Charles R. Yates

Molecular Diagnosis of Thiopurine S-Methyltransferase Deficiency: Genetic Basis for Azathioprine and Mercaptopurine Intolerance

Thiopurine S-methyltransferase (TPM) is a cytosolic enzyme that preferentially catalyzes the S-methylation (that is, inactivation) of such therapeutic agents as mercaptopurine, azathioprine, and thioguanine [1]. These thiopurine medications are currently used to treat many diseases, including cancer [2], autoimmune hepatitis [3], inflammatory bowel disease [4, 5], rheumatoid arthritis [6], multiple sclerosis [7], and autoimmune myasthenia gravis [8]; they are also used as immunosuppressants after organ transplantation [9, 10]. Several clinical studies have shown that patients with low TPM activity are at high risk for severe and potentially fatal hematopoietic toxicity if they are treated with conventional doses of mercaptopurine (for example, 75 mg/m2 body surface area per day) or azathioprine [9, 11-13]. Thiopurine S-methyltransferase activity shows codominant genetic polymorphism [14, 15]. About 90% of white and black persons have high TPM activity, and 10% have intermediate activity caused by heterozygosity at the TPM locus. About 1 in 300 persons inherits TPM deficiency as an autosomal recessive trait. Clinical studies have established an inverse correlation between TPM activity and accumulation of the active thioguanine nucleotide metabolites of mercaptopurine and azathioprine in erythrocytes. Patients with less efficient methylation of these thiopurine medications have more extensive conversion to active thioguanine nucleotides [2, 16]. Patients who have TPM deficiency accumulate higher levels of thioguanine nucleotides in erythrocytes if they receive standard doses of mercaptopurine or azathioprine. This accumulation of nucleotides usually leads to severe hematopoietic toxicity and possibly death [9], but this outcome can be averted if the thiopurine dose is decreased substantially (an 8- to 15-fold reduction) [17-19]. Patients who have intermediate TPM activity that is caused by heterozygosity at the TPM locus accumulate about 50% more thioguanine nucleotides than do patients who have high TPM activity [2]; this places patients with intermediate TPM activity at an intermediate risk for toxicity. Most of these patients are identified only after an episode of severe toxicity occurs. Although prospective measurement of erythrocyte TPM activity has been advocated by some investigators [4, 16], TPM assays are not widely available. Moreover, organ transplant recipients and patients who have recently received a diagnosis of cancer are frequently given transfusions of red blood cells; this precludes measurement of constitutive TPM activity before thiopurine therapy is started. Because thiopurine toxicity can be life threatening in TPM-deficient patients [9] and because of the intermediate risk for toxicity in heterozygous patients, a reliable method to identify patients who have inherited this trait is needed. If the genetic basis for TPM deficiency can be defined and polymerase chain reaction (PCR)-based methods can be developed to detect these inactivating mutations in genomic DNA, it should be possible to diagnose TPM deficiency and heterozygosity on the basis of genotype (as is now possible for other polymorphic enzymes) [17, 18]. To this end, we isolated and characterized two mutant alleles that are associated with TPM deficiency, TPM*2 and TPM*3A [19, 20]. The structures of these alleles are depicted in Figure 1. The molecular defect in TPM*2 is a G238C transversion mutation that leads to an amino acid substitution at codon 80 (Ala80Pro). Heterologous expression of this mutant allele in yeast showed a 100-fold decrease in S-methylation activity. The TPM*3A allele contains two nucleotide transition mutations (G460A and A719G) that lead to the amino acid substitutions Ala154Thr and Tyr240Cys. Heterologous expression of TPM*3A complementary DNA (cDNA) in yeast showed a greater than 200-fold reduction in TPM protein and undetectable activity. Moreover, marked instability of catalytic activity was evident for TPM proteins that were encoded by mutant cDNA containing either of these point mutations alone [20, 21]. We report the development, validation, and application of PCR-based methods for detection of these TPM mutations in the genomic DNA of patients and the elucidation of the polymorphic nature of the TPM gene locus in white persons. We also report a reliable method for the molecular diagnosis of TPM deficiency and heterozygosity that has excellent concordance between genotype and phenotype. Figure 1. Allelic variants at the human thiopurine S-methyltransferase (TPM) locus. Methods Human Patients and Determination of Phenotype Through methods described elsewhere [15], erythrocytes and leukocytes were isolated from the peripheral blood of healthy volunteers and children who had acute lymphoblastic leukemia. The volunteers were unselected blood donors who had been identified during a 2-month period, as described elsewhere [15]. The