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Clinical Chemistry 50: 1528-1534, 2004. First published July 9, 2004; 10.1373/clinchem.2004.034751
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(Clinical Chemistry. 2004;50:1528-1534.)
© 2004 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Rapid, Long-Range Molecular Haplotyping of Thiopurine S-Methyltransferase (TPMT*) *3A, *3B, and *3C

Nicolas von Ahsena, Victor W. Armstrong and Michael Oellerich

1 Department of Clinical Chemistry, George-August University, Göttingen, Germany.

aAddress correspondence to this author at: George-August University, Department of Clinical Chemistry, Robert-Koch-Strasse 40, 37075 Göttingen, Germany. Fax 49-551-39-12504; e-mail nahsen{at}gwdg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 assays will not reflect the metabolic capacity of a deficient patient if that patient has received a blood transfusion from a donor with normal TPMT activity within the previous 3 months. In addition, misclassification is possible because TPMT activity can be induced under therapy with thiopurine drugs. Genotyping is an important diagnostic tool in such situations.

Genotyping can be misleading in rare situations, however. As shown in Fig. 1 , the TPMT*3A allele is defined by the presence of the 460G>A and 719A>G mutations in cis, whereas TPMT*3B (460G>A) and TPMT*3C (719A>G) alleles each harbor only one of these mutations. Conventional genotyping methods cannot discriminate the genotype TPMT*3A/*1 from TPMT*3B/*3C because they do not detect the linkage of mutations that define the underlying allele or haplotype. To date, haplotypes have been studied by pedigree analysis or by molecular biological techniques that are either laborious (cloning) or, in the case of allele-specific amplification PCR (ASA PCR), impossible over long genomic regions. McDonald et al.(5) recently described an elegant method to make haplotyping more practical for the routine molecular diagnostic laboratory, but the published initial application to TPMT genotyping was not focused on the main diagnostic problem, discrimination between TPMT*3A/*1 and TPMT*3B/*3C. Moreover, the method made no use of modern real-time PCR instrumentation for mutation detection.



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  		Figure 1. TPMT genomic structure.

Exons are displayed as closed boxes, introns as lines, and noncoding regions as open boxes. Exon 2 is shown with dashed lines to indicate that it is absent from most transcripts(9). The SNPs that define TPMT*3A, *3B, and *3C are shown.

We therefore developed a method that uses a combination of long-range PCR with intramolecular ligation followed by real-time PCR with hybridization probes for haplotyping. The protocol is tailored for TPMT*3A, *3B, and *3C haplotyping and includes a robust first-round long-range PCR amplification. The method is one ligation step shorter than the protocol of McDonald et al.(5) and is based on ASA PCR followed by hybridization probe genotyping. The method should be of general value for homogeneous probe-based long-range haplotyping.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
This method was designed specifically for the discrimination of TPMT*3A, *3B, and *3C alleles by haplotyping. Conventional genotyping of single-nucleotide polymorphisms (SNPs) in exon 5 (*2), exon 7 (*3B), and exon 10 (*3C) should be the first step(6). For the sake of historical comparability, we keep the original exon assignment although the mutations are actually located on exons 4, 6, and 9, respectively. This is because after the initial studies(7) were performed, TPMT was found to have only nine exons (GenBank accession no. AB045146)(8)(9). Haplotyping is more laborious than conventional SNP genotyping, and in the case of TPMT, it is dependent on previous knowledge of the conventional genotyping result. This is for technical reasons because ASA PCR specificity depends on the underlying mismatches, and the resulting discrimination is not always satisfactory(10).

