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Association of ABCB1 gene polymorphisms and haplotypes with therapeutic efficacy of glucocorticoids in Chinese patients with immune thrombocytopenia
Human Immunology 75 (2014) 317–321
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Association of ABCB1 gene polymorphisms and haplotypes with therapeutic efficacy of glucocorticoids in Chinese patients with immune thrombocytopenia Min Xuan, Huiyuan Li, Rongfeng Fu, Yanhui Yang, Donglei Zhang, Xian Zhang, Renchi Yang ⇑ State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, 288 Nanjing Road, Tianjin 300020, People’s Republic of China
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Article history: Received 17 April 2013 Accepted 14 January 2014 Available online 30 January 2014
a b s t r a c t Resistance to glucocorticoids (GCs) remains a tricky problem complicating the therapy of ITP. Recently, ATP binding cassette gene B1 gene (ABCB1) was reported to be correlated with susceptibility and therapeutic efficacy of autoimmune diseases through P-glycoprotein (Pgp). We investigated three single nucleotide polymorphisms (SNPs) of ABCB1 and their haplotypes by PCR–RFLP (restriction fragment length polymorphism) method in 471 ITP patients and 383 healthy controls, patients were further assigned into GCs-responsive and -non-responsive group according to the therapeutic effects of GCs. We observed a remarkable difference in genotypes of G2677T/A between GCs-responsive and non-responsive group, but not between patients and controls. A frequently expression of T/A allele within G2677T/A was recorded in GCs-responsive group. Furthermore, we found that some haplotypes (CGC, CTC/CAC, CTT/ CAT, TGC, TGT, TTC/TAC and TTT/TAT, in the order of position 1236-2677-3435) were presented significantly differences between non-responsive and responsive group. No difference of C1236T and C3435T polymorphisms was observed between ITP and controls, and between the GCs-responsive and -nonresponsive group. Our findings suggest that ABCB1 polymorphisms, as well as haplotypes derived from C1235T, G2677T/A and C3435T, are associated with inter-individual differences of GCs treatment in ITP. Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Immune thrombocytopenia (ITP) is an autoimmune disease characterized by increased platelet destruction and/or decreased platelet production resulting bleeding [1]. Glucocorticoids (GCs) have been shown potent activity as the first-line approach to ITP treatment through unclear mechanism. With GCs, about 80–90% of patients could achieve remission. However, still a part of patients has no response to these agents. Although mechanisms of interindividual variations in GCs efficacy are not definitely understood yet, nature and/or nurture are both considered to be involved in the pathogenesis, such as fraction bound to cortisol-binding globulin, metabolism by 11b-hydroxysteroid dehydrogenase, expression of glucocorticoid receptors [2], etc. It has been reported extensively that transport proteins can influence drug absorption, distribution, and excretion [3]. P-glycoprotein (Pgp), an important drug transport protein, ⇑ Corresponding author. Fax: +86 22 23909093. E-mail address: [email protected] (R. Yang).
undoubtedly has attracted researcher’s attentions for several years. Pgp is a 170 kDa integral membrane protein expressed in some tumors and nonmalignant tissues (intestine, blood–brain barrier), function as an efflux pump that transports natural compounds [4] and therapeutic agents via ATP hydrolysis to protect cells from a variety of endogenous and exogenous toxins [5,6]. It is worthwhile to mention that Pgp is also involved in determining oral absorption and disposition of drugs by pumping them back into the intestinal lumen [7,8]. Since oral GCs are the gold standard of treatment for ITP, we hypothesized that Pgp might play a crucial role in pharmacokinetics of GCs. Pgp is encoded by the ATP binding cassette gene B1 (ABCB1), also known as the multi-drug resistance gene 1(MDR1) [9], located on chromosome 7q21.1 and consists of 28 introns and exons [10]. ABCB1 polymorphisms may ultimately impact on bioavailability and tissue distribution of drugs by modulating expression of Pgp. Among more than 50 single nucleotide polymorphism (SNPs) identified for ABCB1 [11], three SNPs of C1236T, G2667T/A, and C3435T were frequently studied for their effects on Pgp. Numerous researches on ABCB1 gene have provided evidences for linkage of drug effect in some other autoimmune disorders
http://dx.doi.org/10.1016/j.humimm.2014.01.013 0198-8859/Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
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[12]. Our study focused on the above-mentioned SNPs of ABCB1 and their haplotypes to find out whether they participate in responses to GCs therapy in ITP patients. 2. Materials and methods 2.1. Study population In this case-control study, 471 unrelated ITP patients and 383 healthy controls were recruited (Han ethnic) after providing written, informed consent in accordance with the Declaration of Helsinki and the hospital-based ethics committee. Patients were classified with recent guidelines [13]. All participants were followed-up for at least 6 months after accepting GCs therapy in outpatient departments to evaluate therapeutic outcome. Based on responses to GCs therapy, all patients were grouped into the GCsresponsive and -non-responsive arms. After the 28-day continuous GCs treatment period, patients with platelet counts between 30 and 100 109/L or at least doubling of the baseline were considered to be GCs-response by the definition of maintaining concurrent management of bleeding events [13]. Those patients who did not meet the above criteria were allocated into non-response group. 2.2. Genotyping Purified genomic DNA was isolated from peripheral blood using the Wizard genomic DNA purification kit (Promega, Madison, WI). Table 1 PCR primers, restriction enzymes and fragment size for SNP assays. SNPs
rs ID
Primer sequences
Enzyme
C1236T
rs1128503
F: 50 -TTCACTTCAGTTACCCATC-30 R: 50 -TCTTTGTCACTTTATCCAGC-30
Eco0109I
G2677T
rs2032582
F: 50 -TGCAGGCTATAGGTTCCA GG-30 R: 50 -TTTAGTTTGACTCACCTTCCCG-30
BanI
C3435T
rs1045642
F: 50 -GATCTGTGAACTCTTGTTTTC-30 R: 50 -CTTGTTTTCAGCTGCTTGATGGCAA-30
MboI
The investigated SNPs were genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) methods. The 25 ll PCR mixture contained 50–150 ng of genomic DNA, 10 pM of each primer and Premix Taq (Takara, Dalian, China). The primers, restriction enzymes of corresponding SNPs are listed in Table 1. For PCR amplification, an initial denaturation at 94 °C for 5 min was followed by 35 cycles of 30s at 94 °C, 45s at 58 °C, 45s at 72 °C, and a final extension at 72 °C for 10 min. The productions of PCR were digested respectively with restriction enzymes Eco0109I (C1236T), BanI (G2677T/A), and MboI (C3435T), referring to corresponding optimal temperatures and time of enzymes. Enzymatic cleavage fragments were separated on 1.5% agarose gel (3% agarose gel for G2677T/A), observed under UV light with ethidine bromide staining. To confirm the accuracy of ABCB1 genotyping, amplification products were further sequenced with 36 random samples (Fig. 1). 2.3. Linkage disequilibrium and haplotype analysis Haplotype analysis included C1236T, G2677T/A and C3435T. Haploview software (version 4.2) was used to analyze the potential linkage disequilibrium. Predominant haplotypes were detected by SHEsis software. The criterion for r2 was set at 0.8. Pairwise linkage disequilibrium (LD) between SNPs was measured by Lewontin’s coefficient (D0 value). LD was described as strong (P80% of D0 ), moderate (50 to <80% of D0 ), and weak (<50% of D0 ). The statistical significance of LD between each pair of SNPs was analyzed by Fisher’s exact test. 2.4. Statistical analysis Statistical analyses of data were performed using the SHEsis software. The genotypic distribution and frequencies were evaluated by the Chi-Square test or Fisher’s exact test in case of expected frequencies less than 5. P values less than 0.05 were considered as statistically significant.
