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Nonsense Mutation in the Phosphofructokinase Muscle Proc. Natl. Acad. Sci. USA Vol. 92, pp. 10322-10326, October 1995 Medical Sciences Nonsense mutation in the phosphofructokinase muscle subunit gene associated with retention of intron 10 in one of the isolated transcripts in Ashkenazi Jewish patients with Tarui disease (glycogenosis type VII/phosphofructokinase deficiency/human PFKM gene) OLAVO VASCONCELOS*, KUMARASWAMY SIVAKUMARt, MARINOS C. DALAKASt, MARTHA QUEZADOt, JAMES NAGLE*, MARTA LEON-MONZONt, MARK DUBNICK*, D. CARLETON GAJDUSEK§, AND LEV G. GOLDFARB* *Clinical Neurogenetics Unit and tNeuromuscular Diseases Section, Medical Neurology Branch, §Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, and tLaboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Contributed by D. Carleton Gajdusek, June 28, 1995 ABSTRACT Mutations in the human phosphofructoki- infirmity requires biochemical assays in skeletal muscle (11). nase muscle subunit gene (PFKAM) are known to cause myop- The condition has been described in a number of Ashkenazi athy classified as glycogenosis type VII (Tarui disease). Pre- Jewish families as well as in non-Ashkenazi pedigrees of viously described molecular defects include base substitutions Japanese, Italian, Swiss, and French Canadian origins (12-17). altering encoded amino acids or resulting in abnormal splic- Recent studies have led to the identification of 11 alleles ing. We report a mutation resulting in phosphofructokinase associated with PFK deficiency (Table 1). deficiency in three patients from an Ashkenazi Jewish family. We studied three patients from an Ashkenazi Jewish family Using a reverse transcription PCR assay, PFKM subunit of Polish origin having an inherited myopathy combined with transcripts differing by length were detected in skeletal mus- recurrent compensated hemolysis. Metabolic block in the cle tissue of all three affected subjects. In the longer tran- glycolytic pathway associated with reduced PFK activity and script, an insertion of 252 nucleotides totally homologous to glycogen accumulation in skeletal muscle were diagnostic of the structure of the 10th intron of the PFKM gene was found Tarui disease. Previously reported mutations in the PFKM separating exon 10 from exon 11. In addition, two single base subunit gene were searched and excluded. A study was then transitions were identified by direct sequencing: [exon 6; undertaken to define the molecular defects in the PFKM codon 95; CGA (Arg) to TGA (stop)] and [exon 7; codon 172; transcripts obtained from the skeletal muscle tissue. A disease- ACC (Thr) to ACT (Thr)] in either transcript. Single- causing nonsense mutation at codon 95, exon 6, was identified, stranded conformational polymorphism and restriction en- all affected individuals testing homozygous for the defect.l zyme analyses confirmed the presence of these point substi- tutions in genomic DNA and strongly suggested homozygosity MATERIALS AND METHODS for the pathogenic allele. The nonsense mutation at codon 95 appeared solely responsible for the phenotype in these pa- Subjects. In an Ashkenazi Jewish family with two consan- tients, further expanding genetic heterogeneity of Tarui dis- guineous marriages (Fig. 1), three patients (79-year-old ease. Transcripts with and without intron 10 arising from mother and her 59- and 51-year-old daughters) presented with identical mutant alleles probably resulted from differential fixed muscle weakness developed by the age of 50. Their past pre-mRNA processing and may represent a novel message medical history revealed exercise intolerance and cramps since from the PFKM gene. childhood. On physical examination, muscle weakness (4+/5 to 4/5 on the Medical Research Council scale) affecting the Phosphofructokinase (PFK; ATP:D-fructose-6-phosphate proximal musculature was found. Scleral icterus was seen in 1-phosphotransferase, EC 2.7.1.11) plays a key regulatory role the younger daughter. Ischemic forearm exercise elicited a flat in one of mammals' most important metabolic routes: the lactate response suggesting a metabolic block in the glycolytic glycolytic pathway (1). The human enzyme is an -320-kDa pathway. A decreased PFK activity (-33% of normal) was protein composed of four subunits randomly associated to demonstrated by enzymatic assay in the extracts of skeletal form functional tetramers. Three types of subunits, muscle- muscle obtained at biopsy (18). Focal, mainly subsarcolemmal, type (M), liver-type (L), and platelet-type (P), are encoded by glycogen deposits with proliferation of mitochondria contain- genes on chromosomes lq, 21q, and 10p, respectively (2-5). ing paracrystalline inclusions were seen on electron micros- Variable expression of each locus in different tissues is known copy (data not shown). After informed consent, blood and to reflect developmental and