Alimentary tract and pancreas

Alimentarni trakt i pankreas

ARCH GASTROENTEROHEPATOL 2003; 22 ( No 1 - 2 ): 5 - 9

1Njegica Joji},

2Jelena Uroševi},

1Bo`idar Boji},

2Sonja Pavlovi}.

1Department of Gastroenterology,

Zvezdara Clinical Center,

2Institute for Molecular Genetics and Genetic

Engineering, Belgrade, Serbia

.

Determination of thiopurine

methyltransferase genotype

in the patients with inflammatory

bowel disease before

and during azathioprine

therapy

Key Words:

Thiopurine S-methyltransferase,

inflammatory bowel disease,

azathioprine, leukopenia.

Abstract

Background: Azathioprine (AZA) is very effective in treating chronically active patients with

inflammatory bowel disease (IBD), both Crohn's disease (CD) and ulcerative colitis (UC). Therapy

with AZA is long lasting and generally safe. Some patients (about 2%) develop myelosuppresion.

Low activity of enzyme thiopurine S-methyltransverase (TPMT) due to mutation of TPMT gene is

responsible for bone marrow suppression.

Methods: Polymerase chain reaction - based methods PCR (ARMS) and RFLP method were used

for determination of TPMT gene mutations. All patients were investigated for 3 most frequent

mutant alleles. The numbers of TPMT gene mutations were analyzed in 35 IBD patients and control

group consisted of 100 volunteer blood donors. In the IBD group, patients with myelosuppression

were clinically evaluated.

Results: Thirty-five patients, 24 with CD and 11 with UC enrolled this study. Twenty-seven were

already on AZA therapy lasting from 1 to 84 mounts; 8 patients were examined before AZA administration.

Thirty-three ( 94.2 % ) cases had normal genotype (TPMT*1/TPMT*1) and 2 ( 5.7 % )

were heterozygous ( TPMT*1/TPMT*3 ) with one normal ( TPMT*1 ) an one mutant allele

(TPMT*3 ). Mutant allele TPMT*3 combined two mutations G460A and A719G. Mutant alleles

TPMT*2, TPMT*3B and TPMT*3C were not detected. In the IBD group 14 (40 %) patients had

leukopenia (WBC count <3000/mm3) developed 15 days to 36 mounts after AZA therapy; one

patient with CD developed serious pancytopenia two weeks after starting AZA. Patient with early

AZA related pancytopenia had normal genotype. Thirty (85.7%) patients received concomitant oral

5 - ASA therapy. In the control group, 4 (4 %) volunteers had mutations; 3 ( 1.5 % ) the most frequent

mutant allele TPMT*3A and 1 (0.5 %) very rare, TPMT*3B. Ninety-six ( 96% ) volunteers

had wild type, normal allele TPMT * 1.

Conclusion: Determination of TPMT activity by genotype could identify TPMT homozygotes that

should avoid 6MP/AZA in therapy or TPMT heterozygotes that require reduction in doses. For clinical

use monitoring of WBC counts during therapy was mandatory. Determination of TPMT genotype

should be reserved for selected cases.

Kljucne reci:

Tiopurine S- metiltransveraza,

inflamatorne bolesti creva,

azatioprin, leukopenija

Sazetak

Uvod: Azatioprin (AZA) je veoma efikasan u le~enju hroni~no aktivnih pacijenata sa inflamatornim

bolestima creva (IBC), kako Crohn - ove bolesti (CB) tako i ulceroznog kolitisa (UK). Primena

azatioprina je dugotrajna i bezbedna. Ipak kod oko 2 % pacijenata mo`e da dodje do pojave mijelosupresije

u toku terapije. Sni`ena aktivnost enzima thiopurine S - metiltransveraze (TPMT ) zbog

mutacije TPMT gena je odgovorna za to.

Metod: Odredjivanje prisustva mutacije u TPMT genu vr{eno je PCR (ARMS) i RFLP metodom.

35 pacijenata sa IBC i kontrolna grupa koju je ~inilo 100 dobrovoljnih davalaca krvi testirana su na

tri naj~e{}e mutacije u TPMT genu. Pacijenti kod kojih je do{lo do pojave lekupenije su posebno

klini~ki evaluirani.