children were being treated at St. Jude Childrens Research Hospital or had been referred for evaluation because they could not tolerate chemotherapy. Genotype was determined for all unrelated white patients who had TPM activity that indicated heterozygous or deficient genotypes and for the same number of unrelated persons who had high activity that indicated a homozygous wild-type genotype. We focused our initial studies on white patients because they belong to the ethnic group in which we have identified the largest number of TPM-deficient and heterozygous persons. The activity of TPM in erythrocytes was determined by the radiochemical assay of Weinshilboum and colleagues [22], whose methods we modified, as described elsewhere [15]. The TPM phenotype was assigned on the basis of TPM activity in erythrocytes and according to the criteria of Weinshilboum and Sladek (that is, patients who had <5.0 U/mL of packed red blood cells were considered TPM deficient, those who had 5 to 10 U/mL were considered heterozygous, and those who had >10 U/mL were considered homozygous wild-type) [14]. We used the lowest value of TPM activity in erythrocytes that was measured in each person. We extracted RNA from leukocytes by using the method of Chomczynski and Sacchi [23], and genomic DNA was isolated by chloroform-phenol extractions. The studies were approved by the institutional review board for clinical trials at St. Jude Childrens Research Hospital, and informed consent was obtained from the patients or their guardians. Determination of Intronic Sequences The presence of a TPM-processed pseudogene [24] that could confound PCR-based genotyping methods and the absence of data on the genomic structure of the human TPM gene led us to initially use PCR primers that were complementary to TPM exon sequences to amplify genomic DNA by Expand PCR (Boehringer Mannheim, Indianapolis, Indiana) and thereby identify intronic sequences in the human TPM gene. The final volume for all PCR assays was 50 micro L. Through use of 1 g of placental genomic DNA (Clontech Laboratories, Inc., Palo Alto, California) as a template, PCR was done with primers A (5-GAGTTCTTCGGGGAACATTTCATTG-3) and B (5-CACCTGGATTAATGGCAAC TAATGC-3) in buffer D (Invitrogen, San Diego, California). The buffer contained Tris hydrochloride (pH 8.5), 60 mmol/L; ammonium sulfate, 15 mmol/L; and magnesium chloride, 3.5 mmol/L. The primers had been developed to amplify a fragment of genomic DNA (which included nucleotide 460) for detection of the G460A mutation. The concentration of each oligonucleotide was 0.1 OU/mL (about 0.5 mol/L), and 0.2 L Taq polymerase (Perkin Elmer Cetus, Norwalk, Connecticut) was used. With a Hybaid OmniGene thermocycler (Woodbridge, New Jersey), amplification was done for 30 cycles consisting of denaturation at 94 C for 1 minute, annealing at 55 C for 2 minutes, and extension at 72 C for 1 minute. A final extension step at 72 C for 7 minutes was also done. For the initial cycle, 5 L of deoxynucleoside triphosphates (dNTP, 10 mmol/L) was added after the temperature reached 80 C (following the hot start protocol). An amplified fragment of 138 base pairs was anticipated in the absence of intron sequences; the resulting fragment of 746 base pairs showed the presence of an intervening intron. This fragment was directly cloned into the plasmid pCR-II (Invitrogen). The recombinant plasmid was purified with Qiagen plasmid kits (Chatsworth, California) and sequenced with an automated sequencer using the cycle sequencing reaction and fluorescence-tagged dye terminators (Prism, Applied Biosystems, Foster City, California). The resulting intron sequence and the intron-exon boundary was then used to develop intron-specific primer P460F. Through a similar strategy, Expand PCR was used to amplify intron sequences that flanked the exons containing the G238C mutation (intron 4) and the A719G mutation (intron 9). The resulting intron-containing fragments were directly cloned into the plasmid pCR-II; the plasmid was purified and sequenced as described above. These sequences permitted the development of intron-specific PCR primers P2C and P719F for the detection of G238C and A719G mutations. Detection of TPM Mutations by Polymerase Chain Reaction Detection of G238C We used PCR amplification to determine whether the G238C transversion was present at the TPM locus. Genomic DNA, 400 ng, was amplified under conditions similar to those discussed for the intronic sequence except that 2 L of primer P2W (5-GTATGATTTTAT GCAGGTTTG-3) or P2M (5-GTATGATTTTATGCAGGTTTC-3) was used with primer P2C (5-TAAATAGGAACCATCGGACAC-3) (0.1 OU/mL) in each amplification. Unpurified PCR products were analyzed by electrophoresis in 2.5% MetaPhor gels (MetaPhor Agarose, FMC Bioproducts, Rockland, Maine) stained with ethidium bromide. A DNA fragment was amplified with P2M and P2C primers when C238 (mutant) was present, whereas a DNA fragment was amplified with P2W and P2C primers when G238 (wild-type) was present (Figure 2). Figure 2. Schematic of polymerase chain reac

