Renal Failure, 33(9): 899–903, (2011)Copyright © Informa Healthcare USA, Inc.ISSN 0886-022X print/1525-6049 onlineDOI: 10.3109/0886022X.2011.605980

LABORATORY STUDY

Association between ABCB1 (MDR1) Gene 3435 C>T Polymorphismand Colchicine Unresponsiveness of FMF Patients

Filiz Ozen1, Coskun Silan2, Ahmet Uludag3, Ferhan Candan4, Fatma Silan3, Semra Ozdemir5,Sinem Atik3 and Ozturk Ozdemir1,3

1Department of Medical Genetics, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey; 2Department ofPharmacology, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey; 3Department of MedicalGenetics, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey; 4Department of Internal Medicine,Faculty of Medicine, Cumhuriyet University, Sivas, Turkey; 5Department of Nuclear Medicine, Faculty of Medicine,Cumhuriyet University, Sivas, Turkey

Abstract

The multidrug resistance gene-1 (MDR1, adenosine triphosphate-binding cassette transporter: ABCB1, P-glycoprotein)encodes membrane proteins that play a crucial role in protecting cells from xenobiotics, chemicals, and drugs. The TTgenotype of 3435 codon in exon 26 of MDR1 gene causes overexpression of gene activity and effluxes many chemi-cally diverse compounds across the plasma membrane. We studied the association between C3435T polymorphisms(single nucleotide polymorphism) of MDR1 gene and colchicine-resistant familial Mediterranean fever (FMF) patients.Total genomic DNA samples from 52 FMF patients of colchicine unresponsiveness were used for FMF (MEFV ) andMDR1 genes profile analyses. Target genes were genotyped by multiplex PCR-based reverse-hybridization Strip Assaymethod. The preliminary current results showed increased T allele frequency (0.596) in colchicine unresponsiveness ofFMF patients. The distributions of the CC, CT, and TT genotypes in colchicine nonresponder FMF patients were 17%,46%, and 37%, respectively. Our results indicate that C3435T polymorphism in exon 26 of MDR1 gene is associatedwith colchicine resistance in nonresponder FMF patients during the common therapy protocol.

Keywords: C3435T polymorphism, colchicine resistance, FMF, MDR1

INTRODUCTION

The multidrug-resistant transporter MDR1/P-glyco-protein (P-gly), the gene product of MDR1, is a glyco-sylated membrane protein of 170 kDa, belonging to theadenosine triphosphate (ATP)-binding cassette (ABC)superfamily of membrane transporters.1 MDR1 wasoriginally isolated from resistant tumor cells as partof the mechanism of multidrug resistance, but overthe past decade, it has been elucidated that humanMDR1 is also expressed throughout the body to conferintrinsic resistance to the tissues by exporting unnec-essary or toxic exogenous substances or metabolites.2

A number of various types of structurally unrelateddrugs are substrates for MDR1, other transporters arerecognized as an important class of proteins for regu-lating pharmacokinetics and pharmacodynamics. It is

Address correspondence to Ozturk Ozdemir, Department of Medical Genetics, Faculty of Medicine, Canakkale Onsekiz MartUniversity, 17100 Canakkale, Turkey, Tel.: +90 286 2180018/2079; E-mail: ozturkozdemir@comu.edu.tr

Received 5 April 2011; Revised 10 July 2011; Accepted 12 July 2011

a transmembrane efflux transporter with an importantrole in drug therapy.3 The level of P-gly expression leadsto relevant consequences in terms of efficacy and toxic-ity by modulating drug disposition. A single nucleotidepolymorphism in the exon 26 of the gene C3435T wasrecently associated to P-gly levels, substrate uptake, dif-ferences in MDR1 expression, function, drug response,and disease susceptibility.4,5

Familial Mediterranean fever (FMF) is a genetic dis-order characterized by recurrent attacks of fever andinflammation in the peritoneum, synovium, or pleura,accompanied by pain. Renal failure is most severe com-plication and may develop without overt crises.6 FMF ismostly inherited as autosomal recessive (MIM 249100),but autosomal dominant inheritance is also possible(MIM 134610). FMF is frequent in Turks, Jews, Arabs,

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and Armenians. The studied population has a high rateof FMF carriers.7–9