Fig. 1. PCR–RFLP of G2677T/A polymorphisms. (a) PCR–RFLP-based genotyping result of G2677T/A (lower layer: GG, upper layer: TT/TA/AA, two layers: GA/GT). (b) Confirmation by direct sequencing.
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3. Results The ITP patients enrolled consisted of 304 female (64.5%) and 167 male (35.5%). The clinical features of ITP patients are summarized in Table 2. In this study, we examined three novel SNPs of ABCB1 in a comparatively large population of 854 Han Chinese participants (471 ITP patients and 383 healthy controls) by PCR–RFLP. Among ITP group, 356 patients were allocated into GCs responsive arm and 115 patients presented resistance to standard steroid therapy. The explanation of such a disproportionately large recruited cohort of GCs-non-responsive is probably that most ITP patients referred to our hospital were chronic and/or refractory to treatment. All three genotypic distributions in controls and patients did not deviate from Hardy–Weinberg equilibrium. No significant difference was found in allele and genotype frequencies of ABCB1 polymorphisms between ITP patients and controls referring to gender (data not shown). According to the obtained data (Table 3), Chi-square analysis revealed that there were no statistically difference between variants of C1236T and C3435T in the ITP and control group (both p > 0.05), responsive and non-responsive group (p > 0.05). Table 2 Clinical characteristics of ITP patients.
Number of subjects Sex ratio (male/female) Age at onset (years), mean (SD) Min. age at onset (years) Max. age at onset (years) Under 16 years old (n) Above 16 years old (n)
Responsive
Nonresponsive
Total ITP
356 129/227 36.39 (21.58) 0.3 84 88 268
115 38/77 30.32 (18.32)
471 167/304 34.91 (20.84) 0.3 84 120 351
3 71 32 83
Although there was no statistical difference found between ITP and normal samples in terms of G2677T/A polymorphism, our results demonstrated that homozygously mutated (AA/TT/TA) patients were more sensitive to GCs comparing with the wild-type and heterozygous mutated (GG/GA/GT) subjects, and GG genotype had poor response to GCs than GA/GT genotypes. The frequencies of T and A allele were 7.2 times higher in GCs-response group than those in GCs-non-responsive group. Our results indicated that all three SNPs of ABCB1 were in linkage disequilibrium. The SNPs of C1236T and G2677T/A were found to be in strong linkage disequilibrium ((D0 = 0.86), furthermore, moderate but not such strong linkages were found between the SNPs of C1236T and C3435T (D0 = 0.63), G2677T/A and C3435T (D0 = 0.74). A total of eight haplotypes derived from these three variants were reconstructed (Table 4), in which three haplotypes, TTT/TAT (31.4%), CGC (27.0%) and TGC (20.3%) were most frequently observed in the healthy population, while CGC (19.6%), TGC (19.5%) and TTT/TAT (16.1%) were most found in ITP cohort. Furthermore, there were differences in six haplotypes (CGC, CTC/CAC, CTT/CAT, TGT, TTC/TAC and TTT/TAT, in the order of position 1236-26773435) between patients and controls, and striking differences were also found in some haplotypes (CGC, CTC/CAC, CTT/CAT, TGC, TGT, TTC/TAC and TTT/TAT, in the order of position 1236-2677-3435) between GCs responsive and non-responsive arm.
4. Discussion In the past few years, a number of investigators have reported that correlations between the specific polymorphic variants of ABCB1 and pathogenesis of several diseases, such as epilepsy [14], cancers [15] and some autoimmune disorders (such as SLE, inflammatory bowel diseases, cirrhosis and rheumatoid arthritis) were displayed accompanying by resistance to anti-neoplastic
Table 3 Distribution of the genotype and allele frequencies of ABCB1 in all subjects.
