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Results for “variable methylation”

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PubMed2025

Methylation-based droplet digital polymerase chain reaction shows high concordance with chronic lymphocytic leukemia IGHV somatic mutation status.

OBJECTIVE: Somatic hypermutation at immunoglobulin heavy chain variable (IGHV) genes, an established prognostic and predictive biomarker for chronic lymphocytic leukemia (CLL), is assessed by gene sequencing. We developed a single methylation-specific droplet digital polymerase chain reaction (methyl-ddPCR) to predict IGHV status in patients with CLL. METHODS: The CLL methylation array and IGHV data from the International Cancer Genome Consortium (ICGC) were used for biomarker discovery. Top-ranked candidate regions were manually screened for PCR primer and probe binding sites. A single methyl-ddPCR was evaluated on an internal cohort of CLLs with mutated (M), unmutated (U), and inconclusive IGHV results originally determined by next-generation sequencing (NGS). RESULTS: Analysis of ICGC data identified array probe cg23844018 as a candidate for the PCR. The corresponding CpG site showed high methylation levels in U-CLL and lower levels in M-CLL. On the internal cohort, a single optimal cutoff correctly classified 104 of 115 U- and M-CLLs (90.4%; area under the curve = 0.96). The PCR data correlated with some prognostic fluorescence in situ hybridization and CLL subset groupings. Limited analysis suggests that the PCR may be able to stratify some patients with CLL who have inconclusive results on IGHV NGS testing. CONCLUSIONS: The methyl-ddPCR showed high concordance with CLL IGHV status in an internal cohort.

Humans↗

PubMed2000

Ubiquitous presence of O6-methylguanine in human peripheral and cord blood DNA.

O6-Methylguanine (O6-meG) is a powerful premutagenic lesion that can arise from exposure to methylating agents. Although it has been reported to occur in human DNA, no systematic epidemiological analysis of its occurrence in populations suffering general environmental exposure is available. We report here results from a study of the presence of O6-meG in maternal and cord blood leukocyte DNA of women not knowingly exposed to methylating agents. Using a modification of an already existing method capable of detecting the lesion at levels as low as 16 nmol/molG, the adduct was detected in 31 of 36 maternal and 30 of 36 cord samples, at levels ranging up to 192 nmol/molG. Adduct levels in maternal blood DNA were significantly higher than those in cord blood DNA (P < 0.05), and there was a strong correlation between adduct levels in the two tissues (P < 0.001). In bivariate analysis, no significant association of adduct levels in either tissue and residence air pollution, active and passive smoking status, or eating habits was found. However, intake of fruits/vegetables and of vitamin supplements showed nonstatistically significant trends toward being associated with lower adduct levels in both maternal and cord blood DNA. The same trend was observed after multivariate analysis where all the above variables were controlled for. These findings indicate that premutagenic methylation DNA damage is commonplace in individuals not known to have suffered excessive exposure to environmental methylating agents or their precursors and are compatible with an endogenous origin of this damage, possibly associated with endogenous nitrosation processes.

Adult↗

PubMed2004

Differential methylation status of tumor-associated genes in head and neck squamous carcinoma: incidence and potential implications.