long-range pcr
PCR reactions were carried out in a final volume of 50 µL. The reaction mixture consisted of 1 µL of DNA solution (NucleoSpin® Blood Kit; Macherey&Nagel), 2.5 U of ExpandTM Long Template enzyme mixture (Roche Biochemica), 5 µL of 10x PCR buffer 3 (supplied with the enzyme mixture, includes 2.25 mM Mg2+), and 0.4 mM each of the deoxynucleotide triphosphates (Roche Biochemicals). Amplification primers [0.5 µM each of Int6for (5'-ACGTAGTCGACCTCCACACCCAGGTCCACACATT-3') and Ex10rev (5'-ACGTAGTCGACGGCAACAAGAACGAAACTCC-3')] were placed to avoid amplification of the highly homologous processed TPMT pseudogene [Ref. (11); GenBank accession no. U11424]. PCR-grade water was added to the final volume. DNA quantification before PCR was not necessary. The primers contain a terminal SalI restriction site (underlined) and amplify a 8.944-kb product from the TPMT gene (GenBank accession no. AB045146). PCR was performed on a TGradient cycler (Biometra), with a step-down protocol (35 cycles at 95 °C for 120 s; followed by 15 cycles at 95 °C for 30 s, 62 °C for 30 s, and 68 °C for 600 s; and 20 cycles at 95 °C for 30 s, 60 °C for 30 s, and 68 °C for 600 s). Hotstart was not necessary. PCR products were visualized on a ultraviolet light transilluminator after electrophoresis in 0.8% agarose followed by ethidium bromide staining. Other PCR cyclers [OmniGene cycler (Hybaid), Personal Cycler (Biometra), and the ProGene cycler (Techne)] worked equally well.

restriction digestion and ligation
The flow scheme for the method is shown in Fig. 2 . Briefly, 200 ng of purified long-range PCR reaction product (PCR Purification Kit; Qiagen) was mixed with 25 U of SalI (MBI Fermentas) and 2 µL of 10x reaction buffer and made up to 20 µL with PCR-grade water. The reaction was incubated at 37 °C for 3 h, followed by heat inactivation at 65 °C for 10 min. The reaction product was diluted 1:10 for further use.



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  		Figure 2. Flow scheme (PCR products not to scale) for the TPMT haplotyping method based on ASA PCR with genotype-specific probes.

Tm, melting temperature.

The subsequent 10-µL ligation reaction included ~1 µL of diluted and digested PCR product, 1 µL of 10x ligation buffer without polyethylene glycol, 1.5 µL of T4 DNA ligase (1 U/µL; MBI Fermentas), and PCR-grade water. Incubation was for 30 min at room temperature. The ligation conditions were chosen to maximize intramolecular ligation with a final product concentration of ~0.1 ng/µL(12). The ligation approximates the long-range PCR product ends, including the TPMT mutations of interest close to their ends, from almost 9 kB to <900 bp apart.

rapid-cycle real-time asa pcr
Pure product from the ligation reaction (1 µL) was added to the rapid-cycle real-time ASA PCR in a final volume of 10 µL in LightCycler glass capillaries. Amplification efficiency was enhanced by the addition of double-stranded DNA (dsDNA) fragments to resemble hotstart conditions [Ref. (13); also see the Discussion section]. Fragments 5'-AGCGGATAACAATATCA-3' and 5'-TGATATTGTTATCCGCT-3' (6 µM) were annealed and incubated for 15 min in the PCR mixture excluding amplification primers. The reaction mixture contained 1 U of native Taq DNA polymerase (PanScript; PAN), 1 µL of 10x PCR buffer {200 mM Tris-HCl, 500 mM KCl, pH 8.4 [note that this is not the (NH4)SO4-based buffer supplied with the enzyme]}, 0.2 mM each of the deoxynucleotide triphosphates (Roche Biochemicals), 2.5 mM MgCl2, 25 mL/L dimethyl sulfoxide, and 500 mg/L bovine serum albumin (New England BioLabs). Oligonucleotides were then added (amplification primers at 0.5 µM; hybridization probes and anchor at 0.1 µM), and PCR cycling was commenced. The reaction was set up with TPMT exon 7 460G (wild-type) allele-specific primer Ex7-460Grev (5'-ACCTGGATTAATGGCAACTAAAGC-3') and primer Int9for (5'-AATCCCTGATGTCATTCTT-3'). The final base of the first primer is allele specific; the underlined base introduces an additional mismatch. The hybridization probes target the TPMT exon 10 mutation site [probe TPMT-719G (5'-CTGTAAGTAGACATAACTTTT-FLU-3) and anchor 5'-Cy5.5-AAAAAGACAGTCAATTCCCCAAC-PHO-3', where FLU is fluorescein, and PHO indicates a phosphorylated end]. The probe had the sequence of the mutant antisense strand and was designed by use of the MeltCalc software (http:\\www.meltcalc.com) to give a maximum difference in melting temperature between both genotypes. All oligonucleotides were synthesized by MWG Biotech, including the modifications by Cy5.5, phosphorylation, and fluorescein as indicated.

