Rapid, Long-Range Molecular Haplotyping of Thiopurine S-Methyltransferase

Rapid, Long-Range Molecular Haplotyping of

Thiopurine S-methyltransferase

(TPMT) *3A, *3B, and *3C

Nicolas von Ahsen,* Victor W. Armstrong, and Michael Oellerich

Background: Haplotyping is an important technique in

molecular diagnostics because haplotypes are often

more predictive for individual phenotypes than are the

underlying single-nucleotide polymorphisms (SNPs).

Until recently, methods for haplotyping SNPs separated

by kilobase distances were laborious and not applicable

to high-throughput screening. In the case of thiopurine

S-methyltransferase (TPMT), differentiating among

TPMT*3A, *3B, and *3C alleles is sometimes necessary

for predictive genotyping.

Methods: The genomic region including the two SNPs

that define TPMT*3A, *3B, and *3C alleles was amplified

by long-range PCR. The resulting PCR product was

circularized by ligation and haplotyped by allele-specific

amplification PCR followed by product identification

with hybridization probes.

Results: Critical points were the long-range PCR conditions,

including choice of buffer and primers, optimization

of the ligation reaction, and selection of primers

that allowed for strict allele-specific amplification in the

second-round PCR. Different underlying TPMT haplotypes

could then be differentiated. Results from the

haplotyping method were in full agreement with those

from our standard real-time PCR method: TPMT*1/*3A

(n
20); TPMT*1/*3C (n
4); TPMT*1/*1 (n
6); and

TPMT*3A/*3A (n
6). One TPMT*1/*3A sample failed

to amplify, and no whole blood was available for repeat

DNA isolation.

Conclusions: This method for rapid cycle real-time,

allele-specific amplification PCR-assisted long-range

haplotyping has general application for the haplotyping

of distant SNPs. The procedure is simpler and more

rapid than previous methods. With respect to TPMT,

haplotyping has the potential to discriminate the genotypes

TPMT*1/*3A (intermediate metabolizer) and

TPMT*3B/*3C (poor metabolizer).

© 2004 American Association for Clinical Chemistry

One of the established targets for pharmacogenetic analyses

is thiopurine S-methyltransferase (TPMT;1 EC

2.1.1.67). Although the physiologic function of the enzyme

is still unknown, TPMT is involved in the metabolism

of the thiopurine drugs 6-mercaptopurine, 6-thioguanine,

and azathioprine. Thiopurine drugs are in common

use for the treatment of acute leukemia and autoimmune

disorders such as chronic inflammatory bowel disease,

and for immunosuppression after organ transplantation

(1 ). Approximately 11% of Caucasians have intermediate

TPMT activity, and 1 in 300 individuals has a complete

TPMT deficiency. Patients with absent or low TPMT

activity accumulate pharmacologically active thioguanine

nucleotides and develop life-threatening toxic side effects,

mostly pancytopenia attributable to myelosuppression,

under standard therapy [for a review, see Ref. (2 )].

Intermediate metabolizers often need a dosage reduction

because of side effects. The molecular bases for many of

the deficient phenotypes are known, and TPMT alleles *1

through *15 have been defined, with *1 referring to the

wild-type allele [see Ref. (3 ) and the references therein].

The most common mutations are TPMT*3A in Caucasians

and TPMT*3C in African and Asian populations (4 ). The

TPMT*3B and *2 mutations are much less common,

whereas the remaining TPMT mutations are extremely

rare or private mutations.

It is generally recommended that erythrocyte TPMT

activity be measured before initiation of standard-dose

thiopurine therapy. However, erythrocyte TPMT activity Department of Clinical Chemistry, George-August University, GoЁttingen,

Germany.

*Address correspondence to this author at: George-August University,

Department of Clinical Chemistry, Robert-Koch-Strasse 40, 37075 GoЁttingen,

Germany. Fax 49-551-39-12504; e-mail nahsen@gwdg.de.

Received March 24, 2004; accepted June 10, 2004.

Previously published online at DOI: 10.1373/clinchem.2004.034751

1 Nonstandard abbreviations: TPMT, thiopurine S-methyltransferase; ASA

PCR, allele-specific amplification PCR; SNP, single-nucleotide polymorphism;