PURPOSE: Promoter hypermethylation is one of the major mechanisms in the transcriptional inactivation of certain carcinoma-associated genes. Concurrent methylation analysis of multiple, functionally distinct genes may provide important information on their differential alterations and potential association in head and neck squamous carcinogenesis. EXPERIMENTAL DESIGN: Methylation-specific PCR analysis of the CpG islands of 8 cancer-related genes was performed on 19 cell lines and 32 primary head and neck squamous cell carcinoma (HNSC) specimens with matched histologically normal mucosa and 6 dysplastic lesions. The methylation status and histological features of the specimens were investigated. RESULTS: In histologically normal squamous mucosa, no to low-level methylation (0-22%) was noted in some specimens at all genes except RARbeta2 (50%). Considerable variation in the incidence of methylation of these genes within and between cell lines and tumor specimens was noted. The highest incidences of methylation in the cell lines and primary tumors were noted in RARbeta2 (53%), MGMT (37%), p16 (33%), and DAP-K (25%); low incidence of methylations were noted in E-cadherin (2%), p73 (2%) RASSF1A (10%), and p14 (20%) genes. The incidences of methylation of each gene were almost similar between the HNSC cell lines and primary cancer specimens, although methylation of RASSF1A was observed in cell line (26%), but not in dysplasia and primary tumor. RARbeta, p16, and MGMT genes showed the highest incidences of methylation in premalignant and invasive carcinomas. CONCLUSIONS: Methylation of p16, RARbeta, and MGMT may constitute early events in HNSC tumorigenesis. The infrequent methylation at certain genes suggests a minimal role for this feature in their functional assessment in HNSC. The variability within and between cell lines and tumor specimens supports a heterogeneous and dynamic state of methylation in genes associated with HNSC tumorigenesis.

Adult↗

PubMed1993

Cyanopeptolins, new depsipeptides from the cyanobacterium Microcystis sp. PCC 7806.

Four depsipeptides (peptide lactones), called cyanopeptolins A, B, C and D, have been isolated from the cyanobacterium Microcystis sp. PCC 7806. They possess identical structures consisting of cyclic L-glutamic acid-gamma-aldehyde, L-leucine, N-methyl-phenylalanine, L-valine, L-threonine, L-aspartic acid, hexanoic acid and a variable basic amino acid. This variable amino acid can be L-arginine (cyanopeptolin A), L-lysine (cyanopeptolin B), N epsilon-methyl-L-lysine (cyanopeptolin C) and N epsilon,N epsilon-dimethyl-L-lysine (cyanopeptolin D), respectively. The L-glutamic acid-gamma-aldehyde and the amino group of L-leucine form an unusual 3-amino-6-hydroxy-2-oxo-1-piperidine system. L-Threonine is connected to L-valine via its hydroxy-group forming an ester bonding. The hexanoic acid residue is attached to the N-terminal aspartic acid residue which is not a part of the ring structure. The isolation procedure of the four cyanopeptolins as well as structure elucidation are described. Amino acid analysis, GC/MS analysis, FAB-MS and several NMR techniques were used to reveal the structures.

Amino Acid Sequence↗

PubMed1991

Progesterone administration attenuates excitatory amino acid responses of cerebellar Purkinje cells.

We have previously shown that the sex steroid progesterone plays a modulatory role in amino acid physiology by suppressing excitatory responses of cerebellar Purkinje cells to glutamate and augmenting inhibitory responses of these neurons to GABA. In the present study using the rat, progesterone effects on neuronal responses to the specific excitatory amino acid agonists quisqualate, kainate and N-methyl-D-aspartate were tested using iontophoretic, extracellular single unit recording techniques. In addition, the effect of systemic administration of progesterone on quisqualate-evoked excitation was evaluated in the presence of the GABAA blocker bicuculline. Progesterone consistently attenuated excitatory neuronal responses to local application of all three excitatory amino acids by 40-51%, but exerted variable effects on combined administration of quisqualate and N-methyl-D-aspartate which were dependent on temporal and dose-related factors. Progesterone-induced attenuation of the quisqualate response was found to be mediated primarily by a non-N-methyl-D-aspartate receptor. In addition, bicuculline application did not block progesterone effects on quisqualate excitation, suggesting that the observed steroidal modulation of excitatory amino acid function is not secondary to progesterone-induced potentiation of GABA inhibition.

Amino Acids↗

PubMed1983

Intracisternal A particles: RNA expression and DNA methylation in murine teratocarcinoma cell lines.