Rezultati: U studiju je u{lo 35 pacijenata, 24 sa CB i 11 sa UK. 27 pacijenata je bilo na terapiji AZA

u trajanju od 1 do 84 meseci ; 8 pacijenata je testirano pre zapo~injanja terapije AZA; 33 ( 94.2%)

pacijenta su imala normalan genotip (TPMT*1/TPMT*1) a 2 (5.7 %) su imala mutirani genotip

(TPMT*1/TPMT/*3A). Oba su bili heterozigoti sa jednim normalnim TPMT*1 i jednim mutiranim

alelom TPMT*3A. Mutiran alel TPMT*3 sadr`i dve udru`ene mutacije G460A i A719G. Mutirani

aleli TPMT*2, TPMT*3B i TPMT*3C nisu bili prisutni u grupi pacijenata. 14 ( 40 % ) pacijenata sa

IBC je imalo leukopeniju (WBC < 3,000 mm3) koja se je javila periodu od 15 dana do 36 meseci

nakon uvodjenja AZA u terapiju. Kod jednog pacijenta sa CB do{lo je do pojave pancitopenije 15

dana od primene AZA u dozi od 1mg/kgtt. Ovaj pacijent je imao normalan TPMT genotip. 30

(85.7%) pacijenata je u terapiji pored AZA dobijalo i neki od oralnih preparata 5 -ASA. U kontrolnoj

grupi 96 (96 %) davalaca krvi je imalo normalan TPMT*1 alel. Mutacije su nadjene kod 4 (4%)

osobe. Tri (1,5 %) su imale naj~e{}i mutierani alel TPMT*3A. Jedan (0,5%) davaoc je imao veoma

redak mutirani alel.TPMT* 3B.

Zaklju~ak: Odredjivanjem TPMT genotipa mogu da se identifikuju osobe koje su homozigoti za

mutaciju u TPMT genu i kojima ne treba davati u terapiji 6MP/AZA ili heterozigoti kod kojih se

obavezno redukuje ali ne prekida terapija. Odredjivanje broja leukocita i dalje predstavlja zlatni standard

u pra}enju pacijenata na terapiji 6-MP/AZA.

.

INTRODUCTION

In the last few years the use of 6-Mercaptopurine

(6MP) and its prodrug azathioprine (AZA) for the treatment

inflammatory bowel disease (IBD) has been

increased. This therapy is efficacious in the patients with

chronic active Crohn's disease (CD) and ulcerative colitis

(UC), steroid -dependent and steroid refractory, fistulizing

CD and remission maintenance in both diseases (1). It is

recognized that 6-MP and AZA, even at standard dose can

cause severe myelotoxicity (about 1-2 %) in patients with

TPMT (thiopurine S-methyltransverase) deficiency and

IBD (1,2 ). Allelic variants of the TPMT gene are responsible

for the changes in the enzyme activity and possible

bone marrow suppression (3,4). AZA and 6-MP are purine

analogues. They have antiproliferative effects on the activated

lymphocytes responsible for inflammation (5). Both

drugs are inactive compounds, which must be converted to

6-thioguanine (6-TG), active metabolite responsible for

immunosuppressive effect. AZA is absorbed into plasma

and rapidly converted to 6-MP via nonenzymatic pathway

6-MP is further metabolized by three competitive

enzymes: xanthine oxidase (XO), thiopurine methyltransferase

(TPMT) and hypoxanthine-guanine phosphoribosyltransverase

(HGRT). Only HGRT anabolizes 6-MP into the

6- thioguanine ( 6-TG ) active nucleotide responsible for

the therapeutic activity and drug related leucopoenia.

Reduction in TPMT activity as a result of allelic variants of

TPMT gene may produce bone marrow suppression

because of preferential metabolism of 6-MP to 6-TG.

There are interindividual and interethnic variations in the

TPMT activity as a result of TPMT gene polymorphism.

Most people ( 88 % ) have genotype with two high (normal)

metabolizing alleles and normally metabolize 6-MP

and AZA. 11 % have one high and one low (mutant) allele

and 0.3 % have two low metabolizing alleles (6,7,8).

Mutations occurred alone or in combination on different

alleles of the TPMT gene and may produce functional consequence.

We investigated mutations of TPMT gene in the

IBD patients who were on AZA therapy and its practical

and clinically use in the monitoring myelosuppression.

