Proc. Natl. Acad. Sci. USA Vol. 92, pp. 10322-10326, October 1995 Medical Sciences

Nonsense mutation in the muscle subunit 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-, 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 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- on 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 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 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 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% 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). PCR was performed in a Four alternatively spliced PFKM transcripts have been previ- ously characterized (21, 22). Subtype B transcript accounts for I practically all mRNA produced in mature skeletal muscle. Its exonic organization comprises 23 exons (2-24) plus the 2nd II 1, ,2 intron that is unspliced (23). The translation start codon [ATG (Met)] is located 8 bases downstream of the 5' end of exon 3, while a stop signal (TAA) occurs at position 143 of exon 24. In our study, RT-PCR with oligonucleotides 03F and 24R (Table 2) flanking the translated region on PFKM transcripts pro- duced the expected -2.25-kb fragments in normal controls, IV while in each of the three patients two different species were observed measuring -2250 and -2500 nt (Fig. 2). Sequence analysis of the transcripts revealed complete conservation of VI * the normal exonic alignment except for an insertion of 252 nt interposed between exons 10 and 11 in the longer transcript. VI ][ Comparison of the insert with the GenBank sequences strongly 0 unaffected female = consanguinity suggested retention of the 10th intron. Since information on * affected female -4 proband intronic sequences was limited to few nucleotides at exon- we determined the full structure of intron 0 0 deceased * not tested I intron boundaries, 10 by sequence analysis of genomic clones in control individ- FIG. 1. Complete pedigree of the Ashkenazi Jewish family with uals. Total homology of the normal 10th intron with the insert Tarui disease. Consanguinity has been found in two consecutive in the patients' transcript sequences allowed identification of generations of this family. the 252-nt insert as the retained intron 10 (Fig. 3). By RT-PCR Downloaded by guest on September 26, 2021 10324 Medical Sciences: Vasconcelos et al. Proc. Natl. Acad. Sci. USA 92 (1995) Table 2. Oligonucleotide primers employed in the present study Primer Sequence Position PFK-05F 5'-TACTGAGAATTCCCrGGGAGAGCGTTTCGATGATGC Exon 5 PFK-05R 5'-TACTGAAAGCTTTTCCTTCCCATGAAACCACAT Intron 5 PFK-06F 5'-GGAGGCACGGTGATTGGAAGTGC Exon 6 PFK-06R 5'-ACICCACTCAGAACGGAAGGTGT Exon 6 PFK-07F 5'-TAAGATCACAGATGAGGAGGCTA Exon 7 PFK-07R 5'-CAGTGGTAGTGATGGCATCTAC Exon 7 PFK-15F 5'-AGTGGTTCGCAGACAGTGGCIGTGA Exon 15 PFK-16R 5'-CCCATGACTGCrCCTAGGTCC Intron 15 PFK-03F 5'-TACTGAGCGGCCGCATGACCCATGAAGAGCACCATGCA Exon 3 PFK-23R 5'-TACIGATCTAGACAAAATCTGTCaGGTCCTTCAGC Exon 23 PFK-24R 5'-TACTGATCTAGAGCATGGTCTGAAGTGTCCAAGTC Exon 24 PFK-10F 5'-TCGTCTCAACATCATCATTGTGGCT Exon 10 PFK-11R 5'-CAGAATTCTGTCAAAGGCTGATG Exon 11 PFK-intF 5'-TATGAATGAAGCCAGAGAGGCCTT Intron 10 PFK-intR 5'-GAGGGTTACGCAGAGTCAAAGGAAG Intron 10 Primers 03F, 05F, 05R, 23R, and 24R have 5' adaptors for cohesive ligation (recognition site for Not I and Xba I); F, forward; R, reverse. amplification with lOF and 11R primers annealing at exons each sequenced clone, strongly suggesting homozygosity for adjacent to intron 10, two different products were obtained in the mutated allele. Use of the aberrant stop signal during each of the patients: a 427-bp fragment retaining intron 10 and cytoplasmic translation is predicted to produce severely trun- a 173-bp fragment without it (Fig. 4). The same procedure cated peptides having only 94 amino acids or 12% of the total failed to amplify the longer of these fragments when per- sequence. formed in normal controls. Additionally, to