Two main intracisternal-A-particle-specific RNA species (29 to 30S and 35 to 36S) are variably expressed in particle-producing and particle-nonproducing murine teratocarcinoma cell lines. An analysis of DNA methylation patterns after hybridization with an intracisternal-A-particle-specific probe showed an apparent direct correlation between DNA methylation and RNA expression. However, when the methylation assay was performed on the excised intracisternal A particle genes in one of the particle-rich cell lines (PCC6), some undermethylated sequences were detectable. These results are consistent with the concept that only few of the genes are transcriptionally active.

Animals↗

PubMed2006

Expression of Delta DNMT3B variants and its association with promoter methylation of p16 and RASSF1A in primary non-small cell lung cancer.

Despite the role of DNMT3B in de novo DNA methylation, a correlation between DNMT3B expression and promoter DNA methylation has not being established in tumors. We recently reported DeltaDNMT3B, a subfamily of DNMT3B, with seven variants, as the predominant expression forms in non-small cell lung cancer (NSCLC). We hypothesized that expression of the DeltaDNMT3B variants plays a role in promoter methylation formation during lung tumorigenesis. Expression of seven DeltaDNMT3B variants was measured in 119 NSCLCs and the corresponding normal lungs using reverse transcription-PCR. The expression patterns of DeltaDNMT3B variants were analyzed with the status of p16 and RASSF1A promoter methylation in the tumors as well as in patients' clinical variables, including outcomes. Expression of DeltaDNMT3B variants was detected in 94 of 119 (80%) tumors but in only 22 (18%) of the corresponding normal lungs (P < 0.0001). DeltaDNMT3B1, DeltaDNMT3B2, and DeltaDNMT3B4 were the most frequently detected transcripts in the tumors (62%, 76%, and 46%, respectively). The expression of DeltaDNMT3B variants was associated with p16 and RASSF1A promoter methylation in the tumors, but the strongest association was between DeltaDNMT3B4 and RASSF1A. Forty-two of 46 (91%) tumors with RASSF1A promoter methylation expressed DeltaDNMT3B4 compared with only 13 of 73 (18%) tumors without the promoter methylation (P < 0.0001). Strong associations were also observed between expression of the variants in the tumors and in patients' clinical outcomes. Expression of DeltaDNMT3B variants is common in NSCLC and may play an important role in the development of promoter methylation.

Adult↗

PubMed1992

The hypervariable DXS255 locus contains a LINE-1 repetitive element with a CpG island that is extensively methylated only on the active X chromosome.

The DXS255 locus at Xp11.22 is highly polymorphic due to a 26-bp variable number of tandem repeats (VNTR) motif. In previous studies, one of the MspI sites flanking the VNTR manifested a correlation between methylation and X chromosome inactivation. Here we show, by DNA sequence analysis, that this MspI site is located within the CpG island at the 5' end of a LINE-1 element, which is 2.5 kb from the VNTR. The methylation status of the CpG island was assessed in Southern blotting experiments using the methylation-sensitive enzymes HpaII, HhaI, and BssHII. All these sites were completely methylated on active X chromosomes, consistent with previously reported findings of full methylation of LINE-1 elements throughout the genome. However, on inactive X chromosomes these sites were predominantly unmethylated, although patterns were found to be heterogeneous. The results suggest that LINE-1 elements on the inactive X chromosome are not suppressed by full methylation of their CpG islands. The differential methylation of the DXS255 CpG island provides the basis for a highly informative X inactivation analysis system.

Base Sequence↗

PubMed2005

p53 Aberrations do not predict individual response to fludarabine in patients with B-cell chronic lymphocytic leukaemia in advanced stages Rai III/IV.

