AMPD1 Research Articles

CARDIORESPIRATORY RESPONSES TO EXERCISE AND THE C34T AMPD1 GENE POLYMORPHISM IN THE HERITAGE FAMILY STUDY

An inherited form of AMPD deficiency has been attributed to a non-sense mutation (C to T transition in nucleotide 34) in exon 2 of AMPD1 gene. AMPD deficiency causes AMP accumulation, which can lead to the production of adenosine by AMP 5'-nucleotidase. It is known that adenosine accumulation exerts a vasodilatory response, which could have physiological consequences during exercise. PURPOSE To examine associations of the C34T polymorphism of the AMPD1 gene with cardiorespiratory and performance phenotypes during cycling exercise at absolute and relative power outputs progressing to exhaustion before and after endurance training for 20 weeks in the HERITAGE Family Study cohort (n = 779). Since no Blacks were mutant homozygotes (TT), only Caucasians were considered for analysis (400 normal homozygotes, CC; 97 heterozygotes, CT; and 6 TT). RESULTS Sedentary State. Cycling at the absolute workload of 50 W resulted in higher rate of perceived exertion in TT (p < 0.0001). At the relative intensity of 60% of VO2 max, stroke volume was lower in TT (p < 0.05). Maximal values for power output, SBP, HR, VCO2, and RER were lower in TT (p < 0.05). Response to Training. The cardiorespiratory training response at 50 W and at 60% of VO2 max was similar across C34T-AMPD1 genotypes. However, max ventilation, max VO2, and max VCO2 during exercise increased less in TT (p < 0.01). CONCLUSION The results indicate that subjects with the TT genotype at the C34T AMPD1 gene have diminished exercise capacity and cardiovascular and ventilatory responses to exercise. Endurance training improves submaximal exercise cardiorespiratory and performance phenotypes in carriers of the C34T mutation as much as in non-carriers, but the training response of ventilatory phenotypes during maximal exercise is more limited in TT. Supported by multiple grants from NIH

A G468-T AMPD1 mutant allele contributes to the high incidence of myoadenylate deaminase deficiency in the Caucasian population

Myoadenylate deaminase deficiency is the most common metabolic disorder of skeletal muscle in the Caucasian population, affecting approximately 2% of all individuals. Although most deficient subjects are asymptomatic, some suffer from exercise-induced myalgia suggesting a causal relationship between a lack of enzyme activity and muscle function. In addition, carriers of this derangement in purine nucleotide catabolism may have an adaptive advantage related to clinical outcome in heart disease. The molecular basis of myoadenylate deaminase deficiency in Caucasians has been attributed to a single mutant allele characterized by double C to T transitions at nucleotides +34 and +143 in mRNA encoded by the AMPD1 gene. Polymerase chain reaction-based strategies have been developed to specifically identify this common mutant allele and are considered highly sensitive. Consequently, some laboratories preferentially use this technique over other available diagnostic tests for myoadenylate deaminase deficiency. We previously identified a G468-T mutation in one symptomatic patient who was only heterozygous for the common AMPD1 mutant allele. In this report, nine additional individuals with this compound heterozygous genotype are revealed in a survey of 48 patients with documented deficiency of skeletal muscle adenosine monophosphate deaminase and exercise-induced myalgia. Western blot analysis of leftover biopsy material from one of these individuals does not detect any immunoreactive myoadenylate deaminase polypeptide. Baculoviral expression of the G468-T mutant allele produces a Q156H substitution enzyme exhibiting labile catalytic activity. These combined results demonstrate that the G468-T transversion is dysfunctional and further indicate that AMPD1 alleles harboring this mutation contribute to the high incidence of partial and complete myoadenylate deaminase deficiency in the Caucasian population. Consequently, genetic tests for abnormal AMPD1 expression designed to diagnose patients with metabolic myopathy, and to evaluate genetic markers for clinical outcome in heart disease should not be based solely on the detection of a single mutant allele.

Regulation of skeletal muscle ATP catabolism by AMPD1 genotype during sprint exercise in asymptomatic subjects.