C1236T Allele C T Genotype CC CT TT G2667T/A Allele T/A G Genotype T/A T/A G T/A GG C3435T Allele T C Genotype TT TC CC
Responsive N = 356, n (%)
Non-responsive N = 115, n(%)
ITP N = 471, n(%)
Controls N = 383, n (%)
P-value
OR (95%CI)
273 (38.3) 439 (61.7)
93 (40.4) 137 (59.6)
368 (39.1) 574 (60.9)
269 (37.2) 455 (62.8)
0.571a 0.426b
1.091 (0.806–1.479)a 1.084 (0.888–1.324)b
50 (14.0) 173 (48.6) 133 (37.4)
17 (14.8) 59 (51.3) 39 (33.9)
68 (14.4) 232 (49.3) 171 (36.3)
38 (10.5) 193 (53.3) 131 (36.2)
0.800a 0.206b
573 (80.5) 139 (19.5)
82 (35.7) 148 (64.3)
337 (40.2) 501 (59.8)
297 (40.7) 433 (59.3)
7.77e-016a 0.849b
224 (62.9) 125 (35.1) 7 (2.0)
10 (8.7) 62 (53.9) 43 (37.4)
63 (15.0) 211 (50.4) 145 (34.6)
58 (15.9) 181 (49.6) 126 (34.5)
2.22e-015a 0.944b
145 (63.0) 85 (37.0)
408 (57.3) 304 (42.7)
387 (41.1) 555 (58.9)
336 (44.3) 422 (55.7)
0.124a 0.179b
63 (17.7) 178 (50.0) 115 (32.3)
12 (10.4) 61 (53.0) 42 (36.5)
74 (15.7) 239 (50.7) 158 (33.5)
66 (17.4) 204 (53.8) 109 (28.8)
0.174a 0.319b
CI, confidence interval; OR, odds ratio; ITP, immune thrombocytopenia. a Non-responsive group compared with responsive group. b ITP group compared with controls.
7.44 (5.363–10.321)a 1.020 (0.833–1.248)b
1.271 (0.935–1.726)a 1.141 (0.941–1.385)b
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Table 4 Haplotypes derived from C1236T, G2677T/A and C3435T.
CGC CGT CTC/CAC CTT/CAT TGC TGT TTC/TAC TTT/TAT
Responsive N = 356, n (%)
Non-responsive N = 115, n (%)
ITP N = 471, n (%)
Controls N = 383, n (%)
Pa
Pb
25.2 (3.5) 28.1 (4.0) 160.7 (22.6) 59.0 (8.3) 32.3 (4.5) 53.4 (7.5) 189.8 (26.7) 163.5 (23.0)
83.1 (36.1) 6.8 (2.9) 3.1 (1.4) 0.0 (0.0) 51.3 (22.3) 6.9 (3.0) 7.5 (3.3) 71.3 (3.1)
184.2 (19.6) 57.2 (6.1) 90.3 (9.6) 36.3 (3.8) 183.5 (19.5) 142.0 (15.1) 97.0 (10.3) 151.5 (16.1)
185.4 (27.0) 42.4 (6.2) 19.5 (2.8) 6.7 (1.0) 139.4 (20.3) 42.8 (6.2) 34.6 (5.0) 215.2 (31.4)
0.000382* 0.933887 8.96e008* 0.000359* 0.673113 2.94e008* 0.000127* 3.54e013*
2.33e-015* 0.484933 1.95e013* 6.69e006* 2.22e015* 0.015161* 3.57e014* 0.014136*
a
Non-responsive group compared with responsive group. ITP group compared with controls. P value < 0.05.
b *
agents, antidepressants or immune-suppressants with ABCB1 polymorphisms. But some of these genotyping studies demonstrated conflicting results, which potentially attribute to different ethnic or therapy assessed [16]. It is widely accepted that genetic factors play a pivotal role in several aspects of ITP despite it is an autoimmune disorder, including susceptibility of this disease [17], and the response to GCs treatment varies from patient to patient [18]. Recently, three SNPs of ABCB1 (C1236T, C3435T and G2677T/A) were proved to be associated with GCs anti-inflammatory effects [19], glucocorticoid receptors [20] and related complications [21]. However, little is known about the roles of these SNPs and haplotypes in GCs curative effects during treatment of ITP. Based on Pgp encoded by ABCB1 gene, we assumed that SNPs and haplotypes of ABCB1 may have an impact on the expression and function of Pgp, thereby influence the response to GCs therapy in ITP. C1236T is located in exon 12 within ABCB1. CC genotype of C1236T is more frequent than TT genotype in Caucasians [22], whilst less common in Asians [23], which is exactly shown in our data. A study [24] on HIV-1-infected children suggested that C1236T SNPs can down-regulate lopinavir plasma concentration by altering expression or activity of Pgp. But some investigators held different opinions that C1236T SNPs has nothing to do with drug availability in chronic lymphoproliferative diseases [25]. In the present study, we did not find any correlation between C1236T polymorphism and ITP susceptibility or treatment outcome. The G to T and A transversions at position 2677 in exon 21 are missense mutations, causing an amino acid change in Ala893Ser, which ultimately impact the secondary structure of Pgp [26]. Considering their relationship, the effect on treatment outcome in ovarian cancer was also shown by the results of Gréen et al. [27]. Patients with cancer receiving paclitaxel chemotherapy who carry the G2677T/A TT/TA genotype, showed improved progression free survival and response, possibly through a pharmacokinetic mechanism, but the functional consequences of G2677T/A polymorphism have not been extensively clarified yet [27]. In a study on steroid weaning in a pediatric heart transplant population, Zheng et al. [28] concluded that SNPs of C3435T, accompanied by G2677T/A determined the period of steroid administration after transplantation. However, studies on the impact of SNPs of G2677T/A showed conflicting results on some drugs (such as verapamil, cyclosporine, prazosin and bisantrene). The transport is not affected by known variants of Pgp [29–31], indicating substrate specificity of different Pgp variants. In our study, it was confirmed that homozygous (AA/TT/TA) patients with ITP were tend to easily respond to GCs therapy, whereas carriers of wild-type and heterozygous (GG/GA/GT) displayed a relative GCs resistance. Our results suggested that the presence of these mutated alleles may be a feasible predict factor for successful GCs treatment.