Abnormalities of p53 have been associated with short survival and non-response to therapy in chronic lymphocytic leukaemia (CLL). We have evaluated the rate of response to fludarabine as first-line therapy in 54 patients with advanced stage CLL, analysing the cytogenetic profile, aberrations in p53, including the methylation status of its promoter, and the immunoglobulin heavy-chain variable-region (IGVH) mutation status. According to the advanced stage of the disease in this series, 75% of patients presented genetic aberrations associated with poor prognosis: del(17p) and/or del(11q), and no-mutated IGVH genes. Ten patients (18.5%) had methylation in the promoter region of p53. Eighty-three per cent of patients treated achieved a response, with a high rate of complete remission (47.6%). Although we found a significant correlation between failures and the presence of p53 aberrations (P = 0.0065), either with methylation (P = 0.018) or deletion (P = 0.015), 64% of the patients with aberrations in this gene responded to treatment (11/17), suggesting that fludarabine induces high remission rates, even in these patients. This is the first time that the significance of p53 promoter methylation status is described in this pathology, and our data support that this epigenetic phenomenon could be involved in the pathogenesis and clinical evolution of CLL.

Adult↗

PubMed1989

The magnetic and electronic properties of Methanobacterium thermoautotrophicum (strain delta H) methyl coenzyme M reductase and its nickel tetrapyrrole cofactor F430. A low temperature magnetic circular dichroism study.

Variable temperature magnetic circular dichroism (MCD) spectroscopy has been used to characterize the magnetic and electronic properties of the Ni(II) tetrapyrrole, F430, which is the cofactor of the S-methyl coenzyme M methylreductase enzyme from Methanobacterium thermoautotrophicum (strain delta H). 4-Coordinate forms are found to be diamagnetic (S = 0 ground state), whereas 6-coordinate forms are paramagnetic (S = 1 ground state). MCD studies, together with parallel low temperature UV-visible absorption and resonance Raman investigations, show that the equilibrium distribution of 4-coordinate square-planar and 6-coordinate bis-aquo forms of the native isomer of F430 in aqueous solution is affected by both temperature and the presence of glycerol. In the presence of 50% glycerol, the 12,13-diepimer of F430 is shown to be partially 6-coordinate in frozen solution at low temperature. Low temperature MCD magnetization data allow the determination of the axial zero-field splitting (D) of the S = 1 ground state of bis-ligand complexes of F430. The value of D is sensitive to the nature of the Ni(II) axial ligands: bis-aquo F430, D = +9 +/- 1 cm-1; bis-imidazole F430, D = -8 +/- 2 cm-1. Measurement of D = +10 +/- 1 cm-1 for F430 in the methylreductase holoenzyme argues strongly against histidine imidazole coordination to Ni(II) in the enzyme. The possible existence of alcoholic or phenolic oxygen-containing ligands (serine, threonine, tyrosine, water) to Ni(II) in the enzyme-bound cofactor is discussed.

Circular Dichroism↗

PubMed2000

The mouse tumor cell lines EL4 and RMA display mosaic expression of NK-related and certain other surface molecules and appear to have a common origin.

As a potential means for facilitating studies of NK cell-related molecules, we examined the expression of these molecules on a range of mouse tumor cell lines. Of the lines we initially examined, only EL4 and RMA expressed such molecules, both lines expressing several members of the Ly49 and NKRP1 families. Unexpectedly, several of the NK-related molecules, together with certain other molecules including CD2, CD3, CD4, CD32, and CD44, were often expressed in a mosaic manner, even on freshly derived clones, indicating frequent switching in expression. In each case examined, switching was controlled at the mRNA level, with expression of CD3zeta determining expression of the entire CD3-TCR complex. Each of the variable molecules was expressed independently, with the exception that CD3 was restricted to cells that also expressed CD2. Treatment with drugs that affect DNA methylation and histone acetylation could augment the expression of at least some of the variable molecules. The striking phenotypic similarity between EL4 and RMA led us to examine the state of their TCRbeta genes. Both lines had identical rearrangements on both chromosomes, indicating that RMA is in fact a subline of EL4. Overall, these findings suggest that EL4 is an NK-T cell tumor that may have retained a genetic mechanism that permits the variable expression of a restricted group of molecules involved in recognition and signaling.

Acetylation↗

PubMed2001

Low-threshold N-methyl-D-aspartate receptor function correlates negatively with learning.