Deficiency of myoadenylate deaminase, the muscle isoform of AMP deaminase encoded by the AMPD1 gene, is a common myopathic condition associated with alterations in skeletal muscle energy metabolism. However, recent studies have demonstrated that most individuals harboring this genetic abnormality are asymptomatic. Therefore, 18 healthy subjects with different AMPD1 genotypes were studied during a 30-s Wingate test in order to evaluate the influence of this inherited defect in AMPD1 expression on skeletal muscle energy metabolism and exercise performance in the asymptomatic population. Exercise performances were similar across the AMPD1 genotypes, whereas significant differences in several descriptors of energy metabolism were observed. Normal homozygotes (NN) exhibited the highest levels of AMP deaminase activities, net ATP catabolism, and IMP accumulation, whereas intermediate values were observed in heterozygotes (MN). Conversely, mutant homozygotes (MM) had very low AMP deaminase activities and showed no significant net catabolism of ATP or IMP accumulation. Accordingly, MM also did not show any postexercise increase in plasma ammonia. Unexpectedly, MN consistently exhibited greater increases in plasma ammonia compared with NN despite the relatively lower accumulation of IMP in skeletal muscle. Moreover, time course profiles of postexercise plasma ammonia and blood lactate accumulation also differed across AMPD1 genotypes. Finally, analysis of adenosine in leftover biopsy material revealed a modest twofold increase in MN and a dramatic 25-fold increase in MM.

A common variant of the AMPD1 gene predicts improved survival in patients with ischemic cardiomyopathy, but not non-ischemic cardiomyopathy Yoshikazu Yazaki, Siavash Ghaffari, Joseph B. Muhlestein, Noal I. Hart, Tami L. Bair, Benjamin D. Horne, Dale G. Renlund, Jeffrey L. Anderson, John F. Carlquist. LDS Hospital, Salt Lake City, UT, University of Utah, Salt Lake City, UT

A common variant of the AMPD1 gene predicts improved survival in patients with ischemic cardiomyopathy, but not non-ischemic cardiomyopathy Yoshikazu Yazaki, Siavash Ghaffari, Joseph B. Muhlestein, Noal I. Hart, Tami L. Bair, Benjamin D. Horne, Dale G. Renlund, Jeffrey L. Anderson, John F. Carlquist. LDS Hospital, Salt Lake City, UT, University of Utah, Salt Lake City, UT

A rare case of complete human erythrocyte AMP deaminase deficiency due to two novel missense mutations in AMPD3 Communicated by Mark H. Paalman Online Citation: Human Mutation, Mutation in Brief #395 (2000) Online http://journals.wiley.com/1059-7794/pdf/mutation/395.pdf

Human erythrocyte AMP deaminase (AMPD3) deficiency is a clinically asymptomatic condition characterized by a 50% increase in steady-state levels of ATP in affected cells. The deficiency in Japanese is associated 75% of the time with the same mutation of R573C, and 25% of the time with other heterogeneous mutations of the AMPD3 gene. The heterozygote frequency was estimated at about 1/30. We previously reported five Japanese individuals who had a complete deficiency of AMPD3. Four were homozygotes for the major mutation of R573C; however, one female did not have the R573C allele. To clarify the mutations in her AMPD3 gene, we analyzed the AMPD3 gene and detected a minor mutation, W450R, derived from the mother and a novel mutation,Q712P, derived from the father. The expression study using AMPD3 cDNA containing both mutations showed that each mutation completely reduced the enzyme activity of AMPD3. As the frequency of carriers heterozygous for these mutations seems to be very low, identifying them may lead to a better understanding of the genetic background of populations in Japan. © 2001 Wiley-Liss, Inc.

First missense mutations (R388W and R425H) of AMPD1 accompanied with myopathy found in a Japanese patient

Skeletal muscle AMP deaminase (AMPD: E.C. 3.5.4.6) deficiency is one of the most common inherited defects in the Caucasians, but not in Asians. Although a diagnosis of AMPD1 deficiency is indeed based on the reduced enzymatic activity, its clinical significance is still rather controversial since most subjects are asymptomatic. Alternative splicing of exon 2 in individuals who have inherited this defect is thought to provide a mechanism for phenotypic rescue that may explain the variability of clinical symptoms as we reported earlier. In this report we present the first case with a detectable defect of the AMPD1 gene in a Japanese patient with myopathy. Two missense mutations (R388W and R425H) in exon 9 and exon 10 of the AMPD1 gene were found. Prokaryotic expression showed a comparable amount of the AMPD1 peptides and undetectable AMPD activity in the constructs with these mutations. From this study, we have concluded that this patient is a compound heterozygote for AMPD1 mutant allele. This study also demonstrates the first reported instance of detectable dysfunction of the AMPD1 gene product, suggesting that AMPD1 indeed has a key role in muscle metabolism and function. Hum Mutat 16:467–472, 2000. © 2000 Wiley-Liss, Inc.

