Altered allosteric regulation of muscle 6-phosphofructokinase causes Tarui disease.

Tarui disease is a glycogen storage disease (GSD VII) and characterized by exercise intolerance with muscle weakness and cramping, mild myopathy, myoglobinuria and compensated hemolysis. It is caused by mutations in the muscle 6-phosphofructokinase (Pfk). Pfk is an oligomeric, allosteric enzyme which catalyzes one of the rate-limiting steps of the glycolysis: the phosphorylation of fructose 6-phosphate at position 1. Pfk activity is modulated by a number of regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk displayed several allosteric adenine nucleotide binding sites. Functional studies revealed a reciprocal linkage between the activating and inhibitory allosteric binding sites. Herein, we showed that Asp(543)Ala, a naturally occurring disease-causing mutation in the activating binding site, causes an increased efficacy of ATP at the inhibitory allosteric binding site. The reciprocal linkage between the activating and inhibitory binding sites leads to reduced enzyme activity and therefore to the clinical phenotype. Pharmacological blockage of the inhibitory allosteric binding site or highly efficient ligands for the activating allosteric binding site may be of therapeutic relevance for patients with Tarui disease.

Phosphofructo-1-kinase deficiency leads to a severe cardiac and hematological disorder in addition to skeletal muscle glycogenosis.

Mutations in the gene for muscle phosphofructo-1-kinase (PFKM), a key regulatory enzyme of glycolysis, cause Type VII glycogen storage disease (GSDVII). Clinical manifestations of the disease span from the severe infantile form, leading to death during childhood, to the classical form, which presents mainly with exercise intolerance. PFKM deficiency is considered as a skeletal muscle glycogenosis, but the relative contribution of altered glucose metabolism in other tissues to the pathogenesis of the disease is not fully understood. To elucidate this issue, we have generated mice deficient for PFKM (Pfkm(-/-)). Here, we show that Pfkm(-/-) mice had high lethality around weaning and reduced lifespan, because of the metabolic alterations. In skeletal muscle, including respiratory muscles, the lack of PFK activity blocked glycolysis and resulted in considerable glycogen storage and low ATP content. Although erythrocytes of Pfkm(-/-) mice preserved 50% of PFK activity, they showed strong reduction of 2,3-biphosphoglycerate concentrations and hemolysis, which was associated with compensatory reticulocytosis and splenomegaly. As a consequence of these haematological alterations, and of reduced PFK activity in the heart, Pfkm(-/-) mice developed cardiac hypertrophy with age. Taken together, these alterations resulted in muscle hypoxia and hypervascularization, impaired oxidative metabolism, fiber necrosis, and exercise intolerance. These results indicate that, in GSDVII, marked alterations in muscle bioenergetics and erythrocyte metabolism interact to produce a complex systemic disorder. Therefore, GSDVII is not simply a muscle glycogenosis, and Pfkm(-/-) mice constitute a unique model of GSDVII which may be useful for the design and assessment of new therapies.

Characterization of the enzymatic defect in late-onset muscle phosphofructokinase deficiency. New subtype of glycogen storage disease type VII.

Human phosphofructokinase (PFK) exists in tetrameric isozymic forms, at least in vitro. Muscle and liver contain homotetramers M4 and L4, respectively, whereas red cells contain five isozymes composed of M (muscle) and L (liver) type subunits, i.e., M4, M3L, M2L2, and ML3, and L4. Homozygous deficiency of muscle PFK results in the classic glycogen storage disease type VII characterized by exertional myopathy and hemolytic syndrome beginning in early childhood. The genetic lesion results in a total and partial loss of muscle and red cell PFK, respectively. Characteristically, the residual red cell PFK from the patients consists of isolated L4 isozyme; the M-containing hybrid isozymes are completely absent. In this study, we investigated an 80-yr-old man who presented with a 10-yr history of progressive weakness of the lower limbs as the only symptom. The residual red cell PFK showed the presence of a few M-containing isozymes in addition to the predominant L4 species, indicating that the genetic lesion is a "leaky" mutation of the gene coding for the M subunit. The presence of a small amount of enzyme activity in the muscle may account for the atypical myopathy in this patient.

Heterogeneity of the molecular lesions in inherited phosphofructokinase deficiency.