The intermediate, medial hyperstriatum ventrale (IMHV) is an area of the forebrain of the domestic chick which exhibits great plasticity. Moreover, there is a strong link between plasticity in the IMHV and specific changes in behaviour. The IMHV in vitro is still plastic, and many of its physiological properties are age-dependent, peaking in slices taken from 3- or 4-day-old birds. This 'window' coincides with an important transitional period in a chick's normal behavioural development. It has also been claimed that reversal training is at its most effective in 3- and 4-day-old birds - a proposition which was confirmed by the experiments reported here. A combination of in vivo training followed by in vitro electrophysiology also revealed that the function of low-threshold N-methyl-D-aspartate receptors (one of the age-related variables) is negatively related to the effectiveness of reversal training, when age is held constant.

Age Factors↗

PubMed1990

Characterization of the African HIV-1 isolate CBL-4 (RUT) by partial sequence analysis and virus neutralization with peptide antibody and antisense phosphate-methylated DNA.

The HIV-1 isolate CBL-4 (RUT), originating from Tanzania, was characterized using a comprehensive virus-typing system. This system included sequence analysis of the region coding for the neutralization domain in the third variable region (V3) of the external envelope and of the tat responsive (TAR) region after polymerase chain reaction (PCR) amplification of these sequences from cellular DNA in the CBL-4 (RUT) producer line. Based on independent cluster analysis of TAR and V3 sequences the CBL-4 (RUT) virus was positioned closest to the Z6 (and ELI) African virus family. The V3 amino acid sequence on the surface of the virus particle was confirmed by the inhibition of neutralization of CBL-4 (RUT) by a synthetic peptide derived from the nucleic acid sequence. Using antisense phosphate-methylated DNA covering the TAR loop region of LAV-1/HTLV-IIIB, inhibition of HTLV-IIIB and HTLV-IIIRF infection was seen, whereas no inhibition was observed for CBL-4 (RUT), indicating two or more mismatches in the TAR loop region, a characteristic shared with Z6 virus, but not with ELI. We propose a virus-typing system based on sequence analysis confirmed by virus neutralization with a peptide binding antibody and inhibition by antisense phosphate-methylated DNA to group viruses for laboratory use and vaccine design.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Clonal heterogeneity at allelic methylation sites diagnostic for Prader-Willi and Angelman syndromes.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are developmental disorders resulting from the absence of the paternal or maternal contribution to the 15q11-13 region, respectively. Allele-specific methylation at D15S63 (PW71) has routinely been used as a diagnostic indicator of PWS and AS in DNA samples derived from peripheral blood. Extensive variation in allele-specific methylation patterns, however, has been observed at this site in different tissues, but the frequency or mechanism of this variation has remained uncharacterized. Herein, we have investigated the cellular basis of variation in methylation patterns at four sites of allelic methylation near SNRPN by using DNA samples derived from a panel of primary T lymphocyte clones. Interclonal variability was observed at three of these sites, including the diagnostic PW71 site. Changes in allele-specific methylation patterns occurred at a frequency of about one change in 50% of the cells every 22-25 doublings. In contrast, stable allele-specific methylation was observed in these clonal populations at exon 1 of SNRPN and the androgen receptor locus on the inactive X chromosome, suggesting that methylation at some CpG sites is more faithfully maintained than others. Clonal heterogeneity at PW71 was not an artifact of cell culture because the absence of allelic methylation was also observed in about 20% of the alleles in unstimulated peripheral blood. These results demonstrate that variation in allele-specific methylation at PW71 and other sites in the PWS/AS region appear to depend on the clonal complexity of the particular tissue and on the lack of strict maintenance of methylation within clones.

Alleles↗

PubMed2003

Hypermethylation of RASSF1A promoter is associated with the age at starting smoking and a poor prognosis in primary non-small cell lung cancer.