Although mechanism of G2677T/A affecting GCs sensitivity is unknown, we speculate that SNPs of G2677T/A might be involved in this process by influence expression of Pgp. Several studies reported that C3435T genotype is associated with the timing of Pgp co-translational folding, which attributed in substrate specificity [31]. There are other evidences proposing that a linkage between C3435T and other SNPs (such as in the promoter/enhancer or intronic regions) within ABCB1 gene is likely to regulate the translation of mRNA and affect posttranscriptional modifications [32]. The study by Hoffmeyer et al. [33] initially reported that C3435T can significantly reduce intestinal Pgp expression in TT homozygotes, leading to higher steady-state plasma concentrations after oral digoxin treatment. This implies that SNP of C3435T could alter Pgp activity through molecular mechanisms. But in our study, we did not found an effect of the allelic variants of the C3435T polymorphism on the outcome of GCs treatment in patients with ITP. Previously, the majority of studies only reported that C3435T and G2677T/A are two tightly linked SNPs and can influence Pgp expression [34], suggesting that the C3435T and G2677T/A haplotype may be involved in determining GCs effects. Until today, it should be noted that C1236T polymorphism, one of the most frequent SNPs of ABCB1, also contributes to the bioavailability of drugs. Several researches provided sufficient evidences to prove that C1236T, in combinations with G2677T/A and/or C3435T, play a key role in modifying the function of ABCB1 gene. Besides a missense polymorphism within G2677T/A cannot be distinguished by PCR–RFLP method along (Fig. 1), the haplotype frequencies of controls we observed are approximately consistent with some previous studies [35,36]. Actually, on the basis of data reported in Han ethnic, TTT are more common than TAT and, CAC are more common than CTC. Interestingly, the frequency of CGC is 36.1% in non-responsive arm and only 3.5% in responsive arm. A similar disparity of the haplotype TGC is displayed between these two arms. However, CTC/CAC, CTT/CAT, TTC/TAC or TTT/TAT exhibits a completely reverse trend between the same arms. Which indirectly reflects that carrier with G allele of G2677T/A is more inclined to be resistant to GCs, while patients with T/A allele of G2677T/A are more sensitive to steroids-therapy.
5. Conclusions In summary, our study showed that ITP patients carrying homozygous mutations in G2677T/A respond better to GCs treatment, which implied that ABCB1 polymorphisms may contribute to interindividual variability of GCs responsiveness. Obviously, further functional studies are needed to elucidate the molecular pathways and we hope to find more feasible biomarkers to predict the individual response to GCs therapy.
M. Xuan et al. / Human Immunology 75 (2014) 317–321
Acknowledgments This work was supported in part by grants of National Natural Science Foundation of China (81070397 and 81270581), Ministry of Science and Technology (2011ZX09302-007-04), Ministry of Health (201202017) and Tianjin Municipal Science and Technology Commission (10JCZDJC19700, 12JCQNJC08000). References [1] Cines DB, Blanchette VS. Immune thrombocytopenic purpura. N Engl J Med 2002;346:995. [2] Gross KL, Lu NZ, Cidlowski JA. Molecular mechanisms regulating glucocorticoid sensitivity and resistance. Mol Cell Endocrinol 2009;300:7. [3] Evans WE, McLeod HL. Pharmacogenomics–drug disposition, drug targets, and side effects. N Engl J Med 2003;348:538. [4] Rosenberg MF, Callaghan R, Ford RC, Higgins CF. Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined by electron microscopy and image analysis. J Biol Chem 1997;272:10685. [5] Schinkel AH, Jonker JW. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv Drug Deliv Rev 2003;55:3. [6] Ozen F, Silan C, Uludag A, Candan F, Silan F, Ozdemir S, et al. Association between ABCB1 (MDR1) gene 3435 C > T polymorphism and colchicine unresponsiveness of FMF patients. Ren Fail 2011;33:899. [7] Lin JH, Yamazaki M. Role of P-glycoprotein in pharmacokinetics: clinical implications. Clin Pharmacokinet 2003;42:59. [8] Sparreboom A, van Asperen J, Mayer U, Schinkel AH, Smit JW, Meijer DK, et al. Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine. Proc Natl Acad Sci USA 1997;94:2031. [9] Jeong H, Herskowitz I, Kroetz DL, Rine J. Function-altering SNPs in the human multidrug transporter gene ABCB1 identified using a Saccharomyces-based assay. PLoS Genet 2007;3:e39. [10] Nobili S, Landini I, Giglioni B, Mini E. Pharmacological strategies for overcoming multidrug resistance. Curr Drug Targets 2006;7:861. [11] Ishikawa T, Hirano H, Onishi Y, Sakurai A, Tarui S. Functional evaluation of ABCB1 (P-glycoprotein) polymorphisms: high-speed screening and structureactivity relationship analyses. Drug Metab Pharmacokinet 2004;19:1. [12] Webster JI, Carlstedt-Duke J. Involvement of multidrug resistance proteins (MDR) in the modulation of glucocorticoid response. J Steroid Biochem Mol Biol 2002;82:277. [13] Rodeghiero F, Stasi R, Gernsheimer T, Michel M, Provan D, Arnold DM, et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood 2009;113:2386. [14] Kwan P, Wong V, Ng PW, Lui CH, Sin NC, Poon WS, et al. Gene-wide tagging study of association between ABCB1 polymorphisms and multidrug resistance in epilepsy in Han Chinese. Pharmacogenomics 2009;10:723. [15] Illmer T, Schuler US, Thiede C, Schwarz UI, Kim RB, Gotthard S, et al. MDR1 gene polymorphisms affect therapy outcome in acute myeloid leukemia patients. Cancer Res 2002;62:4955. [16] Li YH, Wang YH, Li Y, Yang L. MDR1 gene polymorphisms and clinical relevance. Yi Chuan Xue Bao 2006;33:93. [17] Carcao MD, Blanchette VS, Wakefield CD, Stephens D, Ellis J, Matheson K, et al. Fcgamma receptor IIa and IIIa polymorphisms in childhood immune thrombocytopenic purpura. Br J Haematol 2003;120:135. [18] Chang NH, Cheung YH, Loh C, Pau E, Roy V, Cai YC, et al. B cell activating factor (BAFF) and T cells cooperate to breach B cell tolerance in lupus-prone New Zealand Black (NZB) mice. PLoS One 2010;5:e11691.
