio; CI, confidence interval.
RMP-resistant and SM-resistant) and their corresponding drugsensitive group were compared. Significant difference was only found between the 2677G>A frequencies of ETB-resistant and ETBsensitive individuals (Data not shown, P ¼ 0.01; OR, 8.20; 95% CI, 1.50e44.00).
of the A-C haplotype in RER versus NoRER (4 versus 1 haplotypes, P < 0.01). However, no significant differences were found in the omnibus test (P > 0.05). On the other hand, no associations were found either in the global test or in the haplotype-specific tests between MDR and NoMDR individuals (P > 0.05).
3.3. Haplotype analysis in TB patients
4. Discussion
Inferred haplotype frequencies from unphased genotypes and results from haplotype analyses are shown in Table 5. Haplotypespecific tests showed a significant difference between frequencies
Nowadays, several genetic factors of the host have been linked to susceptibility to mycobacterial infections [7,24]. Additionally, polymorphisms of several genes, such as SLC11A1, some HLA genes,
Table 4 Genotype and allele frequencies of ABCB1 polymorphisms of RER and NoRER patients. Genotype C1236T CC CT TT Allele frequencies C T C3435T CC CT TT Allele frequencies C T G2677T/A GG GT GA TT TA AA Allele frequencies G T A
RER [n ¼ 22] (%)
NoRER [n ¼ 77] (%)
6 (27.3) 14 (63.6) 2 (9.1)
13 (16.9) 47 (61.0) 17 (22.1)
26 (59.1) 18 (40.9)
73 (47.4) 81 (52.6)
9 (40.9) 10 (45.5) 3 (13.6)
25 (32.5) 35 (45.4) 17 (22.1)
28 (63.6) 16 (36.4)
85 (55.2) 69 (44.8)
6 8 3 3 1 1
(27.3) (36.4) (13.6) (13.6) (4.5) (4.5)
23 (52.3) 15 (34.1) 6 (13.6)
18 43 2 14 0 0
(23.4) (55.8) (2.6) (18.2) (0.0) (0.0)
81 (52.6) 71 (46.1) 2 (1.3)
OR
95% CI
P-value
0.79 0.49
0.28e2.20 0.12e2.10
0.79 0.50
0.70
0.35e1.40
0.39
0.56 4.5 0.64
0.17e1.80 0.60e34.00 0.14e3.00
0.36 0.29 1.00
0.74 11
0.36e1.50 2.00e56.00
1.0 0.004
Statistical comparisons were performed using Fisher bilateral test. Statistically significant data (P < 0.05) is written in bold. RER, patients with tuberculosis simultaneously resistant to ethambutol and rifampin; NoRER, individuals without simultaneous resistance to ethambutol and rifampin; OR, odds ratio; CI, confidence interval.
Please cite this article in press as: Rodríguez-Castillo JA, et al., Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.04.004
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Table 5 Frequencies of ABCB1 inferred haplotypes (G2677T/A-C3435T) of MDR, NoMDR, RER and NoRER patients. Haplotype Omnibus G-C TeT T-C G-T A-C A-T
MDR (%) [n ¼ 76] 35 (46.1%) 26 (34.2%) 5 (6.6%) 5 (6.6%) 4 (5.3%) 1 (1.3%)
NoMDR (%) [n ¼ 122]
P-value
RER (%) [n ¼ 44]
NoRER (%) [n ¼ 154]
P-value
57 (46.7%) 45 (36.9%) 11 (09%) 7 (5.7%) 1 (0.8%) 1 (0.8%)
0.60 0.97 0.70 0.51 0.89 0.07 0.50
21 (47.7%) 13 (29.5%) 3 (6.8%) 2 (4.5%) 4 (9.1%) 1 (2.3%)
71 (46.1%) 58 (37.7%) 13 (8.4%) 10 (6.5%) 1 (0.6%) 1 (0.6%)
0.18 0.81 0.30 0.71 0.55 0.006 0.22
Empirical P-values for global (Omnibus) tests with 4 degrees of freedom were obtained by permutation. Haplotypes A-C and A-T were clustered in a single group for this test. Statistical comparisons for haplotype-specific tests are based on chi-square tests of 1 degree of freedom. Statistically significant data (P < 0.05) is written in bold. MDR, patients with multidrug-resistant tuberculosis; NoMDR, patients without multidrug-resistant tuberculosis; RER, patients with tuberculosis simultaneously resistant to ethambutol and rifampin; NoRER, individuals without simultaneous resistance to ethambutol and rifampin.