selectively amplify Since retention of intron 10 accounted for the unique intron 10 from the patients' transcripts, RT-PCR assays were difference between the isolated transcripts, there still was a done with intF and intR primers annealing within intron 10 possibility that these transcripts have originated from dis- (Table 2). An expected fragment averaging 220 bp was suc- tinct alleles. In order to genotype patients and controls for cessfully produced from all affected, but not the control the observed polymorphisms in exons 6 and 7, SSCP and transcripts (Fig. 4). restriction enzyme analysis were performed. For exon 6, the Two single-base transitions (both C -> T) were identified in SSCP-PCR assay demonstrated a distinct profile of single- longer and shorter RT-PCR fragments in all three patients. stranded fragment migration in patients compared to the One was a known silent polymorphism (12, 21) at position 516 controls and indicated homozygosity for the observed mu- in exon 7 [codon 172; ACC (Thr) to ACI (Thr)]. The other tation (Fig. 5a). Since the exon 7 substitution abolishes a Nla occurred at position 282 in exon 6, changing codon 95 from IV recognition site, restriction analysis was performed and CGA (Arg) to TGA (stop) and thereby creating a premature indicated homozygosity for the ACI allele in all three translational termination signal. To confirm the genomic patients, while all control alleles showed the more common nature of this mutation, PCR amplification of exon 6 was ACC sequence (Fig. Sb). The ACT allele frequency in the carried out with resulting fragments cloned into plasmids. Ten Ashkenazi Jewish population has been estimated at 0.092, recombinant clones from each patient were purified and based on the results of testing of 27 control individuals (54 sequenced. The codon 95 point substitution was present in alleles). a

Mw 1 2 3 cr C/

-3Kb _ -2Kb _ -lKb _

b exon 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 cDNA MEN _ PFK-03F PFK-24R _ I- umummnmmnmm - I I ~~~~~~~~~~~~~~I-2.5Kb intron 10 -2.25Kb CtoT CtoT FIG. 2. Results of RT-PCR analysis of PFKM transcripts amplified with 03F and 24R primers in patients (1, 2, and 3) and healthy controls (C', C"). (a) Agarose gel electrophoresis of RT-PCR fragments visualized with ethidium bromide. (b) Sequence analysis of the longer and shorter RT-PCR fragments. Intron 10 retention was observed in the longer fragment; the C -- T point substitutions were detected in both. Downloaded by guest on September 26, 2021 Medical Sciences: Vasconcelos et al. Proc. Natl. Acad. Sci. USA 92 (1995) 10325 5'-CATCAAGAATIGTTCGTATGAATGAAGCCAGAGAGGCCTTAGAATCCATAGCCCATTCCCTC CTGGCTTCTGAGTCTCCTGACATTGCTTCTCCCCTTGGTCCTTCTGCACATCTCTCCCrGGTTCC CTGCCCCTGATTGCCTCCCACAAAGAACCATTACAAGACAAGAGGCTGAGCTGTCCATGGTTT ACCCAAGTCTCTGCTTGTTITCTTCCTITGACTCTGCGTAACCCTCTCTCTGTCCCTCTGTTGG TCCCTICAG CTGG GTTA-3' FIG. 3. Structure and characterization of intron 10 of the PFKM gene. Exon-intron boundaries are marked with vertical bars: 5' donor site, 3' acceptor site, and the branchpoint are underlined; polypyrimidine tract is shown in bold letters. (A complete cDNA sequence with exon 6 mutation and unspliced intron 10 has been deposited in GenBank.) DISCUSSION exon 6 mutation proved to be sensitive and may be satisfactory for standard population screening. However, lack of specificity This study presents a genetic analysis of three patients origi- requires that positive results are confirmed by sequence anal- nating from an Ashkenazi Jewish family that segregates a ysis. congenital myopathy with compensated hemolysis and de- Retention of intron 10 in just one of the transcript species creased PFK activity in muscle tissue. As the mode of inher- did not seem to bear any pathological role, as the aberrant itance of this disease remains unclear, we have carefully translation stop signal was located upstream to the retained examined the data