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(Clinical Chemistry. 2001;47:548-555.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

High-Throughput Genotyping of Thiopurine S-Methyltransferase by Denaturing HPLC

Elke Schaeffeler1, Thomas Lang1, Ulrich M. Zanger1, Michel Eichelbaum1 and Matthias Schwaba,1

1 Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstrasse 112, D-70376 Stuttgart, Germany.
a Author for correspondence. Fax 49-711-85-92-95; e-mail matthias.schwab{at}ikp-stuttgart.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The thiopurine S-methyltransferase (TPMT) genetic polymorphism has a significant clinical impact on the toxicity of thiopurine drugs, which are used in the treatment of leukemia and as immunosuppressants. To date, 10 mutant alleles are known that are associated with intermediate or low TPMT activity. To facilitate rapid screening of clinically relevant TPMT mutations, we developed a strategy of high-throughput genotyping by applying denaturing HPLC (DHPLC).

Methods: To test the specificity and efficiency of the DHPLC method, 98 DNA samples from a selected population of patients receiving thiopurine therapy or with previous thiopurine withdrawal were analyzed for the most frequent mutant TPMT alleles, *2 and *3A, which contain key mutations in exons 5, 7, and 10 to identify clearly different elution profiles. All fragments were examined by direct sequencing. Additionally, to test the sensitivity of DHPLC analysis, genotyping for the *2 and *3A alleles of all 98 DNA samples was performed by PCR-based methods (PCR-restriction fragment polymorphism analysis and allele-specific PCR).

Results: The presence of mutations discriminating for alleles *2, *3A, *3C, and *3D, as well as various silent and intron mutations, were correctly predicted by DHPLC in 100% of the samples as confirmed by direct sequencing. Comparison with PCR-based methods for alleles *2 and *3 produced an agreement of 100% with no false-negative signals.

Conclusions: DHPLC offers a highly sensitive, rapid, and efficient method for genotyping of the relevant TPMT mutations, discriminating at least for alleles *2 and *3, in clinical and laboratory practice. Additionally, DHPLC allows a simultaneous screening for novel genetic variability in the TPMT gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Thiopurine S-methyltransferase (TPMT)1 is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds such as 6-mercaptopurine, 6-thioguanine, and azathioprine, collectively termed thiopurines. These drugs are used in the treatment of acute lymphoblastic leukemia and rheumatoid arthritis, and as immunosuppressants (1)(2)(3).

The metabolism of these drugs is severely affected by genetic polymorphisms of TPMT. Several case reports and clinical studies have shown that patients with exceptionally low TPMT activity (~1 in 300 individuals) are at high risk of developing severe and potentially fatal hematopoietic toxicity (e.g., pancytopenia), caused by the accumulation of cytotoxic metabolites after treatment with standard doses of thiopurines (2)(4)(5)(6)(7)(8)(9). Additional recent data indicate that patients with heterozygous genotypes, constituting ~10% of Caucasian and African-American populations, are also at greater risk of thiopurine toxicity (10). Prospective determination of erythrocyte TPMT activity is therefore emerging as a routine safety measure before therapy to avoid drug toxicity (11)(12)(13)(14)(15), but there are several limitations with respect to the determination of the constitutive TPMT enzyme activity. For example, if a deficient or heterozygous patient has received transfusions with red blood cells (RBCs) from a homozygous wild-type individual (a rather likely case), TPMT activity cannot be reliably determined within 30–60 days after transfusion (16). Furthermore, thiopurine administration itself may alter TPMT activity in erythrocytes with an increase of enzyme activity (4)(5)(15), and some other clinically important drugs (e.g., sulfasalazine and olsalazine) are partly potent inhibitors of TPMT, leading to possible misclassification especially for heterozygous patients (17)(18)(19)(20).

Several mutant alleles responsible for TPMT deficiency have been described (Fig. 1 ) (21)(22)(23)(24)(25)(26)(27)(28), and the relationship between TPMT geno- and phenotype has been most clearly defined for the clinically relevant TPMT alleles 2, 3A, and 3C in patients and healthy subjects (16)(29). Thus, molecular diagnosis appears to be a feasible approach to predict the TPMT phenotype in up to 95% of individuals (30).



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  		Figure 1. Summary of the currently known allelic variations of the TPMT gene.

{blacksquare} represents exons that encode open reading frame sequences; {square} represents exons outside of the open reading frame. Exons but not intron sizes are proportional to their relative lengths. (C(-178)T), termed as TPMT*1A, is located outside of the open reading frame.