Colchicine is used chiefly in the treatment of FMFbut is also valuable in other inflammatory diseases suchas Behçet’s disease, gout, and recurring pericarditiswith effusion.9,10 Three proteins play pivotal roles incolchicine pharmacokinetics: the colchicine receptor,tubulin, governs the plasma elimination half-life of thedrug; intestinal and hepatic CYP3A4, key to the bio-transformation of colchicine; and P-gly, a cell effluxpump that regulates the tissue distribution of colchicine,as well as its excretion via the biliary tract and kidneys.Pharmacokinetic studies have been performed using aradioimmunology assay to measure blood colchicinelevels. Absorption after oral ingestion varies widely(from 24% to 88% of the dose), the volume of distribu-tion is extremely large (7 L/kg), and binding to albuminis moderate.11 Colchicine is excreted chiefly through theliver and has an elimination half-life of 20–40 h. Withrepeated doses of about 1 mg/day, the steady state isachieved within 8 days and concentrations range from0.3 to 2.5 ng/mL. Studies of associations between phar-macokinetic parameters and pharmacodynamics showthat effects are correlated, not to plasma levels, but tolevels in leukocytes. Adverse events are not uncommon,most notably when colchicine is used in combinationwith drugs that interact with CYP3A4 and/or P-gly,thereby decreasing the renal and/or hepatic eliminationof colchicine. Careful monitoring of this situation iseffective in preventing the development of toxicity.12

The overexpression of P-gly may be associated withchanges in metabolism of sugars as well as energy pro-duction. Structural and ultrastructural characteristicsof multidrug-resistant cells expressing P-gly are typicalfor cells engaged in a metabolically demanding pro-cess of protein synthesis and transport. P-gly-mediatedMDR phenotype is often also associated with alterationsin cytoskeletal elements, microtubule and mitochon-dria distribution, Golgi apparatus, chromatin texture,vacuoles, and caveolae formation.13

P-gly is a complex molecule containing site(s) ableto bind compounds with a molecular weight of sev-eral hundreds and comprising hydrophobic and/or baseregions that capture and transport the numerous unre-lated substances under physiological conditions. Drug-binding sites that are able to recognize substanceswith different chemical structures may have a complexarchitecture in which different parts are responsiblefor binding of different drugs.14 For P-gly substratesand inhibitors, a pharmacophore-based model hasbeen described. The pharmacophores have to containparts with hydrophobic and aromatic characteristicsand functional groups that can act as hydrogen-bonddonors and/or acceptors. Several drugs are known to beP-gly antagonizing agents. They represent a large groupof structurally unrelated substances that can act viadirect interaction with P-gly and inhibition of its trans-port activity, or via possible modulation of processes

(such as phosphorylation) regulating P-gly transportactivity.15

Functional polymorphism of the MDR1 gene(C3435T) correlated with MDR1 in vivo expressionand activity.16 Expression increases in the polymor-phic gene status and makes protein level twice than thewild profile. Intrinsic and/or acquired mutated MDRplays a causal and potentially modifiable role in clini-cal nonresponsiveness to colchicine is a topic of currentinterest.

MATERIAL AND METHODS

Patients, Clinical Diagnosis, and Laboratory AssessmentThe study protocol was approved by the HospitalEthics Committees of Cumhuriyet University Faculty ofMedicine. The informed consents of the patients werealso obtained. Total 52 colchicine unresponsive FMFpatients (29 male and 23 female) with ages ranging from7 to 41 years experiencing at least 2 attacks per month,despite regular colchicine administration, were includedin the current study.

Mutation AnalysisTotal genomic DNA was extracted from 100 µL periph-eral blood samples from patients and healthy controlsby the Invitek kit extraction technique (Invitek, Invisorbspin blood, Germany) and stored at −20◦C until geneticanalysis was performed. MEFV and MDR1 genes weresimultaneously amplified and biotin-labeled in a sin-gle multiplex amplification reaction (Viennelab, StripAssay GmbH, Austria). PCR for both MDR1 gene andMEFV gene was performed in a Perkin Elmer 9600,Singapore and the protocol consisted of an initial melt-ing step of 2 min at 94◦C; followed by 35 cycles of 15s at 94◦C, 30 s at 58◦C, and 30 s at 72◦C; and a finalelongation step of 3 min at 72◦C. The mutation anal-ysis was performed by Strip Assay technique (ViennaLab, Strip Assay GmbH, Austria) which is based on thereverse-hybridization principle automatically.

