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Articles |
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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.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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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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. 1
). 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. 2B
). Sequencing of these
PCR fragments showed the C339T silent mutation in two samples (Fig. 2C
)
and the G292T mutation in one sample (Fig. 2D
), respectively. The third
fragment contained a mutation in intron 5 (C+58T; Fig. 2E
).
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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exon 7
Two sequence variations are known in exon 7: the G460A mutation of
the 3 allele and one silent mutation T474C (1S;
Fig. 1
). 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. 3B
). Direct sequencing of the remaining two samples confirmed for
the one profile the silent mutation 1S (Fig. 3C
), whereas
the other elution profile could be explained by the simultaneous
presence of G460T and T474C (Fig. 3D
and Table 1
). 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. 4B
). Sequencing of these fragments confirmed the A719G transition
of the TPMT3 allele in all cases (Table 1
). 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. 1
) 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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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
).
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. 2–4
, 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. 1
), 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. 1
). 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 3
) 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.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  M. Stanulla, E. Schaeffeler, A. Moricke, S. A. Coulthard, G. Cario, A. Schrauder, P. Kaatsch, M. Dordelmann, K. Welte, M. Zimmermann, et al. Thiopurine methyltransferase genetics is not a major risk factor for secondary malignant neoplasms after treatment of childhood acute lymphoblastic leukemia on Berlin-Frankfurt-Munster protocols Blood, August 13, 2009; 114(7): 1314 - 1318. [Abstract] [Full Text] [PDF]
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represents exons that encode open reading frame sequences; 
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.