The cycling program for amplification of the 805-bp PCR product consisted of initial denaturation at 95 °C for 45 s and 35 cycles of 95 °C for 0 s, 62 °C 1 for s, and 72 °C for 20 s, with the maximum ramp rate. Melting curve data acquisition in channel 3 was performed from 30 to 70 °C at a ramp rate of 0.15 °C/s after the last cycle. Ten additional amplification cycles were used for samples that showed insufficient amplification after 35 cycles.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
long-range pcr
The long-range PCR reaction is very robust under the given reaction conditions. DNA isolated with a standard method based on silica membrane spin technology gave a visible amount of the expected product in 35 of 36 DNA samples investigated. DNA isolated with alternative methods (salting out or phenol–chloroform extraction) performed equally well. According to our experience, successful and reproducible long-range PCR amplification often depends on the PCR cycler used. However, we could run this PCR reaction successfully in four different cyclers from three different manufacturers, which illustrates the robustness of the reaction.

restriction digestion, ligation, and nested pcr
A visible amount of the expected product from the long-range PCR was the best starting point for further sample processing. Only single product purification and quantification steps were needed between the different processing steps. Aliquots were then transferred to the next reaction.

effects of DsDNA addition
The addition of sequence-nonspecific dsDNA gave higher melting peaks for most samples (see Fig. S1 in the Data Supplement that accompanies the online version of this article athttp://www.clinchem.org/content/vol50/issue9/). In some cases the amplification efficiency, as judged by the melting peak maximum height, was not improved further. However, the resulting fluorescence maximum was also never significantly lower than in identical samples run without dsDNA supplementation.

specificity of different asa pcr primers
The primer choice for ASA PCR is limited because the primer 3' end must either match or mismatch a defined position in the DNA strand. As a consequence, there are only two defined positions where the ASA PCR primers must be placed. The most specific amplification was obtained with the reverse primer ending on "C" targeting the exon 7 wild-type allele. The chosen primer cannot amplify TPMT*3A or TPMT*3B alleles. This leads to a haplotyping method in combination with the TPMT*3C genotype-derived hybridization probe. The method cannot unequivocally assign all TPMT genotypes (see Fig. 3 ), but TPMT*1/*3A and TPMT*3B/*3C cases, which cannot be discriminated by conventional genotyping, will be resolved. However, the differences between TPMT*1/*3B and TPMT*1/*3A or TPMT*3B/*3C and TPMT*3C/3C, for example, are evident from the preceding SNP-based genotyping, and such samples are not subject to further testing with this haplotyping method. Typical melting points for the underlying alleles and the corresponding day-to-day precision data are shown in Table 1 . Our analysis included samples from our routine practice. For method validation, we used 20 TPMT*1/*3A samples, 4 TPMT*1/*3C samples, 6 TPMT*1/*1 samples, and 6 TPMT*3A/*3A samples. One TPMT*1/*3A sample failed to amplify, and no whole blood was available for a repeat DNA isolation. We found no discrepant results for samples genotyped with our routine method(6) and with this haplotyping method. The aspect of the resulting melting curves gave no evidence of the PCR-mediated recombination (Fig. 4 ) that may theoretically interfere with this kind of haplotyping method(5).