RESULTS

Colchicine is the standard therapy for preventing febrileattacks, development of amyloidosis by suppressinginflammation in FMF patients. Even though somemutated patients (5–10%) develop amyloidosis dur-ing colchicine treatment due to drug resistance.17

MEFV -mutated FMF patients that develop resistanceto colchicine treatment were studied in the pre-sented results. The mutation types of MEFV geneare shown in Table 1. Heterozygous mutations wereE148Q 19.23%, P369S 1.92%, M680I(G/C) 3.84%,and M694V 48.07; homozygous mutations were E148Q1.92%, M694V 17.3%; combined mutations wereE148Q/M694V 5.76%, M694V/V726A 1.92%. FMFpatients with minor and/or major acute phase reac-tants after colchicine treatment were studied in the

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Table 1. The genotype and mutation type distribution of theMEFV gene in current colchicine unresponsiveness of FMFpatients.

Mutation type Genotype n %

Heterozygous E148Q 10 19.23P369S 1 1.92F479L – –M680I(G/C) 2 3.84M680I(G/A) – –1692del – –M694V 25 48.07M697I – –K695R – –V726A – –A744S – –R761H – –

Homozygous E148Q 1 1.92M694V 9 17.3V726A – –

Combined E148Q /M694V 3 5.76M694V/V726A 1 1.92V726A/E148Q – –

Patients withmutation

52 100

Patients withoutmutation

– –

current study (Table 1). The TT genotype of 3435codon in exon 26 of MDR1 gene causes overexpressionof gene activity and effluxes many chemically diversecompounds across the plasma membrane. We aimed tofind out the T allele frequency in C3435T polymor-phisms of MDR1 gene in current colchicine-resistantFMF patients. The distributions of the CC, CT, and TTgenotypes in 52 colchicine nonresponder FMF patientswere 17% (9 patients), 46% (24 patients), and 37%(19 patients), respectively (Figure 1). Current prelimi-nary results showed that the mutated T allele frequencywas higher than (0.596) the wild C allele (0.404) in thecurrent colchicine unresponsiveness of FMF patients(Table 2).

DISCUSSION

Colchicine is an alkaloid drug that is commonly usedin patients and syndromes such as FMF, gout, Behçet’sdisease, psoriasis, and Sweet’s syndrome. ATP-bindingcassette superfamily B member 1 (ABCB1 or MDR1,P-gly) is a drug transporter that extrudes colchicineout of cells. C3435T polymorphism in ABCB1 gene

CC17%

CT46%

TT37%

Figure 1. The genotype frequencies of MDR1 gene [P-glyco-protein, ATP-binding cassette (ABC) superfamily of membranetransporters] C3435T polymorphism in colchicine unresponsive-ness of FMF patients.

has been demonstrated to alter multidrug resistance inmononuclear cells and may alter response to colchicine.We aimed to investigate the association between MDR1C3435T polymorphism and colchicine-resistant FMFpatients. In many studies, the frequency of 3435Tvariant, most often associated with a decreased P-glyactivity, is significantly higher compared to populationsof African and Asian origin.18–22 Colchicine is a main-stay of treatment in FMF; however, 5–10% of patientsdo not respond to colchicine.

In recent years, it has become apparent that transportproteins play a major role in controlling the distribution,elimination, and potentially the metabolism of somedrugs, including organic cation transporters, organicanion transporters, and P-gly. P-gly has received con-siderable attention in recent years both as a barrierto drug absorption and distribution, and as a poten-tial source for variability in drug pharmacokinetics andpharmacodynamics.

The currently available data on the influence ofMDR1 polymorphisms on drug disposition are very dif-ferent. Hoffmeyer et al.17 reported in accordance withthe reported lower intestinal P-gly expression in the3435TT group, higher maximal digoxin plasma concen-trations during steady state in the TT group in compar-ison to subjects with the CC genotype.16 In accordancewith their own data on MDR1 mRNA expression (andin contrast to findings by Hoffmeyer et al.17), Sakaedaet al. reported higher area under the curve values aftera single oral dose of digoxin 0–4 h in Japanese sub-jects with the 3435CC genotype compared to CT and

Table 2. Mutation distribution, genotype, and allele frequency of the MDR1 gene [P-glycoprotein, ATP-binding cassette(ABC) superfamily of membrane transporters] in the current colchicine unresponsiveness of FMF patients.