A common variant of the AMPD1 gene predicts improved cardiovascular survival in patients with coronary artery disease

OBJECTIVEWe tested whether a common AMPD1 gene variant is associated with improved cardiovascular (CV) survival in patients with coronary artery disease (CAD).BACKGROUNDReduced activity of adenosine monophosphate deaminase (AMPD) may increase production of adenosine, a cardioprotective agent. A common, nonsense, point variant of the AMPD1 gene (C34T) results in enzymatic inactivity and has been associated with prolonged survival in heart failure.METHODSBlood was collected from 367 patients undergoing coronary angiography. Genotyping was done by polymerase chain reaction amplification and restriction enzyme digestion, resulting in allele-specific fragments. Coronary artery disease was defined as ≥70% stenosis of ≥1 coronary artery. Patients were followed prospectively for up to 4.8 years. Survival statistics compared hetero- (+/−) or homozygotic (−/−) carriers with noncarriers.RESULTSPatients were 66 ± 10 years old; 79% were men; 22.6% were heterozygous and 1.9% homozygous for the variant AMPD1(−) allele. During a mean of 3.5 ± 1.0 years, 52 patients (14.2%) died, 37 (10.1%) of CV causes. Cardiovascular mortality was 4.4% (4/90) in AMPD1(−) allele carriers compared with 11.9% (33/277) in noncarriers (p = 0.046). In multiple variable regression analysis, only age (hazard ratio, 1.11/year, p < 0.001) and AMPD1(−) carriage (hazard ratio, 0.36, p = 0.053) were independent predictors of CV mortality.CONCLUSIONSCarriage of a common variant of the AMPD1 gene was associated with improved CV survival in patients with angiographically documented CAD. The dysfunctional AMPD1(−) allele may lead to increased cardiac adenosine and increased cardioprotection during ischemic events. Adenosine monophosphate deaminase-1 genotyping should be further explored in CAD for prognostic, mechanistic and therapeutic insights.

Positive and negative elements mediate control of alternative splicing in the AMPD1 gene.

The second exon of the AMP deaminase (AMPD) 1 gene is alternatively spliced in response to stage-specific signals elaborated during myocyte differentiation. Since inheritance of the mutation in exon 2 of the AMPD1 gene has been recently shown to be associated with a better prognosis of congestive heart failure and the alternative splicing of exon 2 modulates the residual activity of AMPD1 in individuals with this mutant allele, the regulatory mechanism of alternative splicing in the AMPD1 gene is clinically intriguing. Retention or exclusion of exon 2 results from the interplay between negative and positive elements in the primary transcript. Exon 2 is intrinsically defective and difficult to recognize. Herein, we show that this property of exon 2 is the consequence of three defects; a suboptimal 3' splice acceptor site, a suboptimal 5' splice donor site and the small size of the exon. An improvement in any one of these defects relieves the masking of this exon. Further, this defective exon can only be identified in the presence of the adjacent downstream intron.

Control of AMP deaminase 1 binding to myosin heavy chain.

AMP deaminase (AMPD) plays a central role in preserving the adenylate energy charge in myocytes following exercise and in producing intermediates for the citric acid cycle in muscle. Prior studies have demonstrated that AMPD1 binds to myosin heavy chain (MHC) in vitro; binding to the myofibril varies with the state of muscle contraction in vivo, and binding of AMPD1 to MHC is required for activation of this enzyme in myocytes. The present study has identified three domains in AMPD1 that influence binding of this enzyme to MHC using a cotransfection model that permits assessment of mutations introduced into the AMPD1 peptide. One domain that encompasses residues 178-333 of this 727-amino acid peptide is essential for binding of AMPD1 to MHC. This region of AMPD1 shares sequence similarity with several regions of titin, another MHC binding protein. Two additional domains regulate binding of this peptide to MHC in response to intracellular and extracellular signals. A nucleotide binding site, which is located at residues 660-674, controls binding of AMPD1 to MHC in response to changes in intracellular ATP concentration. Deletion analyses demonstrate that the amino-terminal 65 residues of AMPD1 play a critical role in modulating the sensitivity to ATP-induced inhibition of MHC binding. Alternative splicing of the AMPD1 gene product, which alters the sequence of residues 8-12, produces two AMPD1 isoforms that exhibit different MHC binding properties in the presence of ATP. These findings are discussed in the context of the various roles proposed for AMPD in energy production in the myocyte.