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  		Figure 3. Results scheme for the TPMT haplotyping method outlined in Fig. 2Up .

SNPs in exons 5 (*2), 7 (*3B), and 10 (*3C) are discriminated by conventional TPMT genotyping. Only equivocal results (*1/*3A) require further testing to exclude *3B/*3C. The method uses an exon 7 wild-type allele-specific primer and an exon 10 detection probe complementary to the mutant TPMT*3C sequence. Presence of a melting peak at 43 °C indicates the TPMT*1 wild-type allele; a melting peak at 53 °C indicates the TPMT*3C mutant allele; therefore, TPMT*1/*3A will be discriminated from TPMT*3B/*3C. Arrows indicate melting peaks characteristic for TPMT*1 and *3C alleles (A) and absent melting peaks for TPMT*3A and *3B alleles (D).


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  		Table 1. Day-to-day precision of melting points as determined by the TPMT ASA PCR haplotyping method.1



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  		Figure 4. TMPT* haplotyping results.

A representative example of ASA PCR-assisted haplotyping is shown. Note the absence of amplification in the TPMT*3A/*3A sample ({blacksquare}) and in the negative control (*). Only TPMT*3C alleles show a melting peak at high temperature as in the TPMT*1/*3C heterozygous case ( {permzspch063}). Note that, for example, TPMT*1/*1 ({diamond}) and TPMT*1/*3A (+) give rise to almost identical melting profiles. However, these genotypes can easily be resolved by preceding SNP-based genotyping.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The use of haplotyping overcomes certain limitations of SNP-based genotyping. This is important when underlying haplotypes show different effects on the individual phenotype(6)(14)(15)(16). Accordingly, we expect an increasing need for haplotyping in molecular diagnostics.

Identification of TPMT genotypes is of utmost importance for assessing potential intolerance to thiopurine medications. Whether genotyping or phenotyping is preferable for the prediction of possible thiopurine intolerance is still under debate(17). We would also caution that the commercial and/or diagnostic application of these tests may need appropriate patent licensing. Genotyping and phenotyping have their specific advantages and disadvantages, so that a center should have both methods established. TPMT activity is our preferred screening test because it depicts the wide range of possible TPMT activities and is sensitive for all kinds of TPMT mutations. Genotyping is necessary in patients who had received transfusions containing erythrocytes in the preceding 3 months. There is a theoretical chance that transfused donor leukocytes may give rise to an erroneous genotyping result if peripheral blood is used for genotyping. However, since the original study in 1992 on the short-term persistence of donor leukocytes in patients receiving multiple transfusions(18), the number of leukocytes present in a unit of filtered packed erythrocytes has been reduced by at least 3 orders of magnitude by leukocyte depletion. Genotyping appears to be safe even in multiply transfused patients(19), although leukopenic patients were not specifically addressed. If no pretransfusion blood is available for genotyping, we recommend a 24-h safety margin before drawing of blood from leukopenic patients for genotyping; however, this is not based on evidence.

Approximately 95% of the deficiency-causing alleles are defined by TPMT*2, *3A, *3B, and *3C. As a consequence, the sensitivity of genotyping for detecting TPMT deficiencies is high. However, SNP-based genotyping cannot unequivocally predict the phenotype when double heterozygosity for SNPs 460G>A and 719A>G is detected. The interpretation of this common test result [the mutant allele frequency is ~5% in white, African, or African-American populations(20)] is based on a priori probability. It is assumed that the mutations lie in cis, defining a TPMT*1/*3A genotype (intermediate metabolizer). A rare but possible TPMT*3B/*3C genotype results when the mutations lie in trans, and complete TPMT deficiency is the consequence(21). Only haplotyping detects the linkage between SNPs 460G>A and 719A>G and can correctly assign the underlying mutant alleles.