Gene Exon Mutation type (3435C>T transition)

Colchicine unresponsiveness ofFMF patients (n = 52) Allele frequency

Genotype n (%)

MDR1 (ABCB1) 26 Wild CC 9 17 Allele FQHeterozygous mutated CT 24 46 C 0.404Homozygous mutated TT 19 37 T 0.596

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TT.16,23,24 The reasons of those apparent discrepanciesare unknown at the moment.

Since in vitro data indicate that phenytoin is a (mod-erate) P-gly substrate, Kerb et al. studied the potentialimpact of the MDR1 C3435T polymorphism on pheny-toin disposition.25,26 After a single oral dose of pheny-toin, there was a nonsignificant trend to higher plasmaconcentrations in the TT group in comparison tohealthy volunteers with the CT or CC genotypes. More-over, they found that the CC genotype is significantlymore common in volunteers with low-phenytoin plasmaconcentrations.26 Taken together, MDR1 3435 geno-type appears to have some effect on phenytoin plasmaconcentrations, the major determinant of plasma pheny-toin concentration is the CYP2C9 genotype. Thereare also studies with other P-gly substrates, whichdo not find an association between 3435TT geno-type and increased drug concentrations (fexofenadine,loperamide, talinolol).27

Colchicine is the only known drug to prevent FMFattacks and FMF-associated amyloidosis. The dose ofcolchicine administered varied from 1.0 to 3.0 mgdaily, with most patients taking 1.0–1.5 mg; responseconsisted of reductions in the frequency, severity, andduration of attacks. But in some patients colchicine maynot be effective or cannot be well tolerated. Only thearticular manifestations of FMF appeared to respondpoorly to colchicine. Long-term colchicine therapy wasestablished as quite safe, with only mild and infre-quent side effects, and highly effective.28–31 In a study,M694V/M694V homozygous mutation is associatedwith lower response to colchicine treatment.32,33 Wedetected nine patients with M694V/M694V homozy-gous mutation; with these results it should beconsidered that colchicine-unresponsive patients needdifferent mutation analyses and the medical historyto understand the causes of unresponsiveness. Seyahiet al. reported that regular colchicine treatment cannotcontrol FMF attacks in about 5–10% of the patients.34

There are a few studies about MDR1 C3435T poly-morphism and colchicine unresponsive FMF patients’relationship. In our study, T allele frequency (60%)was higher than C allele frequency (40%) in colchicine-unresponsive FMF patients. According to Tufan et al.,patients with the TT genotype for the ABCB1 3435Cto T variant responded better to colchicine in terms oftreatment efficacy and colchicine dose requirements.35

No relationship was detected between MDR1 C3435Tpolymorphism and colchicine response in patientswith Behçet’s disease.36 In addition, our colchicine-unresponsive patient population is more than twice theother studies and it makes our results more reliable.In a study, another mutation of MDR1 (G185V) wasfound to increase colchicine resistance.37 Chen et al.have studied many polymorphisms of MDR1 gene anddetected that the C allele is more frequent than Tallele.38 Similar results were reported in another studyfrom Spain population.39

The clinical response of drugs can be affected withmany environmental and genetic factors, and manyenzymes and proteins are important in a lot of drugs’metabolism and transport. Annereau et al.40 identi-fied 13 differentially expressed genes associated withcolchicine resistance. There may be very different fac-tors that play role in colchicine efficiency during thetherapy. Current results showed us the MDR1 3435C>T polymorphism is associated with colchicine unre-sponsiveness in mutated FMF patients. Studies dealingwith the MDR1 mutations were also pointed out; themononuclear cells that lack of P-gly membranal effluxpump may also provide an explanation for nonrespon-der FMF patients during the colchicine treatment.

CONCLUSION

Fifty-two patients were evaluated as nonresponders inthe current results. Preliminary results revealed an asso-ciation between gene that codes for the drug effluxtransporter ATP-binding cassette superfamily B mem-ber 1 (ABCB1, MDR1) and colchicine response inpatients with FMF. The distributions of ABCB1 CC,CT, and TT genotypes were significantly different andthe T allele frequency was higher than the C allele inthose groups. Results showed that the mutated T allelefrequency was higher than (0.596) the wild C allele(0.404) in the current colchicine unresponsiveness ofFMF patients.

Declaration of interest: Authors report no conflicts ofinterest.

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