Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6457-6461, July 1992 Medical Sciences Molecular basis of AMP deaminase deficiency in skeletal muscle TAKAYUKI MORISAKI*t, MANFRED GROSS*tt, HIROKO MORISAKI*t, DIETER PONGRATZ§, NEPOMUK ZOLLNERt, AND EDWARD W. HOLMES*t¶ *Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, NC 27706; tDepartments of Medicine and Human Genetics, Seymour Gray Molecular Medicine Laboratory, University of Pennsylvania, Philadelphia, PA 19104-4283; tMedizinische Poliklinik der Universitat Munchen, Munich, Federal Republic of Germany; and §Friedrich-Baur-Institut bei der Medizinischen Klinik der Universitat Munchen, Munich, Federal Republic of Germany Communicated by James B. Wyngaarden, April 9, 1992 ABSTRACT AMP deaminase (AMPD; EC 3.5.4.6) is en- inherited defect in the AMPDJ gene since the enzyme defi- coded by a multigene family in mammals. The AMPDI gene is ciency has been reported in several members of the same expressed at high levels in skeletal muscle, where this enzyme family (13, 20). However, acquired deficiency of AMPD has is thought to play an important role in energy metabolism. also been described (14), raising the possibility that the Deficiency of AMPD activity in skeletal muscle is associated absence of AMPD activity may be secondary to other ab- with symptoms of a metabolic myopathy. Eleven unrelated normalities. To determine the molecular basis for this poten- individuals with AMPD deficiency were studied, and each was tially common abnormality we have studied 11 unrelated shown to be homozygous for a mutant allele characterized by individuals with AMPD deficiency. All of these individuals a C -. T transition at nucleotide 34 (codon 12 in exon 2) and are homozygous for the same mutant allele. In randomly at nucleotide 143 (codon 48 in exon 3). The C -* T transition selected Caucasians and African-Americans we have found at codon 12 results in a nonsense mutation predicting a severely this mutant allele in 13% of 144 alleles tested, but we have not truncated AMPD peptide. Consistent with this prediction, no found this mutant allele in 106 DNA samples from Japanese immunoreactive AMPD1 peptide is detectable in skeletal mus- subjects. cle of these patients. This mutant allele is found in 12% of Caucasians and 19% of African-Americans, whereas none of the 106 Japanese subjects surveyed has this mutant allele. We METHODS AND MATERIALS conclude from these studies that this mutant allele is present at Patients. The index case for these studies is an 18-year-old a sufficiently high frequency to account for the 2% reported German female, who first noted calf pain at 4 years of age, incidence of AMPD deficiency in muscle biopsies. The re- usually related to exercise. Persistence of these symptoms stricted distribution and high frequency of this doubly mutated along with weakness of the upper arms eventually led to a allele suggest it arose in a remote ancestor of individuals of muscle biopsy, which exhibited absence of AMPD activity Western European descent. with normal phosphorylase and phosphofructokinase activ- ities. This patient's muscle biopsy, as well as DNA samples AMP deaminase (AMPD; EC 3.5.4.6), an enzyme that cat- from other family members, was studied in detail. Muscle alyzes deamination of AMP to IMP, and the purine nucleo- biopsies or DNA samples from 10 other unrelated individuals tide cycle, of which AMPD is one component, play a central with AMPD deficiency and variable symptoms (Table 1) were role in purine nucleotide interconversion in eukaryotic cells. also analyzed. All patients were identified and referred to us As a consequence, AMPD activity can be a determinant of because muscle biopsies performed for the indicated symp- adenylate energy charge and energy metabolism in the cell (1, toms (Table 1) were found to have reduced levels of AMPD 2). In mammals, AMPD is encoded by a multigene family (3), activity. DNA samples from 59 Caucasians, 13 African- which accounts in part for the tissue-specific and stage- Americans, and 106 Japanese were obtained from normal specific isoforms of AMPD that have been identified (4, 5). volunteers or from investigators who had no knowledge of The activity ofAMPD in skeletal muscle is -100 times higher the donors' medical history. than that of other organs, a consequence of the high level of Protein Analyses. AMPD activity was quantified either by expression of the AMPDJ gene in this tissue (1, 4, 5). a radiochemical assay or by a spectrophotometric assay (5), Since Fishbein et al. (6) first reported 5 patients with and the method employed is noted in the text or figure legend. AMPD deficiency, >100 patients with this enzyme defect Immunoblots were performed with antiserum raised to have been described (7-21). Several centers have reported AMPD purified from rat skeletal muscle (5) using an en- AMPD deficiency in up to 2% of randomly selected muscle hanced chemiluminescence detection system (Amersham). biopsies (6, 7, 9, 12). A review of reported cases of AMPD Nucleic Acid Analyses. Northern