Conventional PCR-based methods [e.g., restriction fragment length polymorphism (RFLP) analysis and allele-specific PCR (ASPCR)] are commonly being used for the detection of TPMT mutations (16)(22), but have the disadvantage of being both time-consuming and labor-intensive methods. Furthermore, additional unknown mutations that may produce low TPMT activity cannot be found simultaneously with the common PCR-based techniques.

Therefore, in the present work we demonstrate the usefulness of denaturing HPLC (DHPLC) for allelic discrimination of clinically relevant TPMT mutations, not only as a screening technique but also as a reliable and efficient genotyping method for known mutations, thus allowing both strategies to be accomplished simultaneously.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
study population
Genomic DNA from peripheral leukocytes was isolated by standard methods from two German patients with severe toxicity (one with pancytopenia, the other with leukopenia) as a result of thiopurine therapy. Measurement of RBC TPMT enzyme activity by a nonradioactive assay in these patients who had received non-RBC transfusions before blood collection revealed very low enzyme activities [<2 nmol 6-methylthioguanine · g-1 hemoglobin (Hb) · h-1], which is indicative of TPMT deficiency (31)(32). Additionally, DNA was acquired from a selected population of 98 Caucasian patients receiving thiopurine treatment (age range, 19–67 years), including several patients withdrawn from thiopurine therapy because of various adverse events. The study protocol was approved by the local ethics committee, and all patients gave written informed consent.

pcr amplification
Fragments of the TPMT gene were amplified using the following oligonucleotide primers, described previously by Otterness et al. (24):

PCRs were performed in a volume of 25 µL, containing 100 ng of genomic DNA, 20 pmol of forward and reverse primer, 200 µM dNTPs, 0.25 U of Taq polymerase and 1x buffer supplied by the manufacturer (Perkin-Elmer). PCRs were initiated by a denaturation step at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 59 °C for 1 min, and 70 °C for 30 s; a final extension was performed at 70 °C for 7 min.

dhplc analysis
Mutation analysis was performed according to the method developed by Oefner and Underhill (33) on an analysis system from Varian (Varian Helix-System®). The PCR products were denatured at 95 °C for 5 min and cooled to 65 °C with a temperature change of 1 °C/min. The samples were kept at 4 °C until 5 µL was applied to a preheated reversed-phase column (Eclipse,dsDNA preparative column; 4.6 x 75 mm; Hewlett Packard). DNA was eluted with the following gradient consisting of buffer A (0.1 mol/L triethylammonium acetate) and buffer B (0.1 mol/L triethylammonium acetate containing 250 mL/L acetonitrile): 60% A-40% B for 30 s; 50% A-50% B for 5.5 min; 25% A-75% B for 10 s; 5% A-95% B for 1 min; and 60% A-40% B for 1.33 min. The mobile phase was heated to the respective denaturing temperatures. The oven temperature at which the detection of the heteroduplex occurred was calculated using the software available at http://insertion.stanford.edu/melt.html (34). The actual running temperature was established by repeatedly injecting the sample 1–2 °C below and above the calculated temperature (for exon 5, 57 °C; for exon 7, 55 °C; for exon 10, 54 °C). Heteroduplex formation was checked by the melting profile of a known sequence variation in exons 5, 7, and 10.

sequencing
PCR products were directly sequenced using infrared-800-labeled primers (MWG Biotech) with the Thermo Sequenase fluorescently labeled primer cycle sequencing reagent set (Amersham Pharmacia Biotech). Sequence analysis was performed on an automated DNA sequencer (LI-COR Inc.) using Base ImagIR data collection and image analysis 4.00 software.

pcr-rflp and aspcr analysis for tpmt2 and 3 alleles
PCR assays to detect the G238C transversion in TPMT2 and the G460A and A719G transitions in TPMT3 were performed as described by Yates et al. (16). ASPCR was used for TPMT2 and PCR-RFLP for TPMT3.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
method optimization
PCR fragments of exons 5, 7, and 10 were amplified from two homozygous wild-type controls as well as from two previously identified TPMT-deficient patients. Phenotyping of these two patients showed low TPMT enzyme activities (<2 nmol 6-methylthioguanine · g-1 Hb · h-1) in agreement with compound heterozygous (2/3A) and homozygous (3A/3A) mutant genotypes detected by sequence analysis. These TPMT alleles contain genetic variations in exons 5, 7, and 10 (see Fig. 1Up ). Each PCR product was subjected to DHPLC, and analysis conditions (gradient and temperature) were optimized for each fragment to yield characteristic and reproducible elution profiles. The corresponding typical elution profiles were compared with patterns of the homozygous wild-type sequence control showing only one single peak of homoduplex DNA. The oven temperature at which the samples are run represents the most critical condition. The optimal running temperature was established by repeatedly injecting the samples 1–2 °C below and above the calculated temperatures. Whereas the optimal conditions for the detection of exon 7 and exon 10 mutations were correctly predicted by the software, detection of the exon 5 mutation required a temperature of 1 °C above the recommended temperature.