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Association of ABCB1 gene polymorphisms and haplotypes with therapeutic efficacy of glucocorticoids in Chinese patients with immune thrombocytopenia Min Xuan, Huiyuan Li, Rongfeng Fu, Yanhui Yang, Donglei Zhang, Xian Zhang, Renchi Yang ⇑ State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, 288 Nanjing Road, Tianjin 300020, People’s Republic of China
a r t i c l e
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Article history: Received 17 April 2013 Accepted 14 January 2014 Available online 30 January 2014
a b s t r a c t Resistance to glucocorticoids (GCs) remains a tricky problem complicating the therapy of ITP. Recently, ATP binding cassette gene B1 gene (ABCB1) was reported to be correlated with susceptibility and therapeutic efficacy of autoimmune diseases through P-glycoprotein (Pgp). We investigated three single nucleotide polymorphisms (SNPs) of ABCB1 and their haplotypes by PCR–RFLP (restriction fragment length polymorphism) method in 471 ITP patients and 383 healthy controls, patients were further assigned into GCs-responsive and -non-responsive group according to the therapeutic effects of GCs. We observed a remarkable difference in genotypes of G2677T/A between GCs-responsive and non-responsive group, but not between patients and controls. A frequently expression of T/A allele within G2677T/A was recorded in GCs-responsive group. Furthermore, we found that some haplotypes (CGC, CTC/CAC, CTT/ CAT, TGC, TGT, TTC/TAC and TTT/TAT, in the order of position 1236-2677-3435) were presented significantly differences between non-responsive and responsive group. No difference of C1236T and C3435T polymorphisms was observed between ITP and controls, and between the GCs-responsive and -nonresponsive group. Our findings suggest that ABCB1 polymorphisms, as well as haplotypes derived from C1235T, G2677T/A and C3435T, are associated with inter-individual differences of GCs treatment in ITP. Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Immune thrombocytopenia (ITP) is an autoimmune disease characterized by increased platelet destruction and/or decreased platelet production resulting bleeding [1]. Glucocorticoids (GCs) have been shown potent activity as the first-line approach to ITP treatment through unclear mechanism. With GCs, about 80–90% of patients could achieve remission. However, still a part of patients has no response to these agents. Although mechanisms of interindividual variations in GCs efficacy are not definitely understood yet, nature and/or nurture are both considered to be involved in the pathogenesis, such as fraction bound to cortisol-binding globulin, metabolism by 11b-hydroxysteroid dehydrogenase, expression of glucocorticoid receptors [2], etc. It has been reported extensively that transport proteins can influence drug absorption, distribution, and excretion [3]. P-glycoprotein (Pgp), an important drug transport protein, ⇑ Corresponding author. Fax: +86 22 23909093. E-mail address: [email protected] (R. Yang).
undoubtedly has attracted researcher’s attentions for several years. Pgp is a 170 kDa integral membrane protein expressed in some tumors and nonmalignant tissues (intestine, blood–brain barrier), function as an efflux pump that transports natural compounds [4] and therapeutic agents via ATP hydrolysis to protect cells from a variety of endogenous and exogenous toxins [5,6]. It is worthwhile to mention that Pgp is also involved in determining oral absorption and disposition of drugs by pumping them back into the intestinal lumen [7,8]. Since oral GCs are the gold standard of treatment for ITP, we hypothesized that Pgp might play a crucial role in pharmacokinetics of GCs. Pgp is encoded by the ATP binding cassette gene B1 (ABCB1), also known as the multi-drug resistance gene 1(MDR1) [9], located on chromosome 7q21.1 and consists of 28 introns and exons [10]. ABCB1 polymorphisms may ultimately impact on bioavailability and tissue distribution of drugs by modulating expression of Pgp. Among more than 50 single nucleotide polymorphism (SNPs) identified for ABCB1 [11], three SNPs of C1236T, G2667T/A, and C3435T were frequently studied for their effects on Pgp. Numerous researches on ABCB1 gene have provided evidences for linkage of drug effect in some other autoimmune disorders
http://dx.doi.org/10.1016/j.humimm.2014.01.013 0198-8859/Ó 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
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[12]. Our study focused on the above-mentioned SNPs of ABCB1 and their haplotypes to find out whether they participate in responses to GCs therapy in ITP patients. 2. Materials and methods 2.1. Study population In this case-control study, 471 unrelated ITP patients and 383 healthy controls were recruited (Han ethnic) after providing written, informed consent in accordance with the Declaration of Helsinki and the hospital-based ethics committee. Patients were classified with recent guidelines [13]. All participants were followed-up for at least 6 months after accepting GCs therapy in outpatient departments to evaluate therapeutic outcome. Based on responses to GCs therapy, all patients were grouped into the GCsresponsive and -non-responsive arms. After the 28-day continuous GCs treatment period, patients with platelet counts between 30 and 100 109/L or at least doubling of the baseline were considered to be GCs-response by the definition of maintaining concurrent management of bleeding events [13]. Those patients who did not meet the above criteria were allocated into non-response group. 2.2. Genotyping Purified genomic DNA was isolated from peripheral blood using the Wizard genomic DNA purification kit (Promega, Madison, WI). Table 1 PCR primers, restriction enzymes and fragment size for SNP assays. SNPs
rs ID
Primer sequences
Enzyme
C1236T
rs1128503
F: 50 -TTCACTTCAGTTACCCATC-30 R: 50 -TCTTTGTCACTTTATCCAGC-30
Eco0109I
G2677T
rs2032582
F: 50 -TGCAGGCTATAGGTTCCA GG-30 R: 50 -TTTAGTTTGACTCACCTTCCCG-30
BanI
C3435T
rs1045642
F: 50 -GATCTGTGAACTCTTGTTTTC-30 R: 50 -CTTGTTTTCAGCTGCTTGATGGCAA-30
MboI
The investigated SNPs were genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) methods. The 25 ll PCR mixture contained 50–150 ng of genomic DNA, 10 pM of each primer and Premix Taq (Takara, Dalian, China). The primers, restriction enzymes of corresponding SNPs are listed in Table 1. For PCR amplification, an initial denaturation at 94 °C for 5 min was followed by 35 cycles of 30s at 94 °C, 45s at 58 °C, 45s at 72 °C, and a final extension at 72 °C for 10 min. The productions of PCR were digested respectively with restriction enzymes Eco0109I (C1236T), BanI (G2677T/A), and MboI (C3435T), referring to corresponding optimal temperatures and time of enzymes. Enzymatic cleavage fragments were separated on 1.5% agarose gel (3% agarose gel for G2677T/A), observed under UV light with ethidine bromide staining. To confirm the accuracy of ABCB1 genotyping, amplification products were further sequenced with 36 random samples (Fig. 1). 2.3. Linkage disequilibrium and haplotype analysis Haplotype analysis included C1236T, G2677T/A and C3435T. Haploview software (version 4.2) was used to analyze the potential linkage disequilibrium. Predominant haplotypes were detected by SHEsis software. The criterion for r2 was set at 0.8. Pairwise linkage disequilibrium (LD) between SNPs was measured by Lewontin’s coefficient (D0 value). LD was described as strong (P80% of D0 ), moderate (50 to <80% of D0 ), and weak (<50% of D0 ). The statistical significance of LD between each pair of SNPs was analyzed by Fisher’s exact test. 2.4. Statistical analysis Statistical analyses of data were performed using the SHEsis software. The genotypic distribution and frequencies were evaluated by the Chi-Square test or Fisher’s exact test in case of expected frequencies less than 5. P values less than 0.05 were considered as statistically significant.