tissue-specific glycolytic require- skeletal muscle specimens were collected for genetic evalua- ments muscle and liver assemble tion. (6, 7). Skeletal exclusively M4 Reverse Transcription (RT) and Sequencing. Total RNA (muscle) and L4 (liver) homotetramers. Red blood cells and from frozen muscle of patients and normal controls was platelets, conversely, may harbor up to five isozymes by in cesium chloride For 5 combining subunits in hybrid forms (8). As a result, tissues with extracted by centrifugation (19). RT, restricted subunit utilization are vulnerable to ,ug of total RNA from patients and 1 ,tg from controls were particularly denatured at 70°C for 10 min in the presence of oligo(dT)- mutations. primers. Ten millimolar Tris (pH 8.3) RT buffer, 50 mM Muscle PFK deficiency (Tarui disease; glycogenosis type MgCl2, 1 mM (each) dNTP, and 2.5 units of RT polymerase VII) is an inherited disorder characterized by exercise intol- (Life Technologies, Grand Island, NY) were added and incu- erance, cramps, and myoglobinuria with signs of hemolytic bated at and for 60 and 20 anemia and hyperuricemia (9, 10). Partial PFK deficiency is 42°C 70°C min, respectively. routinely found in erythrocytes, but conclusive diagnosis of the Abbreviations: PFK, phosphofructokinase; SSCP, single-stranded conformational polymorphism; RT, reverse transcription; PCR, poly- The publication costs of this article were defrayed in part by page charge merase chain reaction. payment. This article must therefore be hereby marked "advertisement" in IThe sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U24183). 10322 Downloaded by guest on September 26, 2021 Medical Sciences: Vasconcelos et al. Proc. Natl. Acad. Sci. USA 92 (1995) 10323 Table 1. Pathogenic molecular defects in the PFKM subunit gene on chromosome lq PFK No. Gene defect Transcript defect activity,* % Ref. or source 1 G -> T substitution at the 5' 75-bp in-frame deletion in 1-3 12 donor site of intron 15 exon 15 2 G -> A substitution at the 5' In-frame deletion of exon 5 None 13 donor site of intron 5 3 Base deletion in exon 22 Frame-shift mutation None 13 4 A -* C substitution at the 3' 5-bp or 12-bp deletions in exon -1.8 14 acceptor site of intron 6 7 5 G -> C substitution at Codon 39 alteration from CGA -3.3 14 position 116 in exon 4 (Arg) to CCA (Pro) 6 A -> C substitution at Codon 543 alteration from 6 14 position 1623 in exon 18 GAC (Asp) to GCC (Ala) 7 Promoter region intact Lack of gene expression 6 14 8 G -> A substitution at the 5' Deletion of exon 19 None 15 donor site of intron 19 9 G - T substitution at Codon 39 alteration from None 16 position 116 in exon 4 CGA (Arg) to CTA (Leu) 10 G -- A substitution at Codon 209 alteration from None 17 position 636 in exon 8 GGC (Gly) to GAC (Asp) 11 G -* A substitution at Codon 696 alteration from -~8 17 position 2087 in exon 22 CGU (Arg) to CAU (His) 12 G -* A substitution at Codon 100 alteration from -8 17 position 299 in exon 6 CGA (Arg) to CAA (Gln) 13 C -* T substitution at Codon 95 alteration from -33 This study position 282 in exon 6 CGA (Arg) to UGA (stop) *PFK activity estimated by the chromatography assay. Conversion to double-stranded cDNA was achieved by PCR 10-,tl volume, with 50 ng of genomic template, 1 ,tCi of with 5' adaptor primers (Table 2) using 2 ,ul of the RT reaction [a-32P]dCTP (1 Ci = 37 GBq), and oligomers 6F and 6R as the starting template. Generated fragments encompassing (Table 2). Reaction conditions were 30 cycles of 1 min at -2.25 kb of the PFKM coding region were made cohesive- 94°C, 1 min at 55°C, and 1 min at 72°C. The amplification ended by digestion with Not I and Xba I endonucleases and product was diluted 1:25 with 0.1% SDS/10 mM EDTA, and cloned into the Not I/Xba I site of pBluescript II Sk + /- an aliquot mixed 1:1 with loading buffer, denatured at 95°C phagemid (Stratagene). For sequencing, sequential Exo III for 3 min, and chilled on ice before being loaded onto a 6% deletions were performed, yielding partially overlapping sub- acrylamide/10% glycerol gel. Electrophoresis at 60 W for 5 clones covering the transcripts. Both strands were sequenced h was followed by autoradiography (DuPont) at -70°C for 3 using Prism cycle-sequencing protocol (Applied Biosyste,ms- h. Testing for the transition in exon 7 was performed by Perkin-Elmer) and contigs assembled. The resulting cDNA restriction enzyme analysis. PCR-amplified exon 7 fragments sequences were aligned for comparative study with the human were incubated with Nla IV (New England Biolabs) for 3 h PFKM cDNA sequences retrieved from GenBank. at 37°C and resolved in a 3% agarose gel stained with Genotyping. Patients and controls were screened for the ethidium bromide. point mutation in exon 6 using a modified version of a previously reported single-stranded confirmational polymor- RESULTS phism (SSCP)-PCR protocol (20).
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