As shown in Figs. 2A , 3A , and 4A , samples with the wild-type sequence eluted as a single peak, whereas multiple peaks indicative of heteroduplexes appeared in the heterozygous samples containing the 2 and 3 alleles (Figs. 2B , 3B , and 4B ). Homozygous nucleotide exchanges can sometimes be distinguished because of a slight shift in the elution time compared with the reference. The addition of an approximately equal amount of wild-type DNA to the samples (1:1) before the denaturation step allows homozygous alterations to be detected reliably. This was done routinely for all samples to identify homozygous sequence variations. Therefore, all samples were analyzed first without mixing with an equal amount of wild-type DNA to detect heterozygotes, and then wild-type DNA was mixed with each sample to detect homozygous mutants.



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  		Figure 2. DHPLC detection of TPMT mutations in exon 5.

Elution profiles associated with sequence variations in exon 5: (A), wild type; (B), G238C; (C), C339T; (D), G292T; (E), intron 5, C+58T. The depicted chromatograms show up to five overlaid elution profiles to demonstrate the reproducibility of DHPLC.



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  		Figure 3. DHPLC detection of TPMT mutations in exon 7.

Elution profiles associated with sequence variations in exon 7: (A), wild type; (B), G460A; (C), T474C; (D), G460A and T474C. The depicted chromatograms show up to five overlaid elution profiles to demonstrate the reproducibility of DHPLC.



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  		Figure 4. DHPLC detection of TPMT mutations in exon 10.

Elution profiles associated with sequence variations in exon 10: (A), wild type; (B), A719G. The depicted chromatograms show up to five overlaid elution profiles to demonstrate the reproducibility of DHPLC.

validation of genotyping by dhplc
To test the specificity of the DHPLC method for the detection of alleles 2 and 3, we analyzed DNA from 98 patients and compared DHPLC results with direct sequencing.

PCR fragments of exons 5, 7, and 10 were amplified from all 98 patients and subjected to DHPLC analysis using the established gradient and temperature conditions. All PCR fragments, including those with a clearly different elution profile, were examined by direct sequencing.

exon 5
In addition to the G238C transition discriminating for the TPMT2 allele, further polymorphisms are known in exon 5, one silent mutation (C339T) and one mutation that produces a stop codon (G292T), which discriminates for the TPMT3D allelic variant (Fig. 1Up ). By comparing the DHPLC chromatograms of the 98 samples, we identified three profiles that could be clearly distinguished from the 2 profile in exon 5 (G238C; Fig. 2BUp ). Sequencing of these PCR fragments showed the C339T silent mutation in two samples (Fig. 2CUp ) and the G292T mutation in one sample (Fig. 2DUp ), respectively. The third fragment contained a mutation in intron 5 (C+58T; Fig. 2EUp ). Additionally, seven patients were homozygous for this mutation. Thus, with the help of DHPLC we could accurately differentiate the three known sequence variations within exon 5 and one intron mutation in the flanking region (Table 1 ).


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  		Table 1. Summary of mutation analysis by DHPLC and direct sequencing in 100 DNA samples.1

exon 7
Two sequence variations are known in exon 7: the G460A mutation of the 3 allele and one silent mutation T474C (1S; Fig. 1Up ). Comparison of the DHPLC chromatograms from the exon 7 fragment of the 98 DNA samples revealed three kinds of profiles. One corresponded to the G460A mutation (Fig. 3BUp ). Direct sequencing of the remaining two samples confirmed for the one profile the silent mutation 1S (Fig. 3CUp ), whereas the other elution profile could be explained by the simultaneous presence of G460T and T474C (Fig. 3DUp and Table 1Up ). This elution profile showed a complex pattern and therefore could clearly be distinguished from the profiles described above. Three patients were homozygous for TPMT1S.

exon 10
In exon 10 we observed only one kind of elution profile (Fig. 4BUp ). Sequencing of these fragments confirmed the A719G transition of the TPMT3 allele in all cases (Table 1Up ). Two patients had a homozygous genotype for both mutations G460A and A716G, which discriminates for TPMT3A. Additional elution profiles that discriminate for the 4, 7, and 8 alleles (Fig. 1Up ) were not present.