Clinical heterogeneity and molecular mechanisms in inborn muscle AMP deaminase deficiency.

Lack of the muscle-specific isoform of AMP deaminase (myoadenylate deaminase deficiency) can cause a metabolic myopathy, with exercise-induced muscle symptoms such as early fatigue, cramps and/or myalgia. It is the most common muscle enzyme defect in man, found in about 2-3% of all muscle biopsies. The genetic basis of the inherited defect is the nonsense mutation C34-T in the AMPD1 gene encoding myoadenylate deaminase. The mutation results in a premature stop of the enzyme synthesis. In a healthy German population, the frequency of the mutant allele was 0.1, and 1% of this population is expected to be homozygous for the mutation. In people with muscle symptoms, the allele frequency was significantly higher (0.145). The correlation between allele frequency and muscle symptoms underscores the clinical significance of this defect. However, the vast majority of homozygous subjects do not develop a metabolic myopathy. This clinical heterogeneity may be due to molecular genetic factors such as alternative splicing of the exon harbouring the mutation, or due to metabolic conditions such as pathways compensating for the defect. The real basis for the high percentage of asymptomatic homozygous subjects remains to be revealed.

Human adenosine receptors

Myo-adenylate deaminase deficiency (mADD) is the most common enzyme deficiency restricted to skeletal muscle, with a frequency of 1–2% in frozen muscle biopsies and complaints of easy fatigue and muscle cramping on exertion. A double C > T transition at coding bases 34 in exon 2 and 143 in exon 3 is the main cause of mADD. A 1-day assay using allele-specific oligomers and no isotope would be valuable for single cases.Downstream primers with penultimate mismatch and 3′ terminus matching the mutant or the normal base in exons 2 and 3 are used with a common upstream primer for each exon, to give amplimers of 150 bp for exon 2 and 200 bp for exon 3. A short common primer further downstream in exon 3 provides a competing 300-bp amplimer whose product contribution is readily controlled by adjusting the annealing temperature. The entire procedure could be done in 1 day: DNA isolation, polymerase chain reaction (PCR), electrophoresis in agarose gel with ethidium bromide, and visualization by ultraviolet light. Deficient individuals have bands only with the mutant primers, normal persons have bands only with the normal primers, and heterozygotes (carriers) show bands with both primer sets. The empty slots show the 300-bp competing band, proving the PCR amplified the correct template. Allele-specific oligomers PCR results were verified by dot blots and by restriction endonuclease analysis of exon 2.A simple and reliable allele-specific PCR assay using DNA from blood (or muscle) is now available for the diagnosis of individual cases of mADD caused by the common double-mutant AMPD1 gene, including the rare instances arising from homologous recombination between the two mutations.

Reciprocol developmental patterns of AMPD3 gene expression determine adult rat tissue specificity of isoform C

Reciprocol developmental patterns of AMPD3 gene expression determine adult rat tissue specificity of isoform C

Molecular analysis of mouse Ampd3 gene encoding heart-type isoform of AMP deaminase

Purine metabolism is one mechanism to maintain cellular ATP concentration. The pathway to degrade adenosine nucleotide directly involves in stabilization of the energy charge during ATP consumption. AMP deaminase (E.C.3.5.4.6.; AMPD), which catalyzes the hydrolytic deamination of AMP and converts it to IMP, is thought to play an important role in purine metabolism in all eukaryotic cells. In higher eukaryotes, AMPD has multiple isoforms encoded by a family of AMPD multigenes.1 There are three genes in human: AMPD1, AMPD2, AMPD3, whose products are predominant in muscle (M), liver (L) and erythrocyte (E), respectively.2–4 In rodent, two genes, Ampd1 and Ampd2 that are equivalent to the human AMPD1 and AMPD2, respectively, have been known,2–5 but the gene corresponding to AMPD3 has not been reported. From the analogy of human AMPD, this hypothetical gene has been inferred to code for heart(H)-type AMPD, but molecular studies with rodent heart AMPD has not been done. In this study, mouse cDNA and the gene encoding H-isoform of AMPD have been isolated and characterized for the first time.