Genetic polymorphism of thiopurine S-methyltransferase: clinical importance and molecular mechanisms.

Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurines such as mercaptopurine and thioguanine. TPMT activity exhibits genetic polymorphism, with about 1 in 300 inheriting TPMT-deficiency as an autosomal recessive trait. If treated with standard dosages of thiopurines. TPMT-deficient patients accumulate excessive thioguanine nucleotides (TGN) in hematopoietic tissues, leading to severe hematopoietic toxicity that can be fatal. However, TPMT-deficient patients can be successfully treated with a 10-15-fold lower dosage of these medications. The human gene encoding polymorphic TPMT has been cloned and characterized, and two mutant alleles have recently been isolated from TPMT-deficient and heterozygous patients (TPMT*2, TPMT*3), permitting development of PCR-based methods to identify TPMT-deficient and heterozygous patients prior to therapy. TPMT*3 is the predominant mutant allele in American whites, accounting for about 75% of mutations in this population. Ongoing studies aim to better define the influence of TPMT activity on thiopurine efficacy, to identify additional mutant alleles and determine their frequency in different ethnic groups, to elucidate the mechanism(s) for loss of function of mutant proteins, to identify potential endogenous substrates and to define the molecular mechanisms of TPMT regulation. Together, these advances bold the promise of improving the safety and efficacy of thiopurine therapy.

The Effect of CYP3A5 and MDR1 Polymorphic Expression on Cyclosporine Oral Disposition in Renal Transplant Patients

Variability in CYP3A (CYP3A4/5) and P‐glycoprotein (human MDR1 gene product) activity underlies interindividual differences in oral cyclosporine (CsA) bioavailability. Racial differences in polymorphic expression of CYP3A5 and MDR1 may explain observed interracial variability in oral bioavailability. Our objective was to evaluate the effect of CYP3A5 and MDR1 polymorphic expression on CsA oral disposition. Steady‐state plasma concentration profiles (n = 19) were sampled in renal transplant recipients receiving concentration‐adjusted CsA maintenance therapy. CsA plasma concentrations were measured by fluorescence polarization immunoassay. CYP3A5 and MDR1 genotypes were determined by real‐time polymerase chain reaction. Noncompartmental pharmacokinetic analysis and nonlinear mixed‐effects modeling (NONMEM) were performed to assess the effect of genotype on CsA pharmacokinetics. MDR1 C3435T genotype was identified as the best predictor of CsA systemic exposure. CsA oral clearance was significantly higher in subjects who carried at least one 3435T allele compared to homozygous wild‐type individuals (40.0 ± 2.2 vs. 26.4 ± 3.1 L/h, p = 0.007). MDR1 C3435T genotype accounted for 43% of the interindividual variability of CsA oral clearance in the study population after accounting for interoccasion variability. The authors were unable to independently assess whether CYP3A5 correlated with any CsA pharmacokinetic parameter since all CYP3A5 nonexpressors were also 3435T allele carriers. MDR1 3435T allele carriers have enhanced oral clearance compared to individuals with the CC genotype. The frequency of the 3435T allele is lower in African Americans compared to Caucasians. Thus, the MDR1 C3435T genotype offers a potential mechanistic basis to explain interracial differences in CsA oral bioavailability. Further studies are needed to explore the relationship between CYP3A5 and MDR1 genotype and phenotype.