TLR7, TLR8 and INFG, have been associated with increased susceptibility to pulmonary tuberculosis [25]. Nevertheless, only a few reports exist regarding the role of host genetics in factors that may lead to the development of resistant forms of TB. In this regard, Takahashi et al. found a possible association between two linked polymorphisms of the gene SLC11A1 and both, MDR-TB and cavity formation in the lung [9]. In the present study, a novel association has been proposed between the 2677G>A allele of human ABCB1 and combined resistance to RMP and ETB. P-gp is an efflux pump involved in uptake, distribution and excretion of many drugs. Polymorphisms of this protein have shown to differently affect its function against different compounds. In addition, some of these polymorphisms have been associated to pharmacoresistance to several anti-cancer and antiepileptic drugs, and to higher risk of interactions or toxicity by cardiovascular drugs [18e20]. In 2007, it was demonstrated that both RMP and ETB are substrates of P-gp but not INH [21]. It is currently unknown whether SM and PZA are substrates for P-gp. In this study, prevalence of MDR-TB in the 99 patients with pulmonary TB was 38.4%. This rate is by far higher than the estimated in Mexico, which is 2.4% for new cases and 6.5% for previously treated patients [6]. This difference could be explained by the fact that patients were recruited from a TB clinic that is also a referral center for MDR-TB cases, leading to selection of higher proportion of MDR individuals. As expected, MDR-TB cases were more common among relapse cases, which include true relapses and reinfections [26e28]. Nevertheless, none of the studied polymorphisms showed significant association to MDR-TB in our sample. This finding is consistent with results of a previous study conducted by Lian, Wu and Zhou [29]. They reported no differences among P-gp mRNA levels among peripheral blood mononuclear cells from patients with MDR-TB, Non-MDR and healthy subjects. Even though these results suggest no relationship between P-gp and MDR-TB, data is lacking about differences in its expression on cell's membrane surface or functionality of P-gp in relation to the resistance status of TB patients. Although the investigation of factors that may contribute to generation of MDR-TB cases is of particular interest due to its clinical relevance [2], we also wanted to evaluate if ABCB1 polymorphisms are associated specifically to simultaneous resistance to RMP and ETB since pharmacokinetics of these drugs is more likely to be affected by P-gp. Interestingly, we observed for the first time an association between the A allele of G2677T/A and the presence of RER. G2677T/A is a triallelic nonsynonymous polymorphism whose 2677G>T and 2677G>A alleles respectively cause 893Ala/ Ser and 893Ala/Thr substitutions in P-gp [30]. In a previous study, Sakurai et al. expressed variant forms of human P-gp polymorphisms in insect Sf9 cells. Among the studied polymorphisms, G2677T/A had the greatest impact on both the activity and substrate specificity of P-gp. Enhanced ATP-ase activity of P-gp and
increased efflux of several drugs was observed in cells transfected with 2677G>A [16]. Nonetheless, whether G2677T/A or other polymorphisms of ABCB1 have any effect on P-gp activity against RMP or ETB remains to be elucidated. In a study conducted in African subjects, no difference was found in RMP pharmacokinetic profiles of 57 individuals with different genotypes of C1236T, C3435T and G2677T [31]. Unfortunately, only 2677G and 2677G>T alleles but not 2677G>A were assessed and such data cannot be contrasted properly with our results. Another similar study with 16 healthy controls and 72 TB patients evaluated the C3435T polymorphism in ABCB1 gene, and it was not associated to the area under the curve of RMP [32]. Notably, in our study C3435T was neither associated to resistance status in TB patients. Haplotype-specific tests in our sample showed a possible association between the A-C haplotype (Formed by the 2677G>A and 3435C alleles) and RER. Nonetheless, the omnibus test failed to show a significant association, suggesting that the single effect of the 2677G>A allele may account for the association found at the haplotype level. No additional significant effects of the haplotypes were detected on the RER or MDR status. Although the present work provides the first insight into an association between the 2677G>A allele and resistant-TB, limitations of this study should be noted. 1) The high rate of MDR-TB patients attending the clinic led to an increased proportion of drug-resistance in our sample compared to the general population with TB, potentially introducing bias. 2) Due to its small sample size, the study had low power to resolve additional associations. 3) The absolute number of drug-resistant TB patients might have been too small to effectively dissect the effect of the allele involved. Given the association found between 2677G>A and ETBmonoresistance in the post-hoc analysis, whether the association found for RER was caused secondary to single association with ETBmonoresistance remains a possibility. Further studies with higher power might show a stronger association of G2677T/A with either RMP or ETB-monoresistance or even with MDR-TB. Therefore, if this association is confirmed in future studies, use of alternative regimens could be a reasonable approach for the treatment of these cases. 5. Conclusion This study showed a possible association between RER-TB and the 2677G>A allele of human ABCB1. This finding suggests that 893Ala/Thr substitution in P-gp, could be a facilitator for the development of drug resistant TB. Further research is needed to better understand that relationship. Acknowledgment We thank Susana Covarrubias for technical support.
Please cite this article in press as: Rodríguez-Castillo JA, et al., Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.04.004
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Please cite this article in press as: Rodríguez-Castillo JA, et al., Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.04.004