MATERIAL AND METHODS

From January 2002 to April 2003, 35 patients with

proven IBD were analyzed, 24 with CD and 11 with UC.

Twenty-seven were already on oral AZA therapy lasting

from 1 to 84 months. Eight patients were examined before

AZA administration. All patients had complete blood

counts measured weekly for the first month and then

monthly for the next months. TPMT gene mutations were

analyzed in the IBD patients and control group consisted of

100 volunteer blood donors. Polymerase chain reaction -

based method ( PCR ) Amplification Refractory Mutation

System ( ARMS ) and Restriction Fragment Length

Polymorphism ( RFLP ) method were used for determination

of TPMT gene mutations (6,7). This strategy allows

identifying known mutations of the gene. DNA sample

were investigated for normal and three most frequent

mutant alleles. Point mutations of the gene were known to

occur either on the same allele or on the different alleles.

The most frequent mutant allele in white population

TPMT*3A combined two mutations G460A and A719G on

the same allele (6, 8, 9). Identification of genotype can predict

TPMT phenotype and recognize the patients at higher

risk of myelotoxicity during AZA/6-MP therapy (6). Some

of these TPMT alleles are functional as *1, *1S, and *1A,

or nonfunctional as *2, *3A, *3B, *3C, *3D, *4, *5, *6,

*7. Individuals may be homozygous or heterozygous for

one or two functional or nonfunctional mutations, which

predict their TPMT genotype associated with enzyme

activity. For statistical analysis descriptive statistics, frequencies

and group analysis were performed.

Figure 1. Azathioprine ( AZA ) metabolism.

RESULTS

Between January 2002 and April 2003 there were 35

patients with IBD who had TPMT genotyping ( 24 with

CD and 11 with UC, 22 females, 13 males, ages 19 - 48,

median 31 ). Thirty-three ( 94,2 % ) patients were homozygous

with two wild type ( normal ) TPMT*1/TPMT*1 alleles

corresponding to high enzyme activity. Two ( 5,7 % )

patients, one with CD and one with UC were heterozygous

( TPMT*1/TPMT*3 ) with one wild type TPMT*1 and one

mutant allele TPMT*3A ( Table 1 ). This mutant allele

combines the two mutations: G460A, which results guanine

to adenine substitutions at codon 460 ( G460 ? A ),

and A719G adenine to guanine substitution at codon 719

( A719 G ) ( Figure 2 ). These mutations correspond to

intermediate enzyme activity. Genotype

TPMT*1/TPMT*2, TPMT*1/TPMT*3B and

TPMT*1/TPMT*3C were not detected in IBD group.

Frequency of TPMT*1 normal allele were 97.1 % and of

TPMT*3 mutant allele 2.9 % . We also determined TPMT

genotype in the control group consisting of 100 volunteer

blood donors. 96 ( 96 % ) volunteer had normal genotype

TPMT *1/TPMT *1. Four ( 4 % ) had mutant genotypes,

3 ( 3 % ) TPMT *1/TPMT *3 and 1 (1 % ) very rare

TPMT*1/TPMT*3B genotype. Frequency of TPMT* 1

normal allele in the control group were 98%, mutant alleles

TPMT*3 1.5 % and TPMT *3B 0.5 %. Twenty-seven

IBD patients received AZA therapy in dose of 1 -

2.5mg/kgtt lasting from 1 to 84 mounts. Eight patients

were examining before AZA therapy. In the IBD group 14

patients had leukopenia ( WBC < 3,000/mm3 ) developed

15 days to 36 mounts after starting AZA. Three patients

had serious leukopenia ( WBC < 2,500/ mm3 ) receiving

doses of AZA between 1.5 - 2mg/kgtt, two, eight, nine

and 16 mounts after starting therapy ( Table 2 ). Thirty

patients received concomitant 5- ASA therapy in dose of

1.5 - 4 g/day. Only one patient with CD had pancitopenia

two weeks after AZA therapy at dose of 1mg/kgtt. This

patient stopped AZA immediately. She didn't have any

concomitant medication during AZA therapy and her

TPMT genotype was normal ( TPMT*1/TPMT*1 ). She

was on Sulphasalazine ( SASP) 3g/day one year ago and

stop with them three mont before starting AZA During

SASP therapy leukopenia was not developing. Her hematologist

confirms normal cellularity of bone marrow.