Table 2 summarizes the frequencies of TPMT genotypes in our study population. Additionally, all patients with heterozygous genotypes 1/3A (n = 9), 1S/3A (n = 2), 1/3C (n = 1), and 1/3D (n = 1) were phenotyped for TPMT retrospectively. The individual TPMT activities of these patients are shown in Table 3 , including whether or not the patient was on azathioprine/6-mercaptopurine therapy at time of phenotyping.


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  		Table 2. Frequency of TPMT genotypes in the study population.1


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  		Table 3. TPMT enzyme activity and medication data of all patients with heterozygosity for TPMT (n = 13).

To test the sensitivity of DHPLC method for genotyping of the TPMT2 and 3 alleles, the DHPLC results of all 98 individuals were compared with PCR-based genotyping data (PCR-RFLP and ASPCR) for the 2 and 3 mutations. The evaluation was performed as a blind study by independent researchers for each technique. All locus-specific variations within a fragment found by PCR-RFLP and ASPCR were correctly defined in 100% of cases by DHPLC, and no false-negative signals were detected.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hepatic drug metabolism is one of the major determinants for the efficacy and toxicity of drugs and is influenced by physiologic, environmental, and genetic factors. The polymorphism of TPMT has considerable clinical importance because it can be responsible for the toxicity of thiopurine drugs, producing potentially life-threatening complications (8)(9). High-throughput genotyping for the clinically relevant alleles offers the possibility of predicting the individual TPMT phenotype before commencing thiopurine therapy (30)(35).

To date, 10 mutant alleles responsible for the TPMT deficiency and several silent and intronic mutations have been described. TPMT3A appears to be the most frequent inactivating mutant allele in Caucasians (3.2–5.7%), whereas TPMT2 and TPMT3C, both associated with low activity, are less frequent (0.2–0.5% and 0.2–0.8%, respectively) (16)(36)(37)(38). On the other hand, TPMT3C is the most frequent mutant TPMT allele in Asian and African populations (30) so that TPMT3C may be the most common TPMT mutant allele on this planet. Additionally, rare inactivating variants (TPMT3B, 3D, 4, 5, 6, 7, and 8) as well as some intronic and silent mutations have been reported, although mostly in single individuals (16)(24)(27) (Fig. 1Up ).

Usually, PCR-based methods (e.g., PCR-RFLP and ASPCR) have been used for the detection of TPMT mutations (16)(22). The technical disadvantages of these methods include the number of PCR tests that have to be performed as well as the gel-casting requirements and hazardous dyes for staining DNA. Thus, these PCR-based methods cannot be easily automated and do not seem to be a rapid and convenient technique in clinical laboratory practice. Additionally, these methods do not offer the possibility to identify novel mutations.

DHPLC has been used before as a highly automated screening method for the detection of novel polymorphisms. This method is based on the differential retention of homo- and heteroduplex DNA molecules by ion-pair chromatography under conditions of partial heat denaturation. Heteroduplex molecules are generated in heterozygous samples when homologous DNA single strands carrying point mutations or small insertions and deletions reanneal. Under conditions of partial denaturation, heteroduplex molecules are generally eluted ahead of homoduplexes, producing at least one additional peak. The elution profiles of such samples are distinct from those having homozygous sequences, making the identification of samples harboring polymorphisms or mutations an easy and straightforward procedure. Homozygous mutants can be detected by adding an approximately equal amount of wild-type DNA to the mutant species. Previous studies have shown that DHPLC is not only a highly sensitive and automated high-throughput method that can be used as a powerful screening method to detect polymorphisms (39)(40)(41) but can also be used for genotyping (40)(42)(43)(44). The sensitivity of the DHPLC method is dependent on the HPLC conditions used for analysis. A further critical step is the analysis of elution profiles. For maximum sensitivity, any deviation from the elution profile of control samples must be considered. The sensitivity of DHPLC has been investigated in several studies, which report accuracies for detection of mutations up to 100%. However, in some cases different mutations yielded similar elution profiles, which led to misinterpretation (44)(45)(46)(47). Therefore, a thorough evaluation of this method is necessary, including a comparison with already established genotyping methods (e.g., RFLP and single-strand conformation polymorphism analysis) if DHPLC is used for genotyping.