Cigarette smoking is the most common cause of lung cancer. The greatest risk of lung cancer is among those who started smoking early in life and continued throughout their lives. However, the molecular mechanisms responsible for the susceptibility to lung cancer in the young smoker are not clear. Recently, several groups have reported that the hypermethylation of CpG islands is associated with exposure to tobacco smoke. We studied the association between the age at starting smoking and hypermethylation of p14, p16, and RASSF1A promoters in 204 primary non-small cell lung cancer patients. We also examined whether hypermethylation of the RASSF1A promoter is an independent prognostic factor. Methylation rates in the 204 samples were detected in 9% for p14, 27% for p16, and 32% for RASSF1A. There was no relationship between the hypermethylation of p14 and p16 and the age at starting smoking. However, hypermethylation of the RASSF1A promoter was found to be significantly associated with the age at starting smoking (P = 0.001). No relationship was found between the methylation status of the RASSF1A promoter and other smoking variables, such as pack-years, smoking status, and the duration of smoking. The age at starting smoking in patients with hypermethylation of RASSF1A was earlier than that of patients without hypermethylation of the RASSF1A promoter (19 +/- 8 versus 25 +/- 7; P = 0.001). Young smokers who started smoking before age 19 were 4.23 times [95% confidence interval (CI) = 1.03-9.67; P = 0.001] more likely to have hypermethylation of the RASSF1A promoter than smokers who started smoking after the age of 19. Furthermore, hypermethylation of the RASSF1A promoter was found to be associated with a poor prognosis in non-small cell lung cancer patients at stages 1 and 2 (P = 0.02 and 0.01, respectively; Log-rank test). The hazard of failure was approximately 3.27 times higher for patients with hypermethylation of the RASSF1A promoter than for those without hypermethylation of the RASSF1A promoter (95% CI = 1.42-8.71; P = 0.01). Young smokers who started the habit before the age of 19 also had a poorer prognosis than those who started after the age of 19 (hazard ratio = 2.14, 95% CI = 1.22-9.11; P = 0.02). Our results suggest that starting cigarette smoking at an early age is associated with hypermethylation of the RASSF1A promoter and that hypermethylation of the RASSF1A promoter may be an independent prognostic factor in primary non-small cell lung cancer.

Adult↗

PubMed1977

Activation of protein kinase and glycogen phosphorylase in isolated rat liver cells by glucagon and catecholamines.

In liver cells isolated from fed female rats, glucagon (290nM) increased adenosine 3':5'-monophosphate (cyclic AMP) content and decreased cyclic AMP binding 30 s after addition of hormones. Both returned to control values after 10 min. Glucagon also stimulated cyclic AMP-independent protein kinase activity at 30 s and decreased protein kinase activity assayed in the presence of 2 muM cyclic AMP at 1 min. Glucagon increased the levels of glycogen phosphorylase a, but there was no change in total glycogen phosphorylase activity. Glucagon increased glycogen phosphorylase a at concentrations considerably less than those required to affect cyclic AMP and protein kinase. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine, potentiated the action of glucagon on all variables, but did not increase the maximuM activation of glycogen phosphorylase. Epinephrine (1muM) decreased cyclic AMP binding and increased glycogen phosphorylase a after a 1-min incubation with cells. Although 0.1 muM epinephrine stimulated phosphorylase a, a concentration of 10 muM was required to increase protein kinase activity. 1-Methyl-3-isobutyl xanthine (0.1 mM) potentiated the action of epinephrine on cyclic AMP and protein kinase. (-)-Propranolol (10muM) completely abolished the changes in cyclic AMP binding and protein kinase due to epinephrine (1muM) in the presence of 0.1mM 1-methyl-3-isobutyl xanthine, yet inhibited the increase in phosphorylase a by only 14 per cent. Phenylephrine (0.1muM) increased glycogen phosphorylase a, although concentrations as great as 10 muM failed to affect cyclic AMP binding or protein kinase in the absence of phosphodiesterase inhibitor. Isoproterenol (0.1muM) stimulated phosphorylase and decreased cyclic AMP binding, but only a concentration of 10muM increased protein kinase. 1-Methyl-3-isobutyl xanthine potentiated the action of isoproterenol on cyclic AMP binding and protein kinase, and propranolol reduced the augmentation of glucose release and glycogen phosphorylase activity due to isoproterenol. These data indicate that both alpha- and beta-adrenergic agents are capable of stimulating glycogenolysis and glycogen phosphorylase a in isolated rat liver cells. Low concentrations of glucagon and beta-adrenergic agonists stimulate glycogen phosphorylase without any detectable increase in cyclic AMP or protein kinase activity. The effects of alpha-adrenergic agents appear to be completely independent of changes in cyclic AMP protein kinase activity.