In vivo evidence for a novel pathway of vitamin D3 metabolism initiated by P450scc and modified by CYP27B1

We define previously unrecognized in vivo pathways of vitamin D3 (D3) metabolism generating novel D3‐hydroxyderivatives different from 25‐hydroxyvitamin D3 [25(OH)D3] and 1,25(OH)2D3. Their novel products include 20‐hydroxyvitamin D3 [20(OH)D3], 22(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3, 1,20(OH)2D3,1,20,23(OH)3D3, and 17,20,23(OH)3D3 and were produced by placenta, adrenal glands, and epidermal keratinocytes. We detected the predominant metabolite [20(OH)D3] in human serum with a relative concentration ~20 times lower than 25(OH)D3. Use of inhibitors and studies performed with isolated mitochondria and purified enzymes demonstrated involvement of the steroidogenic enzyme cytochrome P450scc (CYP11A1) as well as CYP27B1 (1α‐hydroxylase). In placenta and adrenal glands with high CYP11A1 expression, the predominant pathway was D3 → 20(OH)D3 → 20,23(OH)2D3 → 17,20,23(OH)3D3 with further 1α‐hydroxylation, and minor pathways were D3 → 25(OH)D3 → 1,25(OH)2D3 and D3 → 22(OH)D3 → 20,22(OH)2D3. In epidermal keratinocytes, we observed higher proportions of 22(OH)D3 and 20,22(OH)2D3. We also detected endogenous production of 20(OH)D3, 22(OH) D3, 20,23(OH)2D3, 20,22(OH)2D3, and 17,20,23(OH)3D3 by immortalized human keratinocytes. Thus, we provide in vivo evidence for novel pathways of D3 metabolism initiated by CYP11A1, with the product profile showing organ/cell type specificity and being modified by CYP27B1 activity. These findings define the pathway intermediates as natural products/endogenous bioregulators and break the current dogma that vitamin D is solely activated through the sequence D3 → 25(OH)D3 → 1,25(OH)2D3.—Slominski, A. T., Km, T.‐K., Shehabi, H. Z., Semak, I., Tang, E. K. Y., Nguyen, M. N., Benson, H. A. E., Korik, E., Janjetovic, Z., Chen, J., Yates, C. R., Postlethwaite, A., Li, W., Tuckey, R. C. In vivo evidence for a novel pathway of vitamin D3 metabolism initiated by P450scc and modified by CYP27B1. FASEB J. 26, 3901–3915 (2012). www.fasebj.org

Structural Determinants of P-Glycoprotein-Mediated Transport of Glucocorticoids

AbstractPurpose. The aim of this study was to determine requisite structural features for P-glycoprotein-mediated transport of a series of structurally related glucocorticoids (GCs). Methods. Transport experiments were conducted in wild-type and stably transfected MDR1 LLC-PK cell line. Transport efficiency (Teff = Peff, B→A / Peff, A→B) in both cell lines was compared as a measure of passive diffusion and P-glycoprotein-mediated transepithelial transport for each steroid. Three-dimensional structure-activity relationships were built to determine how specific structural features within the steroids affect their P-gp-mediated efflux. Results. Mean (± SD) Teff in LLC-PK cells was 1.1 ± 0.17, indicating that differences in structure and partition coefficient did not affect drug flux in the absence of P-glycoprotein. Teff in L-MDR1 cells ranged from 3.6 to 26.6, demonstrating the importance of glucocorticoid structure to P-glycoprotein transport. The rank order of Teff in MDR1 cells was: methylprednisolone> prednisolone > betamethasone > dexamethasone/prednisone > cortisol. There was no correlation between individual Teff values and partition coefficient. 3D-QSAR models were built using CoMFA and CoMSIA with a q2 (r2) of 0.48 (0.99) and 0.41 (0.95), respectively. Conclusions. Nonpolar bulky substituents around the C-6α position, as well as a hydrogen-bond donor at position C-11, enhance P-glycoprotein affinity and cellular efflux, whereas bulky substituents at C-16 diminish transporter affinity.