Fig. 1. PCR–RFLP of G2677T/A polymorphisms. (a) PCR–RFLP-based genotyping result of G2677T/A (lower layer: GG, upper layer: TT/TA/AA, two layers: GA/GT). (b) Confirmation by direct sequencing.
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3. Results The ITP patients enrolled consisted of 304 female (64.5%) and 167 male (35.5%). The clinical features of ITP patients are summarized in Table 2. In this study, we examined three novel SNPs of ABCB1 in a comparatively large population of 854 Han Chinese participants (471 ITP patients and 383 healthy controls) by PCR–RFLP. Among ITP group, 356 patients were allocated into GCs responsive arm and 115 patients presented resistance to standard steroid therapy. The explanation of such a disproportionately large recruited cohort of GCs-non-responsive is probably that most ITP patients referred to our hospital were chronic and/or refractory to treatment. All three genotypic distributions in controls and patients did not deviate from Hardy–Weinberg equilibrium. No significant difference was found in allele and genotype frequencies of ABCB1 polymorphisms between ITP patients and controls referring to gender (data not shown). According to the obtained data (Table 3), Chi-square analysis revealed that there were no statistically difference between variants of C1236T and C3435T in the ITP and control group (both p > 0.05), responsive and non-responsive group (p > 0.05). Table 2 Clinical characteristics of ITP patients.
Number of subjects Sex ratio (male/female) Age at onset (years), mean (SD) Min. age at onset (years) Max. age at onset (years) Under 16 years old (n) Above 16 years old (n)
Responsive
Nonresponsive
Total ITP
356 129/227 36.39 (21.58) 0.3 84 88 268
115 38/77 30.32 (18.32)
471 167/304 34.91 (20.84) 0.3 84 120 351
3 71 32 83
Although there was no statistical difference found between ITP and normal samples in terms of G2677T/A polymorphism, our results demonstrated that homozygously mutated (AA/TT/TA) patients were more sensitive to GCs comparing with the wild-type and heterozygous mutated (GG/GA/GT) subjects, and GG genotype had poor response to GCs than GA/GT genotypes. The frequencies of T and A allele were 7.2 times higher in GCs-response group than those in GCs-non-responsive group. Our results indicated that all three SNPs of ABCB1 were in linkage disequilibrium. The SNPs of C1236T and G2677T/A were found to be in strong linkage disequilibrium ((D0 = 0.86), furthermore, moderate but not such strong linkages were found between the SNPs of C1236T and C3435T (D0 = 0.63), G2677T/A and C3435T (D0 = 0.74). A total of eight haplotypes derived from these three variants were reconstructed (Table 4), in which three haplotypes, TTT/TAT (31.4%), CGC (27.0%) and TGC (20.3%) were most frequently observed in the healthy population, while CGC (19.6%), TGC (19.5%) and TTT/TAT (16.1%) were most found in ITP cohort. Furthermore, there were differences in six haplotypes (CGC, CTC/CAC, CTT/CAT, TGT, TTC/TAC and TTT/TAT, in the order of position 1236-26773435) between patients and controls, and striking differences were also found in some haplotypes (CGC, CTC/CAC, CTT/CAT, TGC, TGT, TTC/TAC and TTT/TAT, in the order of position 1236-2677-3435) between GCs responsive and non-responsive arm.
4. Discussion In the past few years, a number of investigators have reported that correlations between the specific polymorphic variants of ABCB1 and pathogenesis of several diseases, such as epilepsy [14], cancers [15] and some autoimmune disorders (such as SLE, inflammatory bowel diseases, cirrhosis and rheumatoid arthritis) were displayed accompanying by resistance to anti-neoplastic
Table 3 Distribution of the genotype and allele frequencies of ABCB1 in all subjects.