Animals↗

PubMedDate not supplied

Durene-capped porphyrin complexes of iron(II). Binding of imidazoles, and spectroscopic trends within Fe(porp)B(L) species (B = imidazole base; L = RNC, CO, O2).

Solution equilibria are presented for in situ reactions of the type FeII(porp) + B----FeII(porp)B, where porp represents the dianion of some durene-capped porphyrins with variable length linking methylene straps, and B is 1-methyl, 1,2-dimethyl, or 1,5-dicyclohexylimidazole. Increasing distortion of the porphyrin skeleton has no effect on coordination of B at the unhindered side of the capped porphyrin. Increasing skeleton distortion gives rise to regular trends within the visible spectra of the free base porphyrins, the hemin (FeIII) chlorides, and six-coordinate FeII(porp)B(L) species (L = RNC, CO, O2), while the visible spectra of FeII(porp) and of FeII(porp)B species are independent of the nature of porp and B, which may be related to greater flexibility of the lower coordination number systems. v(CO) data for the FeII(porp)(CO)B species are discussed briefly.

Benzene Derivatives↗

PubMed2006

Efficient screening method of the thiopurine methyltransferase polymorphisms for patients considering taking thiopurine drugs in a Chinese Han population in Henan Province (central China).

BACKGROUND: Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzed the S-methylation of thiopurine drugs. TPMT activity exhibits an interindividual variability, mainly as a result of genetic polymorphism. Patients with intermediate or deficient TPMT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. We determined a cut-off concentration of the TPMT activity assay less than which genotyping of the TPMT gene should be performed. In addition, the influence of hemodialysis on TPMT activity in uremic patients was examined. METHODS: In 248 healthy subjects and 30 uremic patients, PCR-based methods were used to analyze the most common functional mutations TPMT2, 3A, 3B and 3C. A HPLC assay was used to measure erythrocyte TPMT activity in the whole population. RESULTS: Seven TPMT3C heterozygotes were identified, while TPMT2, 3A and 3B alleles were not detected in 248 healthy subjects. The frequency of TPMT3C allele was 1.4% (7/496). The TPMT activity in healthy subjects was normally distributed, ranged from 6.09 to 28.65 nmol/h/ml pRBC with a mean of 16.03 +/- 4.16 nmol/h/ml pRBC. The cut-off for high TPMT activity and intermediate TPMT activity was 10.07 nmol/h/ml pRBC. There were 19 intermediate activity healthy subjects (7.7%) and 229 high activity healthy subjects (92.3%), and no TPMT deficiency subject was found. All of the 229 healthy subjects with high activity had no mutant alleles, while 7 of the 19 subjects with intermediate activity had a mutant allele. Phenotypes were in good agreement with genotypes for 95% of subjects. The uremic patients were all homozygous for the wild-type allele whose TPMT activity was activated significantly before hemodialysis compared with TPMT activity after hemodialysis. CONCLUSIONS: We defined the cut-off values for the TPMT phenotyping assay at 10.07 nmol/h/ml pRBC, less than which additional genotyping elucidates the individual risk for drug therapy. In uremic patients, TPMT activity is increased by some uremic factors, and dialysis shifted their TPMT activity close to that of a healthy control group.

Adult↗