Pharmacokinetics of immunosuppressants: a perspective on ethnic differences.

Despite recent advancements in solid organ transplantation, African-American renal allograft recipients continue to exhibit poorer prognosis in long-term clinical outcome and graft survival compared to Caucasian patients. The role of immunosuppressants in post-transplant outcome is crucial, and associations between exposure-related pharmacokinetic parameters and clinical outcome have been made for several drugs in this class. Thus, ethnic differences in the pharmacokinetics of immunosuppressants are potentially a key factor in the observed differences in post-transplant outcome between African-Americans and Caucasians. Ethnic differences in pharmacokinetics of mycophenolate mofetil and azathioprine based on the current literature are either absent or only of minor relevance. Cyclosporine, tacrolimus, sirolimus and everolimus, however, have all been described to exhibit ethnicity-specific differences in bioavailability and/or dose-adjusted systemic exposure, although currently available reports are controversial for some of these drugs. Oral bioavailability of these drugs in African-Americans was between 20 and 50% lower than in Caucasians or Non-African-Americans, leading to higher dose requirements in African-Americans to maintain similar average concentrations of the respective immunosuppressant. Since all four drugs undergo extensive metabolism and are substrates for CYP3A isoenzymes as well as the drug transporter P-glycoprotein, interethnic variability in activity of these enzymes/transporter may provide a common mechanism for the observed ethnic differences. These ethnic differences are most likely mediated via several non-genetic as well as genetic factors, including known genetic variations that impair transporter/enzyme activity in genes such as CYP3A4, CYP3A5 and ABCB1 (MDR1). Appreciation of differences in immunosuppressant pharmacokinetics and dose requirements between African-Americans and Caucasians in clinical practice is expected to improve post-transplant immunosuppressive pharmacotherapy and may thus contribute to equalize prognostic outcome for all transplant patients.

Isolation of a human thiopurine S‐methyltransferase (TPMT) complementary DNA with a single nucleotide transition A719G (TPMT*3C) and its association with loss of TPMT protein and catalytic activity in humans

Thiopurine S‐methyltransferase (TPMT) is a cytosolic enzyme that catalyzes the S‐methylation of mercaptopurine, azathioprine, thioguanine and most of their nucleotide metabolites. TPMT exhibits genetic polymorphism, with about 10% of individuals having intermediate TPMT activity because of heterozygosity at the TPMT locus and about 1 in 300 inheriting TPMT deficiency as an autosomal recessive trait. Although several mutant alleles have now been associated with inheritance of TPMT deficiency in humans, the expression of only TPMT*2 and TPMT*3A has been established by isolation and characterization of complementary DNA (cDNA) from individuals with low TPMT activity.

Systemic Inflammation-Associated Glucocorticoid Resistance and Outcome of ARDS

Abstract: Dysregulated systemic inflammation with excess activation of pro‐inflammatory transcription factor nuclear factor‐κB (NF‐κB)—activated by inflammatory signals—compared to the anti‐inflammatory transcription factor glucocorticoid receptor‐α (GRα)—activated by endogenous or exogenous glucocorticoids (GCs)—is an important pathogenetic mechanism for pulmonary and extrapulmonary organ dysfunction in patients with acute respiratory distress syndrome (ARDS). Activation of one transcription factor in excess of the binding (inhibitory) capacity of the other shifts cellular responses toward increased (dysregulated) or decreased (regulated) transcription of inflammatory mediators over time. Recent data indicate that failure to improve in ARDS (unresolving ARDS) is frequently associated with failure of the activated GRs to downregulate the transcription of inflammatory cytokines despite elevated levels of circulating cortisol, a condition defined as systemic inflammation‐associated acquired GC resistance; it is potentially reversible with prolonged GC supplementation.