pertaining to the surveyed genealogy. The disease in this pedigree has classical features of autosomal intron, presumably preventing its participation in protein recessive inheritance since the defective gene is located on an coding. Most commonly, mutations causing splicing defects autosomal chromosome, and all patients originated from culminating with complete intron retention occur within highly consanguineous marriages. The high inbreeding rate seen in conserved intronic sequences such as splice junction sites (24, this family (two consecutive consanguineous marriages) con- 25) and less frequently in branch-point sites or in the poly- ferred to the pedigree a false impression of an autosomal pyrimidine tract (26), putatively disrupting important signaling dominant transmission pattern. The affected mother harbored sequences necessary for appropriate nuclear release of mature two copies of the defective allele, greatly concentrating its mRNA molecules. Point mutations and single base insertions frequency in the proband's generation. into nonconserved intronic sequences also promote defective The causal relationship between the characterized exon 6 splicing, probably by altering the intron's secondary structure CGA (Arg) to IGA (stop) mutation and the clinical pheno- (27). In our patients, sequence analysis of the unspliced intron type in our patients is highly probable since (i) this mutation and neighboring exons has failed to uncover any abnormality changes dramatically the predicted structure of the encoded that could disturb the normal splicing mechanism. Alterna- protein, (ii) it was present in all tested transcripts, which is in tively, retention of intron 10 in some of the cytoplasmic mRNA agreement with the autosomal recessive pattern ofinheritance, species of the affected subjects could have resulted from and (iii) this transition was not observed in wild-type alleles. differential pre-mRNA processing. This hypothesis seems This newly characterized defect represents a causative factor of more suitable because (i) retention of intron 10 did not occur Tarui disease. The SSCP-PCR assay employed to detect the in all PFKM subunit transcripts originating from homozygous loci and (ii) complete integrity of all signaling conserved a intronic sequences was demonstrated. Among the mRNA types arising from the PFKM gene, type B, the predominant C' C" form expressed in skeletal muscle cells, has intron 2 retained 1 2 3 4 S 6 7 8 9 10 -- m - MW I II a C

I 2 3 C nd'

369bp _ 246bp _- I23bhp - b patients patients C exon 10 exon II ,' I I4 " A 7 b _ tntronlOintron 10 cDNA - : _oo intF intR_4 _-10F hR_ mm1_ I I 427bp (longu) I 173bp (short)

FIG. 4. RT-PCR analysis of intron 10 amplified with primers 1OF 246 _ - and 11R from the adjacent exons and primers intF and intR from 123 ; inside the intron 10. Three patients (lanes 1-6) and two healthy a unrelated controls (lanes 7-10) were tested. (a) Agarose gel electro- phoresis: a single band, 220 bp, is visible indicating the presence of FIG. 5. SSCP and restriction analysis of genomic sequences of intron 10 amplified with primers intF and intR in the patients (lanes exons 6 and 7 amplified with primer sets 6F and 6R and 7F and 7R, 1, 3, and 5) but not in the controls (lanes 7 and 9). Two bands, 427 bp respectively. (a) Mutation in codon 6 has altered the pattern of and 173 bp, were produced with primers 1OF and 11R in the patients, fragment migration in patients compared to the control (C). nd, confirming the existence of intron 10-plus and intron 10-minus tran- nondenatured. (b) The point substitution in exon 7 abolishes a Nla IV script species (lanes 2, 4, and 6). The control transcripts did not contain site that would normally cut the 154-bp PCR product into 45-bp and intron 10 (lanes 8 and 10). (b) Sequence analysis of the longer and 109-bp fragments. All three patients (lanes 1-3 before the exposure shorter RT-PCR