C1236T Allele C T Genotype CC CT TT G2667T/A Allele T/A G Genotype T/A T/A G T/A GG C3435T Allele T C Genotype TT TC CC
Responsive N = 356, n (%)
Non-responsive N = 115, n(%)
ITP N = 471, n(%)
Controls N = 383, n (%)
P-value
OR (95%CI)
273 (38.3) 439 (61.7)
93 (40.4) 137 (59.6)
368 (39.1) 574 (60.9)
269 (37.2) 455 (62.8)
0.571a 0.426b
1.091 (0.806–1.479)a 1.084 (0.888–1.324)b
50 (14.0) 173 (48.6) 133 (37.4)
17 (14.8) 59 (51.3) 39 (33.9)
68 (14.4) 232 (49.3) 171 (36.3)
38 (10.5) 193 (53.3) 131 (36.2)
0.800a 0.206b
573 (80.5) 139 (19.5)
82 (35.7) 148 (64.3)
337 (40.2) 501 (59.8)
297 (40.7) 433 (59.3)
7.77e-016a 0.849b
224 (62.9) 125 (35.1) 7 (2.0)
10 (8.7) 62 (53.9) 43 (37.4)
63 (15.0) 211 (50.4) 145 (34.6)
58 (15.9) 181 (49.6) 126 (34.5)
2.22e-015a 0.944b
145 (63.0) 85 (37.0)
408 (57.3) 304 (42.7)
387 (41.1) 555 (58.9)
336 (44.3) 422 (55.7)
0.124a 0.179b
63 (17.7) 178 (50.0) 115 (32.3)
12 (10.4) 61 (53.0) 42 (36.5)
74 (15.7) 239 (50.7) 158 (33.5)
66 (17.4) 204 (53.8) 109 (28.8)
0.174a 0.319b
CI, confidence interval; OR, odds ratio; ITP, immune thrombocytopenia. a Non-responsive group compared with responsive group. b ITP group compared with controls.
7.44 (5.363–10.321)a 1.020 (0.833–1.248)b
1.271 (0.935–1.726)a 1.141 (0.941–1.385)b
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Table 4 Haplotypes derived from C1236T, G2677T/A and C3435T.
CGC CGT CTC/CAC CTT/CAT TGC TGT TTC/TAC TTT/TAT
Responsive N = 356, n (%)
Non-responsive N = 115, n (%)
ITP N = 471, n (%)
Controls N = 383, n (%)
Pa
Pb
25.2 (3.5) 28.1 (4.0) 160.7 (22.6) 59.0 (8.3) 32.3 (4.5) 53.4 (7.5) 189.8 (26.7) 163.5 (23.0)
83.1 (36.1) 6.8 (2.9) 3.1 (1.4) 0.0 (0.0) 51.3 (22.3) 6.9 (3.0) 7.5 (3.3) 71.3 (3.1)
184.2 (19.6) 57.2 (6.1) 90.3 (9.6) 36.3 (3.8) 183.5 (19.5) 142.0 (15.1) 97.0 (10.3) 151.5 (16.1)
185.4 (27.0) 42.4 (6.2) 19.5 (2.8) 6.7 (1.0) 139.4 (20.3) 42.8 (6.2) 34.6 (5.0) 215.2 (31.4)
0.000382* 0.933887 8.96e008* 0.000359* 0.673113 2.94e008* 0.000127* 3.54e013*
2.33e-015* 0.484933 1.95e013* 6.69e006* 2.22e015* 0.015161* 3.57e014* 0.014136*
a
Non-responsive group compared with responsive group. ITP group compared with controls. P value < 0.05.
b *
agents, antidepressants or immune-suppressants with ABCB1 polymorphisms. But some of these genotyping studies demonstrated conflicting results, which potentially attribute to different ethnic or therapy assessed [16]. It is widely accepted that genetic factors play a pivotal role in several aspects of ITP despite it is an autoimmune disorder, including susceptibility of this disease [17], and the response to GCs treatment varies from patient to patient [18]. Recently, three SNPs of ABCB1 (C1236T, C3435T and G2677T/A) were proved to be associated with GCs anti-inflammatory effects [19], glucocorticoid receptors [20] and related complications [21]. However, little is known about the roles of these SNPs and haplotypes in GCs curative effects during treatment of ITP. Based on Pgp encoded by ABCB1 gene, we assumed that SNPs and haplotypes of ABCB1 may have an impact on the expression and function of Pgp, thereby influence the response to GCs therapy in ITP. C1236T is located in exon 12 within ABCB1. CC genotype of C1236T is more frequent than TT genotype in Caucasians [22], whilst less common in Asians [23], which is exactly shown in our data. A study [24] on HIV-1-infected children suggested that C1236T SNPs can down-regulate lopinavir plasma concentration by altering expression or activity of Pgp. But some investigators held different opinions that C1236T SNPs has nothing to do with drug availability in chronic lymphoproliferative diseases [25]. In the present study, we did not find any correlation between C1236T polymorphism and ITP susceptibility or treatment outcome. The G to T and A transversions at position 2677 in exon 21 are missense mutations, causing an amino acid change in Ala893Ser, which ultimately impact the secondary structure of Pgp [26]. Considering their relationship, the effect on treatment outcome in ovarian cancer was also shown by the results of Gréen et al. [27]. Patients with cancer receiving paclitaxel chemotherapy who carry the G2677T/A TT/TA genotype, showed improved progression free survival and response, possibly through a pharmacokinetic mechanism, but the functional consequences of G2677T/A polymorphism have not been extensively clarified yet [27]. In a study on steroid weaning in a pediatric heart transplant population, Zheng et al. [28] concluded that SNPs of C3435T, accompanied by G2677T/A determined the period of steroid administration after transplantation. However, studies on the impact of SNPs of G2677T/A showed conflicting results on some drugs (such as verapamil, cyclosporine, prazosin and bisantrene). The transport is not affected by known variants of Pgp [29–31], indicating substrate specificity of different Pgp variants. In our study, it was confirmed that homozygous (AA/TT/TA) patients with ITP were tend to easily respond to GCs therapy, whereas carriers of wild-type and heterozygous (GG/GA/GT) displayed a relative GCs resistance. Our results suggested that the presence of these mutated alleles may be a feasible predict factor for successful GCs treatment.