Promoter and Intronic Sequences of the Human Thiopurine S-Methyltransferase (TPMT) Gene Isolated from a Human Pacl Genomic Library

AbstractPurpose. To isolate and characterize the polymorphic human thiopurine S-methyltransferase (TPMT) gene. Methods. The human TPMT gene was isolated by PCR screening of a phage artificial chromosome (PAC) library, using exon- and intron-specific primers, then mapped and sequenced. Results. Two separate PAC1 clones were isolated that contained the same 25 kb gene with 9 exons encompassing the entire TPMT open reading frame. Structural characterization revealed distinct differences when compared to a TPMT gene previously isolated from a chromosome 6-specific human genomic library; the 5′-flanking region (putative promoter) contains 17 additional nucleotides located at position-77 upstream from the transcription start site, in addition to several nucleotide sequence differences, and intron 8 is only 1.6 kb, 5 kb shorter than previously reported. Southern and PCR analyses of genomic DNA from 18 unrelated individuals revealed only the TPMT gene structure corresponding to the PAC clones we isolated. Analysis of the TPMT promoter activity using the 5′-terminal region confirmed transcriptional activity in human HepG2 and CCRF-CEM cells. The 5′-flank is 71% GC rich and does not contain consensus sequences for TATA box or CCAAT elements. FISH analysis demonstrated the presence of the TPMT-homologous sequence on the short arm of chromosome 6 (sublocalized to 6p22). Conclusions. These findings establish the genomic structure of the human TPMT gene, revealing differences in the promoter and intronic sequences compared to that previously reported and providing a basis for future studies to further elucidate its biological function and regulation.

Cytochrome P450scc-dependent metabolism of 7-dehydrocholesterol in placenta and epidermal keratinocytes.

The discovery that 7-dehydrocholesterol (7DHC) is an excellent substrate for cytochrome P450scc (CYP11A1) opens up new possibilities in biochemistry. To elucidate its biological significance we tested ex vivo P450scc-dependent metabolism of 7DHC by tissues expressing high and low levels of P450scc activity, placenta and epidermal keratinocytes, respectively. Incubation of human placenta fragments with 7DHC led to its conversion to 7-dehydropregnenolone (7DHP), which was inhibited by dl-aminoglutethimide, and stimulated by forskolin. Final proof for P450scc involvement was provided in isolated placental mitochondria where production of 7DHP was almost completely inhibited by 22R-hydroxycholesterol. 7DHC was metabolized by placental mitochondria at a faster rate than exogenous cholesterol, under both limiting and saturating conditions of substrate transport, consistent with higher catalytic efficiency (k(cat)/K(m)) with 7DHC as substrate than with cholesterol. Ex vivo experiments showed five 5,7-dienal intermediates with MS spectra of dihydroxy and mono-hydroxy-7DHC and retention time corresponding to 20,22(OH)(2)7DHC and 22(OH)7DHC. The chemical structure of 20,22(OH)(2)7DHC was defined by NMR. 7DHP was further metabolized by either placental fragments or placental microsomes to 7-dehydroprogesterone as defined by UV, MS and NMR, and to an additional product with a 5,7-dienal structure and MS corresponding to hydroxy-7DHP. Furthermore, epidermal keratinocytes transformed either exogenous or endogenous 7DHC to 7DHP. 7DHP inhibited keratinocytes proliferation, while the product of its pholytic transformation, pregcalciferol, lost this capability. In conclusion, tissues expressing P450scc can metabolize 7DHC to biologically active 7DHP with 22(OH)7DHC and 20,22(OH)(2)7DHC serving as intermediates, and with further metabolism to 7-dehydroprogesterone and (OH)7DHP.