fragments; intron 10 was present in the longer and lanes 5-7 after cutting) have the rare exon 7 variant in both fragment. chromosomes. The control (lanes 4 and 8) was restricted. Downloaded by guest on September 26, 2021 10326 Medical Sciences: Vasconcelos et al. Proc. Natl. Acad. Sci. USA 92 (1995) in its 5' untranslated terminus (23). Although PFKM tran- 1. Bloxham, D. P. & Lardy, H. A. (1973) The (Academic, scripts retaining intron 10 have not yet been isolated from New York), Vol. 8, pp. 239-278. skeletal muscle of healthy individuals, it is possible that such 2. Vora, S., Durham, S., de Martinville, B., George, D. L. & species exist transiently in mature cells or during various stages Francke, U. (1982) Somatic Cell Genet. 8, 95-104. of cell differentiation and could be overexpressed under 3. Hilliker, C. E., Darville, M. I., Aly, M. S., Chikri, M., Szpirer, C., A with a base change Marynen, P., Rousseau, G. G. & Cassiman, J.-J. (1991) Genomics pathological conditions. Swedish patient 10, 867-873. in the last nucleotide of exon 13 and retention of part of intron 4. Van Keuren, M., Drabkin, H., Hart, I., Harker, D., Patterson, D. 13, and retention of intron 10 and part of intron 16 on the other & Vora, S. (1986) Hum. Genet. 74, 34-40. allele was described in a recent review (28). 5. Vora, S., Miranda, A. F., Hernandez, E. & Francke, U. (1983) The lack of PFK activity in individuals with glycogenosis Hum. Genet. 63, 374-379. type VII has been generically correlated with two basic 6. Tsai, M. Y., Gonzales, F. & Kemp, R. G. (1975) in Isozymes, ed. mechanisms: (i) failure of M-subunit synthesis and (ii) synthe- Market, C. L. (Academic, New York), Vol. 2, pp. 818-835. sis of inactive proteins (29). According to the first mechanism, 7. Davidson, M., Collins, M., Byrne, J. & Vora, S. (1983) Biochem. lack of enzyme activity is linked to the failure of PFKM gene J. 214, 703-710. expression in muscle tissue. This situation is not applicable to 8. Vora, S., Seaman, C., Durham, S. & Piomelli, S. (1980) Proc. Natl. our patients, since 33% of residual activity was found and two Acad. Sci. USA 77, 62-66. transcripts were well characterized in their skeletal muscle 9. Tarui, S., Okuno, G., Ikura, Y., Tanaka, T., Suda, M. & Nish- cells. By the second mechanism, defective PFKM-type sub- ikawa, M. (1965) Biochem. Biophys. Res. Commun. 19, 517-523. the tetramers the second 10. Layzer, R. B., Rowland, L. P. & Ranney, H. M. (1967) Arch. units make nonfunctional. Although Neurol. 17, 512-523. mechanism seems to more closely resemble the situation, it 11. Siegel, G., Agranoff, B., Albers, R. W. & Molinoff, P. (1989) probably does not explain the partial enzyme activity existent Basic Neurochemistry: Disease of Carbohydrate, , and in muscle tissue of our patients. In reality, the residual activity Mitochondrial (Raven, New York), 4th Ed. encountered in our study continues to be unexplained since it 12. Nakajima, H., Kono, N., Yamasaki, T., Hotta, K., Kawachi, M., did not seem to result from partial ability of truncated protein. Kuwajima, M., Noguchi, T., Tanaka, T. & Tarui, S. (1990) J. Biol. The termination codon identified in all three homozygous Chem. 265, 9392-9395. patients appeared to be linked to a decreased concentration of 13. Raben, N., Sherman, J., Miller, F., Mena, H. & Plotz, P. (1993) steady-state cytoplasmic mRNA. RT reaction with PFKM J. Biol. Chem. 268, 4963-4967. subunit transcripts in affected individuals required as much as 14. Tsujino, S., Servide, S., Tonin, P., Shanske, S., Azan, G. & five times the amount of total RNA template normally used in DiMauro, S. (1994) Am. J. Hum. Genet. 54, 812-819. healthy controls. A similar phenomenon has been described in 15. Hamaguchi, T., Nakajima, H., Noguchi, T., Ono, A., Kono, N., other human disorders including cystic fibrosis (30). The Tarui, S., Kuwajima, M. & Matsuzawa, Y. (1994) Biochem. reasons nonsense and frame-shift mutations may induce Biophys. Res. Commun. 