Cloning and expression of cDNA encoding heart-type isoform of AMP deaminase

Mouse cDNA for the heart-type (H) isoform of AMP deaminase (EC 3.5.4.6., AMPD) has been isolated and characterized. The cDNA for the Ampd gene expressed in heart is predicted to encode a peptide of 766 amino acids with a molecular mass of 88 kDa. The coding region of this cDNA was quite homologous to the human AMPD3 gene which encodes the erythrocyte-type (E) isoform of AMPD, although the H-isoform of rodent AMPD was reported to be immunologically distinct from the E-isoform of human AMPD. The non-coding region of the isolated cDNA is not homologous to human AMPD3, while rodent Ampd1 has similarity to human AMPD1 in the non-coding region as well as in the coding region. The transcripts for the cloned cDNA were expressed in heart, slow-twitch skeletal muscle and non-muscle tissues. Prokaryotic expression showed that this cDNA encodes a catalytically active enzyme which reacts to the specific antibody raised to the H-isoform of AMPD. © 1997 Elsevier Science B.V. All rights reserved.

Cloning, sequence and characterization of the human AMPD2 gene: evidence for transcriptional regulation by two closely spaced promoters

AMP deaminase (AMPD) is manifest through a multigene family in higher eukaryotes, including man. The human AMPD1 and AMPD3 genes have been cloned and partially characterized. This study describes the cloning, chromosomal localization, partial-sequence and characterization of the human AMPD2 gene. Composed of nineteen exons and eighteen intervening sequences spanning nearly 14 kb of genomic DNA, the human AMPD2 gene is positioned on the short arm of chromosome 1 near the p13.3 boundary. Two alternative 5′ exons (1A and 1B) are remotely located upstream, whereas the other seventeen are compressed into the 3′ terminal one-half of the gene. Transient transfections of human retinal pigment epithelial (RPE) cells using heterologous constructs containing 5′ flanking and 5′ untranslated sequences cloned upstream of a luciferase reporter gene show that promoter activities are associated with exons 1A and 1B. Inspection of genomic DNA sequence reveals that AMPD2 promoter regions lack readily identifiable TATA boxes and are G + C-rich, particularly in the region of multiple transcription initiation sites in exon 1A. The regulation and evolution of the entire human AMPD multigene family are discussed.

GNAI3, GNAT2, AMPD2, GSTM are clustered in 120 kb of Chinese hamster chromosome 1q.

We studied a polygenic region located on Chromosome (Chr) 1q in Chinese hamster cells that is coamplified along with the AMPD2 gene. Previous sequence analysis identified both members of the GSTM family and the GNAI3 gene within a cloned 120-kb region surrounding the AMPD2 locus. We show here that the GNAT2 gene, which is inactive in the fibroblastic cells, lies within the 20 kb separating the transcriptionally active GNAI3 and AMPD2 genes. We map most gene ends by sequence comparison with human homologs; one is inferred from the presence of an unmethylated CpG island. This Chinese hamster locus corresponds to a region of conserved linkage between human Chr 1 (locus 1p13) and mouse Chr 3 (position 52.5 cM), where Gnai-3 and Gnat-2 have been mapped. The AMPD2 gene is presently unlocalized in human genome; its proposed position on mouse Chr 3 is at 53.4 cM. Our results, obtained by physical mapping, strongly suggest that the order and possibly the tight linkage of these genes are conserved on all three genomes.

Characterization of the human AMPD3 gene reveals that 5′ exon useage is subject to transcriptional control by three tandem promoters and alternative splicing