Our experience during implementation of this TPMT haplotyping method is of general value for those interested in the use of this methodology for other genes or polymorphisms. It is advisable to try combinations of several primer pairs and buffers for optimization of the initial long-range PCR. Efficient amplification is the best starting point for successful haplotyping. We chose SalI, a standard enzyme used for cloning, as restriction enzyme because it costs ~7 times less per unit than does NotI, which was used in the initial description of the method. Appropriate sample dilution before ligation is critical to favor intramolecular over intermolecular ligation(12). Today, many ligase buffers contain polyethylene glycol, which favors intermolecular ligation. Such buffers must not be used for this methodology. Significant intermolecular ligation leads to less distinctive genotyping results because, for example, a TPMT*1/*3A genotype could transform to a mixture of ligation-generated pseudo-TPMT*3B and pseudo-TPMT*3C alleles. However, we found this not to be of practical relevance under the conditions that we recommend.

Wittwer et al.(22) previously indicated that kinetic primer annealing under rapid cycling conditions provides favorable mismatch discrimination and increases the annealing range of successful ASA PCR. We found that experimental fine tuning for the best annealing temperature is still important to achieve optimum discrimination between the alleles to be amplified. The use of an exon 7 wild-type reverse primer with a C-A mismatch with the other allele was superior to an exon 7 mutant reverse primer leading to a T-G mismatch. The choice of the best primer still largely relies on trial and error. Not only do the unpublished stabilities of terminal mismatches in DNA play a role, but also the inherent substrate specificity of the Taq polymerase [see also Ref. (10)]. We introduced an additional mismatch 2 bases from the 3' terminus of the allele-specific primer. This is an established practice to further increase single-nucleotide discrimination(23).

The addition of dsDNA fragments to the ASA PCR reaction improved the amplification efficiency without compromising allele specificity. dsDNA supplementation was initially recommended to avoid mispriming in a high background of nonspecific DNA(13). The principle is that DNA polymerases bind to short dsDNA fragments added before the amplification primers. The polymerases then cannot extend primers under low stringency conditions. The dsDNA fragments are designed to melt below 50 °C so that above this temperature during PCR cycling, the polymerase is liberated and active. To our knowledge, this is the first application of this technique for improved ASA PCR. In contrast, an alternative method to improve the reliability of ASA PCR by use of a competing depository oligonucleotide was not helpful under our rapid-cycle PCR conditions [data not shown and Ref. (24)].

This haplotyping method, although technically demanding, has several advantages if the fact that the alternatives are also labor intensive is kept in mind. The construction of pedigrees is difficult because not all relatives may be available for analysis and more samples must be genotyped. Methods based on the cloning of large genomic regions are also technically demanding, and not all routine laboratories have facilities with the necessary biosafety levels. In addition, analysis of reverse transcription-PCR products requires RNA of appropriate amounts and quality, which is often not readily available, particularly for the many samples from pediatric patients.

In summary, we present a method for real-time PCR-assisted haplotyping. Our approach, although streamlined for TPMT haplotyping, is general and simpler than the initial protocol. Consequently, less hands-on time is needed, and the method is faster, less expensive, and more robust than the initial method. Haplotyping itself will become more important in the field of molecular diagnostics because it has the potential to explain a higher proportion of individual phenotypic variations than does SNP-based genotyping. With respect to TPMT, haplotyping has the potential to discriminate the genotypes TPMT*1/*3A (intermediate metabolizer) and TPMT*3B/*3C (poor metabolizer).


   Acknowledgments
 
We gratefully acknowledge the excellent technical assistance of Reiner Andag and Sandra Hartung. The MeltCalc software is copyrighted by Ekkehard Scütz and Nicolas von Ahsen. Dr. von Ahsen is the co-inventor (US patent 6,475,737) of an algorithm for automated selection of hybridization probes for genotyping as implemented in the MeltCalc software.


   Footnotes
 
1 Nonstandard abbreviations: TPMT, thiopurine S-methyltransferase; ASA PCR, allele-specific amplification PCR; SNP, single-nucleotide polymorphism; and dsDNA, double-stranded DNA.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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clinchem.2004.034751v1
50/9/1528    most recent
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