202, 444-449. why 16. Sherman, J. B., Raben, N., Nicastri, C., Argov, Z., Nakajima, H., intranuclear transcript instability are not clear. Several param- Adams, E. M., Eng, C. M., Cowan, T. M. & Plotz, P. H. (1994) eters such as the transcription rate, the efficiency of pre- Am. J. Hum. Genet. 55, 305-313. mRNA processing and transport into cytoplasm, and the 17. Raben, N., Exelbert, R., Spiegel, R., Sherman, J. B., Nakajima, stability of the produced mature mRNA are thought to be H., Plotz, P. & Heinisch, J. (1995) Am. J. Hum. Genet. 56, individually or simultaneously affected (31). Moreover, if 131-141. ribosomal translation of these defective transcripts occurs in 18. Sivakumar, K, Vasconcelos, O., Goldfarb, L. G. & Dalakas, our patients, it is predicted to produce severely truncated M. C. (1995) Ann. Neurol., in press. PFKM subunits consisting of only "12% of the normal amino 19. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular acid content. Even if random tetramerization of such patho- Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, logically shortened motifs takes place, it is very unlikely that Plainview, NY), 2nd Ed., pp. 7.17-7.22. the assembled protein would carry catalytic properties since 20. Orita, M., Suzuki, Y., Sekiya, T. & Hayashi, K (1989) Genomics the active sites and regulatory centers would be absent. 5, 874-879. Various factors may contribute to the complexity of clinical 21. Nakajima, H., Yamasaki, T., Noguchi, T., Tanaka, T., Kono, N. & Tarui, S. (1990) Biochem. Biophys. Res. Commun. 166, 637- expression: developmental mosaic formation, multiple chro- 641. mosomal loci interaction, environmental influences, and par- 22. Sharma, P. M., Reddy, G. R., Babior, B. M. & McLachlan, A. ticularly tissue-specific . In other human (1990) J. Biol. Chem. 265, 9006-9010. disorders where stop codons were similarly found as the 23. Valdez, B. C., Chen, Z., Sosa, M. G., Younathan, E. S. & Chang, underlying defect (32), some level of "phenotypic rescue" S. H. (1989) Gene 76, 167-169. attributed to increased nuclear release of mature mRNA 24. Kousaku, 0. & Suzuki, K (1988) Biochem. Biophys. Res. Com- species skipping the exon harboring the mutation is believed to mun. 153, 463-469. occur. In our patients, successful removal of the mutated exon 25. Sameshima, Y., Akiyama, T., Mori, N., Mizoguchi, H., Toyo- 6 has not been observed in the isolated transcripts. In fact, even shima, K., Sugimura, T., Terada, M. & Yokota, J. (1990) Bio- if exon 6 had been skipped, no degree of "rescue" would have chem. Biophys. Res. Commun. 173, 697-703. been achieved because the resultant coding frame would have 26. Ohshima, Y. & Gotoh, Y. (1987) J. Mol. Biol. 195, 247-259. shifted, leading to a premature termination of translation 27. Chow, V. T. K, Quek, H. H. & Tock, E. P. C. (1993) CancerLett. within exon 7, thereby preventing a "functional protein" from 73, 141-148. of 28. Raben, N. & Sherman, J. B. (1995) Hum. Mutat. 6, 1-6. being synthesized. To the contrary, complete retention 29. Vora, S: (1983) Isozymes: Curr. Top. Biol. Med. Res. 11, 3-23. intron 10 had happened in one of the identified transcripts. 30. Hamosh, Y., Trapnell, B. C., Zeitlin, P. L., Montrose-Rafizadeh, However, rather than a proper rescue attempt, the retention C., Rosenstein, B. J., Crystal, R. G. & Cutting, G. R. (1991) J. appeared primarily related to an overexpression of an un- Clin. Invest. 88, 1880-1885. known PFKM gene message in skeletal muscle. It is anticipated 31. Cooper, D. N. & Krawczak, M. (1993) Human Gene Mutation that future efforts will elucidate these and other important (BIOS, Oxford). points and improve our understanding of the molecular dy- 32. Morisaki, H., Morisaki, T., Newby, K. L. & Holmes, E. W. (1993) namics of this debilitating disorder. J. Clin. Invest. 91, 2275-2280. Downloaded by guest on September 26, 2021