Although mechanism of G2677T/A affecting GCs sensitivity is unknown, we speculate that SNPs of G2677T/A might be involved in this process by influence expression of Pgp. Several studies reported that C3435T genotype is associated with the timing of Pgp co-translational folding, which attributed in substrate specificity [31]. There are other evidences proposing that a linkage between C3435T and other SNPs (such as in the promoter/enhancer or intronic regions) within ABCB1 gene is likely to regulate the translation of mRNA and affect posttranscriptional modifications [32]. The study by Hoffmeyer et al. [33] initially reported that C3435T can significantly reduce intestinal Pgp expression in TT homozygotes, leading to higher steady-state plasma concentrations after oral digoxin treatment. This implies that SNP of C3435T could alter Pgp activity through molecular mechanisms. But in our study, we did not found an effect of the allelic variants of the C3435T polymorphism on the outcome of GCs treatment in patients with ITP. Previously, the majority of studies only reported that C3435T and G2677T/A are two tightly linked SNPs and can influence Pgp expression [34], suggesting that the C3435T and G2677T/A haplotype may be involved in determining GCs effects. Until today, it should be noted that C1236T polymorphism, one of the most frequent SNPs of ABCB1, also contributes to the bioavailability of drugs. Several researches provided sufficient evidences to prove that C1236T, in combinations with G2677T/A and/or C3435T, play a key role in modifying the function of ABCB1 gene. Besides a missense polymorphism within G2677T/A cannot be distinguished by PCR–RFLP method along (Fig. 1), the haplotype frequencies of controls we observed are approximately consistent with some previous studies [35,36]. Actually, on the basis of data reported in Han ethnic, TTT are more common than TAT and, CAC are more common than CTC. Interestingly, the frequency of CGC is 36.1% in non-responsive arm and only 3.5% in responsive arm. A similar disparity of the haplotype TGC is displayed between these two arms. However, CTC/CAC, CTT/CAT, TTC/TAC or TTT/TAT exhibits a completely reverse trend between the same arms. Which indirectly reflects that carrier with G allele of G2677T/A is more inclined to be resistant to GCs, while patients with T/A allele of G2677T/A are more sensitive to steroids-therapy.
5. Conclusions In summary, our study showed that ITP patients carrying homozygous mutations in G2677T/A respond better to GCs treatment, which implied that ABCB1 polymorphisms may contribute to interindividual variability of GCs responsiveness. Obviously, further functional studies are needed to elucidate the molecular pathways and we hope to find more feasible biomarkers to predict the individual response to GCs therapy.
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Acknowledgments This work was supported in part by grants of National Natural Science Foundation of China (81070397 and 81270581), Ministry of Science and Technology (2011ZX09302-007-04), Ministry of Health (201202017) and Tianjin Municipal Science and Technology Commission (10JCZDJC19700, 12JCQNJC08000). References [1] Cines DB, Blanchette VS. Immune thrombocytopenic purpura. N Engl J Med 2002;346:995. [2] Gross KL, Lu NZ, Cidlowski JA. Molecular mechanisms regulating glucocorticoid sensitivity and resistance. Mol Cell Endocrinol 2009;300:7. [3] Evans WE, McLeod HL. Pharmacogenomics–drug disposition, drug targets, and side effects. N Engl J Med 2003;348:538. [4] Rosenberg MF, Callaghan R, Ford RC, Higgins CF. Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined by electron microscopy and image analysis. J Biol Chem 1997;272:10685. [5] Schinkel AH, Jonker JW. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv Drug Deliv Rev 2003;55:3. [6] Ozen F, Silan C, Uludag A, Candan F, Silan F, Ozdemir S, et al. Association between ABCB1 (MDR1) gene 3435 C > T polymorphism and colchicine unresponsiveness of FMF patients. Ren Fail 2011;33:899. [7] Lin JH, Yamazaki M. Role of P-glycoprotein in pharmacokinetics: clinical implications. Clin Pharmacokinet 2003;42:59. [8] Sparreboom A, van Asperen J, Mayer U, Schinkel AH, Smit JW, Meijer DK, et al. Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine. Proc Natl Acad Sci USA 1997;94:2031. [9] Jeong H, Herskowitz I, Kroetz DL, Rine J. Function-altering SNPs in the human multidrug transporter gene ABCB1 identified using a Saccharomyces-based assay. PLoS Genet 2007;3:e39. [10] Nobili S, Landini I, Giglioni B, Mini E. Pharmacological strategies for overcoming multidrug resistance. Curr Drug Targets 2006;7:861. [11] Ishikawa T, Hirano H, Onishi Y, Sakurai A, Tarui S. Functional evaluation of ABCB1 (P-glycoprotein) polymorphisms: high-speed screening and structureactivity relationship analyses. Drug Metab Pharmacokinet 2004;19:1. [12] Webster JI, Carlstedt-Duke J. Involvement of multidrug resistance proteins (MDR) in the modulation of glucocorticoid response. J Steroid Biochem Mol Biol 2002;82:277. [13] Rodeghiero F, Stasi R, Gernsheimer T, Michel M, Provan D, Arnold DM, et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood 2009;113:2386. [14] Kwan P, Wong V, Ng PW, Lui CH, Sin NC, Poon WS, et al. Gene-wide tagging study of association between ABCB1 polymorphisms and multidrug resistance in epilepsy in Han Chinese. Pharmacogenomics 2009;10:723. [15] Illmer T, Schuler US, Thiede C, Schwarz UI, Kim RB, Gotthard S, et al. MDR1 gene polymorphisms affect therapy outcome in acute myeloid leukemia patients. Cancer Res 2002;62:4955. [16] Li YH, Wang YH, Li Y, Yang L. MDR1 gene polymorphisms and clinical relevance. Yi Chuan Xue Bao 2006;33:93. [17] Carcao MD, Blanchette VS, Wakefield CD, Stephens D, Ellis J, Matheson K, et al. Fcgamma receptor IIa and IIIa polymorphisms in childhood immune thrombocytopenic purpura. Br J Haematol 2003;120:135. [18] Chang NH, Cheung YH, Loh C, Pau E, Roy V, Cai YC, et al. B cell activating factor (BAFF) and T cells cooperate to breach B cell tolerance in lupus-prone New Zealand Black (NZB) mice. PLoS One 2010;5:e11691.
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