Previous work has identified multiple human AMPD3 transcripts proposed to differ by mutually exclusive alternative splicing of three exons located at, or near, the 5′ end of the gene. In this study, we perform a more comprehensive evaluation of human AMPD3 gene expression. Combined Northern blot and RNase protection analyses show that alternative mRNAs are widely expressed in human tissues and cells, but at variable relative abundances. Sequencing of human genomic clones, together with human-mouse somatic cell hybrid analysis, demonstrates that the entire gene is comprised of seventeen exons spanning approx. 60 kilobases on the short arm of chromosome 11 in the region p13-pter. Together, RT-PCR and additional RNase protection analyses establish that exons 1a, 1b, and 1c are 5′ terminal sequences in alternative transcripts. Transient transfection experiments show fusion constructs containing proximal flanking and 5′ untranslated sequence from each of these exons are able to direct expression of a reporter luciferase gene in mammalian cell lines. These combined results reveal that AMPD3 gene expression is subject to transcriptional control by three tandem promoters. Differential regulation of the exon 1b promoter in skeletal myocytes, as compared to retinal pigment epithelial cells, is proposed to be mediated by skeletal muscle-specific basic helix-loop-helix protein/E-box interactions. Finally, an internal splice acceptor site in exon 1c is shown to be used alternatively to retain the 3′ portion of this exon in mature AMPD3 transcripts initiating upstream in exon 1b.

Characterization of human AMP deaminase 2 (AMPD2) gene expression reveals alternative transcripts encoding variable N-terminal extensions of isoform L.

AMP deaminase (AMPD) is a highly regulated enzymic activity and multiple isoforms of this enzyme are coded for by a multigene family in mammalian species, including man. Isoform L (liver) is the main activity present in adult human liver and is the protein product of the AMPD2 gene, which is widely expressed in non-muscle tissues and cells. A previous report described almost the full-length cDNA sequence and part of the human AMPD2 gene and also presented Northern blot evidence for multiple transcripts in brain. This study was performed to further characterize the AMPD2 gene and its expression in human tissues. AMPD2 genomic and human cerebellum cDNA clones were isolated, sequenced and used as probes in RNase protection analyses which together demonstrated the following: (1) an intervening sequence near the 5'-end of the published AMPD2 cDNA, which affects the predicted N-terminal amino acid sequence of isoform L; (2) alternative transcripts resulting from exon shuffling at, or near, the 5'-end of the AMPD2 gene that exhibit tissue-specific patterns of relative abundance; (3) predicted usage of three different initiation codons to confer variable N-terminal extensions on isoform L polypeptides; and (4) an extension of a 3' untranslated sequence in some AMPD2 transcripts. In addition, reverse transcriptase PCR and additional RNase protection analyses were used to map the 5'-ends of two mutually-exclusive exon 1 sequences, both of which contain multiple transcription-initiation sites. These results are discussed in relation to predicted isoform L diversity across human tissues and cells.

Identification of Functional Domains in AMPD1 by Mutational Analysis

AMP deaminase (AMPD) is a complex allosteric enzyme encoded by a multigene family in higher eukaryotes. The amino terminus of each AMPD gene is unique, while the mid and carboxy termini have been highly conserved among all the AMPD genes. Mutational analyses of the AMPD1 gene demonstrate that the catalytic site and a regulatory site, likely an ATP binding site, are located in the highly conserved carboxy terminus. Deletion mutants and a normal splice variant of AMPD1 demonstrate that the amino terminus has a profound influence on catalytic activity of AMPD and by inference from prior studies this region also influences binding of AMPD1 to myosin. Results of these studies suggest a regulatory model in which alternative splicing in the amino terminal region of AMPD1 generates isoforms of AMPD that exhibit differential sensitivity to effector molecules such as ATP.

The highly conserved Chinese hamster GNAI3 gene maps less than 60 kb from the AMPD2 gene and lacks the intronic U6 snRNA present in its human counterpart.

The identity of a gene coamplified with the adenylate deaminase 2 gene (AMPD2) in coformycin-resistant cells was determined by analysis of its genomic sequence. Sequence comparisons reveal a significant homology with the 3' terminal part of the gene encoding the alpha i3 subunit of Gi proteins from several species (GNAI3). Identification of the gene was confirmed by Western blot analysis of its products. A precise sequence comparison was performed with the human genomic sequence. It showed that conservation remains important in noncoding exons as well as in introns. However, sequences corresponding to combined U6 snRNA and E protein pseudogene, previously identified inside intron 7 of the human gene, were not found in the Chinese hamster gene. GNAI3 is mapped to a region of conserved linkage between human chromosome 1 (locus 1p13) and mouse chromosome 3 (at 48.4 cM). The Chinese hamster GNAI3 gene maps to chromosome 1 within a 120-kb fragment that also comprises the AMPD2 and GSTM genes.