hybridization of RNA deficiency noted that 88% of these individuals with AMPD extracted from skeletal muscle was performed as described deficiency describe exercise-related symptoms, including from this laboratory (3). First-strand cDNA (22) was synthe- muscle aches, cramps, and early fatigue (1). Symptoms are sized from patient-derived RNA samples using an oligonu- variable, however, with some reports of asymptomatic indi- cleotide complementary to bases 2239-2258 (5'-TTGGTT- viduals and descriptions ofother patients who exhibit a range TACTTTTTTTTATTC-3') in this 2.3-kilobase (kb) mRNA of neuromuscular disorders. In the few patients studied in (the sequence of human AMPD is reported in ref. 23). detail (6, 17, 18), the deficiency of AMPD activity has been Single-strand cDNA was amplified by the polymerase chain restricted to skeletal muscle, consistent with high-level ex- reaction (PCR) (24, 25) in two separate reactions; oligonu- pression of the AMPD1 gene being restricted to skeletal cleotides corresponding to bases -20 to -1 (5'-AATCAAG- muscle (3, 4). GATCCCAGCAACA-3') and 881-900 (5'-CACCTTCCTG- The molecular basis for AMPD deficiency is not known, CAGTTATAAA-3') were used to synthesize the 5' region; but in some individuals it is presumed to be the result of an Abbreviation: AMPD, AMP deaminase. The publication costs of this article were defrayed in part by page charge ITo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Medicine, University of Pennsylvania, 100 Centrex, 3400 Spruce in accordance with 18 U.S.C. §1734 solely to indicate this fact. Street, Philadelphia, PA 19104-4283. 6457 Downloaded by guest on September 30, 2021 6458 Medical Sciences: Morisaki et al. Proc. Natl. Acad Sci. USA 89 (1992) Table 1. AMPD-deficient patients AMPD activity,* Age at onset of Patient Sex Age, years units/g Clinical presentation symptoms, years lt 9 18 5.4 Calf pain and muscle weakness 4 2 9 16 12.5 Progressive weakness and cramps in legs 11 3 25 18.8 Rhabdomyolysis after viral infection 25 4 d 31 13.5 Pain in both legs after exercise 30 5 d 32 8.8 Easy fatigue and pain in both legs 29 6 45 6.2 Pain in both arms after exercise 41 7 9 45 4.4 Pain in arms and shoulders after exercise 33 8 d 51 3.4 Right shoulder pain after exercise 50 9 d 53 6.6 Weakness and pain in legs during exercise 50 10 d 62 8.3 Muscle pain after exercise 55 11 9 68 0 Pain in arms and legs aggravated by exercise 55 *AMPD activity normal range is 60-300 units/g of noncollagen protein. tIndex case. oligonucleotides corresponding to bases 881-900 (5'- To evaluate the significance of the missense mutation at TTATAACTGCAGGAAGGTG-3') and bases 2239-2258 (5'- position 143, wild-type and mutant cDNAs were ligated into TTGGTTTACTTTTTTTTATTC-3') were used to synthe- a prokaryotic expression vector (pKK233-2) and E. coli size the 3' region. This approach was selected to reduce the lysates were assayed for AMPD activity. The 5' terminal 133 probability of errors induced by PCR oflong DNA sequences nucleotides of the mutant cDNA were replaced with the (26) and to facilitate functional analysis of individual muta- wild-type sequence to remove the nonsense mutation at tions that might be found in the 5' and 3' regions of this position 34, resulting in a cDNA that had the single C -+ T transcript. PCR products were subcloned into pBS (Strata- mutation at position 143. Prokaryotes do not exhibit detect- gene) for sequencing by the dideoxynucleotide chain- able AMPD activity and any activity in the lysate from E. coli termination method (27). Thirty independently isolated pBS transformed with the expression vector is presumably de- subclones were pooled for initial sequencing to obviate rived from the plasmid introduced into E. coli. This was PCR-induced errors. Mutations identified by this screening confirmed by immunoprecipitation of the AMPD activity procedure were subsequently confirmed by sequencing this using an antiserum specific for the AMPDJ gene product (5). region in individual clones. The PCR products were also E. coli transformed with the wild-type cDNA exhibit AMPD subcloned into a prokaryotic vector, pKK233-2 (Pharmacia), activity in the range of 2-8 munits/mg of protein, and the for expression in Escherichia coli. This approach permits a AMPD activity in lysates from E. coli transformed with the rapid functional analysis of mutations since prokaryotes do cDNA mutated only at position 143 is not detectably differ- not exhibit AMPD activity. Genomic DNA, isolated from ent. patients and volunteers by standard methods (22), was am- Although the mutation at position 143 appears to have little plified by PCR with oligonucleotides corresponding to intron effect on catalytic activity of AMPD, the nonsense mutation 1 and intron 2 (5'-GCAATCTACATGTGTCTACC-3') and at position 34 in this patient would be expected to give rise to 5'-ATAGCCATGTTTCTGAATTA-3') for evaluation of a severely truncated peptide, only 11 amino acid residues exon 2; oligonucleotides corresponding to intron 2 and intron compared to 747 residues in the control.