Patients with mutant genotype ( TPMT*1/TPMT*3 ) had

WBC counts between 2.600 - 3.000/ mm3 during AZA

therapy. Patient with CD and mutant genotype received

AZA in dose of 125mg/day for 11 monts. Patient with UC

and mutant genotype was on the same AZA dose for 24

mounts. Both patients had concomitant medication, one

with 5-ASA in dose of 1,5g/day and second one with SASP

in dose of 3g/day. Daily AZA dose was reduced in both

patients but not stopped. 5-ASA and SASP therapy was

started three and five years ago but patients never developed

leukopenia. None of the 8 patients examining before

starting AZA therapy developed TPMT gene mutation.

Figure 2. Detection of G460A mutation in TPMT gene

with PCR RFLP1 method.

Table 1. Distribution of TPMT genotype in IBD patients and control group.

Table 1. AZA dosage and WBC count in IBD patients

with normal and mutant TPMT genotype

DISCUSSION

In the last few years 6MP/AZA is widely used for the

treatment of chronic active IBD, mostly CD. This therapy

is long lasting and safe. About 2 % of patients with TPMT

enzyme deficiency are intolerant to AZA and may develop

leukopenia during the treatment (1.2). Bone marrow suppression

is due to excessive accumulation of cytotoxic

metabolites as a result of higher 6-MP conversion to 6-

thioguanine nucleotides (10,11). These patients should not

receive 6-MP or AZA for IBD treatment. This was the reason

for TPMT analysis in IBD patients (12). Colombel et

al. confirmed that myelosuppression occurred within 1.5

months in TPMT homozygous patients, but much more

later in heterozygous or in patients without mutations (3).

TPMT enzyme activity may be determined by genotype or

phenotype which means by direct measurement of red

blood cell (RBC) enzyme activity . In the beginning genotyping

was the only commercially available test. Today

determination of TPMT phenotype is also commercially

available and may provide a more accurate assessment of

enzyme activity (4). To make a choice, one or the other

method depends on laboratory possibilities. We investigated

TPMT genotyping in 35 IBD patients during AZA therapy

and found that only 2 (5.7%) patients were heterozygous

with one wild (TPMT*1) and one mutant (TPMT*3)

allele that correspond to intermediate TPMT activity (3,4).

In the control group 4 (4%) individuals had TPMT gene

mutations. Frequency is similar as in a European control

population where 10 % had mutations (12). Fourteen IBD

patients during AZA therapy developed leukopenia (WBC

< 3.000 mm3), only two patients had TPMT gene mutations.

They were 11 and 24 mounts on AZA (2mg/kgtt),

without serious leukopenia during monitoring. Both

patients were heterozygous with nonfunctional TPMT*3A

alleles and intermediate enzyme activity. This was the reason

to reduce daily AZA dose.Mutations that correspond to

alleles *2, *3A and *3C are most frequent in Europe (12).

All patients in our study with leukopenia also had concomitant

oral 5 -ASA therapy that has been shown to

inhibit TPMT activity and increase the serum 6-TG levels

and predispose patients with AZA therapy to leukopenia

overlooked etiology was possible in the majority of these

patients.

In conclusion: determination of TPMT activity by

genotype could identify TPMT homozygotes that should

avoid 6MP/AZA in therapy or TPMT heterozygotes that

require reduction in doses and careful introduction of the

other drugs that may interfere with myelotoxicity during

AZA therapy. For clinical use monitoring of the WBC and

platelet counts during 6MP/AZA therapy was mandatory.

Determination of genotype or phenotype is much more

expensive and should be reserved for selected patients

only.

(13,14). Only one patient developed serious pancytopenia

15 days after AZA administration at dose of 1mg/kgtt.

This patient had normal TPMT genotype. She was on

SASP for two years and stopped with them three mounts

before being treated with AZA . She also received metronidazol

eight monts before AZA therapy. Both drugs may

cause myelosuppression (13,15). Her hematologist

excluded bone marrow irregularity. She did not have any

viral infection known to induce neutropenia or trombopenia

( 16,17). TPMT phenotype or direct measurement of

enzyme activity may provide a more accurate assessment

of metabolic activity and toxicity in these patients (18).

Finally, no obvious cause of bone marrow suppression or

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