In the present study we established high-throughput genotyping for the clinically relevant inactivating TPMT alleles using the DHPLC technique. Initially, DHPLC conditions for genotyping of the 2 and 3A alleles were tested as a straightforward procedure; subsequently, the specificity of the DHPLC method was evaluated in a selected population of 98 patients.

The patient population was highly selected because it included only patients presently on azathioprine/6-mercaptopurine therapy or patients who had previously withdrawn from thiopurine treatment because of serious side effects. Thus, to evaluate the DHPLC method for genotyping, it could be expected that various functional mutations of TPMT that may be associated with side effects of thiopurine therapy were present in this population.

With respect to the DHPLC results of all 98 samples, we did not miss any alteration seen in cycle sequencing. In all three exons, unique sequence variations could be detected with the DHPLC technique for alleles 2 and 3 in 100%, and two silent mutations (1S and C339T) as well as one intron mutation [intron 5 (C+58T)] could be characterized. In all cases, the identified elution profiles were consistently reproducible, as shown by the overlaying of several profiles in Figs. 2–4Up Up Up , so that the DHPLC method has been demonstrated to be highly robust. Additionally, discrimination between double heterozygotes for mutations located on one exon, as shown in the present study for exon 7, is an additional advantage.

Further elution profiles discriminating between the TPMT4, 7, and 8 alleles, which include additional mutations in exon 10 (Fig. 1Up ), were not found. To our knowledge, these alleles have been described only in single cases (30), so that detection of these mutations seems to be very unlikely in our small study population.

Using the established DHPLC procedure, we could not identify the 5 and 6 alleles of TPMT because the responsible mutations are located in exons 4 (T146C) and 8 (A539T), respectively (Fig. 1Up ). Because these two alleles are extremely rare genetic variants that have been described only in one heterozygous individual of undefined ethnicity and one Korean subject (24), we believe that screening for these mutations may be unnecessary.

DHPLC, as well as conventional PCR-based methods and direct sequencing, respectively, cannot clearly distinguish between two genotypes: TPMT3A/1, which discriminates for an intermediate phenotype, and TPMT3B/3C, which is responsible for deficiency. None of the present genotyping methods can determine when the G460A mutation is on the same allele as the A719G mutation (1/3A) vs when these two mutations are on opposite alleles (3C/3B). Because the TPMT3B allele is described only in single cases (24), the established genotyping methods seem to be sufficient to provide a simple and reliable strategy for predicting TPMT phenotype. To confirm this assumption, we retrospectively phenotyped all patients in our study with a heterozygous genotype. None of these patients had shown deficient TPMT activity (<2 nmol 6-methylthioguanine · g-1 Hb · h-1). Five patients were on azathioprine/6-mercaptopurine therapy (see Table 3Up ) at the time of phenotyping. Thus, a correlation between phenotype and genotype of 100% could not be expected for these patients because an increase of TPMT activity during TPMT treatment is a well-known phenomenon in up to 30% of individuals (4)(5)(15).

According to genotype/phenotype correlation studies in different populations, ~95% of patients with TPMT deficiency and low enzyme activity are predicted correctly (30). Nevertheless, to achieve a phenotypical predictive power >99% by genotyping, which has been shown for other drug-metabolizing enzymes [e.g., cytochrome P450 2D6 (48)], the established DHPLC technique offers the advantage of simultaneously detecting novel mutations in the three exons 5, 7, and 10.

In conclusion, we have developed and validated a DHPLC method that allows determination of at least alleles 2 and 3 and various silent and intronic mutations in exons 5, 7, and 10 as a highly sensitive and accurate technique. Because of the different elution profiles, the nature of the sequence variations can be predicted independently of sequencing. Compared with PCR-based methods, DHPLC requires less manual handling and time. Thus, this technique can be used as an automated method for genotyping of TPMT and can be applied as a suitable procedure for large-scale genotyping in clinical and laboratory practice to bring genetic analysis closer to the goal of high-throughput mutation screening.


   Acknowledgments
 
This work was supported by the BMBF (FKZ 01 GG 9846) and the Robert-Bosch Foundation, Stuttgart, Germany. We sincerely thank Richard M. Weinshilboum for supplying control PCR fragments of the *2 and *3A variants and Andrea Zwicker for excellent technical assistance.


   Footnotes
 
1 Nonstandard abbreviations: TPMT, thiopurine S-methyltransferase; RBC, red blood cell; RFLP, restriction fragment length polymorphism; ASPCR, allele-specific PCR; DHPLC, denaturing HPLC; and Hb, hemoglobin.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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