Human phosphofructokinase (PFK; EC 2.7.1.11) exists in tetrameric isozymic forms. Muscle and liver contain the homotetramers M4 and L4, whereas erythrocytes contain five isozymes composed of M (muscle) and L (liver) subunits, i.e., M4, M3L, M2L2, ML3, and L4. Inherited defects of erythrocyte PFK are usually partial and are described in association with heterogeneous clinical syndromes. To define the molecular basis and pathogenesis of this enzymopathy, we investigated four unrelated individuals manifesting myopathy and hemolysis (glycogenosis type VII), isolated hemolysis, or no symptoms at all. The three symptomatic patients showed high-normal hemoglobin levels, despite hemolysis and early-onset hyperuricemia. They showed total lack of muscle-type PFK and suffered from exertional myopathy of varying severity. In the erythrocytes, a metabolic crossover was evident at the PFK step: the levels of hexose monophosphates were elevated and those of 2,3-diphosphoglycerate (2,3-DPG) were depressed, causing strikingly increased hemoglobin-oxygen affinity. In all cases, the residual erythrocyte PFK consisted exclusively of L4 isozyme, indicating homozygosity for the deficiency of the catalytically active M subunit. However, presence of immunoreactive M subunit was shown in cultured fibroblasts by indirect immunofluorescence with monoclonal anti-M antibody. The fourth individual was completely asymptomatic, had normal erythrocyte metabolism, and had no evidence of hemolysis. His residual erythrocyte PFK showed a striking decrease of the L4, ML3, and M2L2 isozymes, secondary to a mutant unstable L subunit. Identical alterations of erythrocyte PFK were found in his asymptomatic son, indicating heterozygosity for the mutant unstable L subunit in this kindred. These studies show that, except for the varying severity of the myopathic symptoms, glycogenosis type VII has highly uniform clinical and biochemical features and results from homozygosity for mutant inactive M subunit(s). The absence of anemia despite hemolysis may be explained by the low 2,3-DPG levels. The hyperuricemia may result from hyperactivity of the hexose monophosphate shunt. In contrast, the clinically silent carrier state results from heterozygosity for mutant M or L subunit. Of the two, the M subunit appears to be more critical for adequate glycolytic flux in the erythrocyte, since its absence is correlated with hemolysis.

Tarui disease and distal glycogenoses: clinical and genetic update.

Phosphofructokinase deficiency (Tarui disease) was the first disorder recognized to directly affect glycolysis. Since the discovery of the disease, in 1965, a wide range of biochemical, physiological and molecular studies have greatly contributed to our knowledge concerning not only phosphofructokinase function in normal muscle but also on the general control of glycolysis and glycogen metabolism. Studies on phosphofructokinase deficiency vastly enriched the field of glycogen storage diseases, making a relevant improvement also in the molecular genetic area. So far, more than one hundred patients have been described with prominent clinical symptoms characterized by muscle cramps, exercise intolerance, rhabdomyolysis and myoglobinuria, often associated with haemolytic anaemia and hyperuricaemia. The muscle phosphofructokinase gene is located on chromosome 12 and about 20 mutations have been described. Other glycogenoses have been recognised in the distal part of the glycolytic pathway: these are infrequent but some may induce muscle cramps, exercise intolerance and rhabdomyolysis. Phosphoglycerate Kinase, Phosphoglycerate Mutase, Lactate Dehydrogenase, beta-Enolase and Aldolase A deficiencies have been described as distal glycogenoses. From the molecular point of view, the majority of these enzyme deficiencies are sustained by "private" mutations.

Late-onset muscle phosphofructokinase deficiency.

A 75-year-old man had a 10-year history of slowly progressive limb weakness without cramps or myoglobinuria. Clinical, morphologic, and biochemical studies showed muscle phosphofructokinase (PFK) deficiency. Erythrocyte PFK activity in his asymptomatic daughter was 63% of normal, compatible with a carrier state. The chronic myopathic variant of muscle PFK deficiency appears to be transmitted as an autosomal recessive trait and may be due to a distinct genetic defect.

Muscle phosphofructokinase deficiency in two generations.

Phosphofructokinase (PFK) is the key regulatory enzyme of glycolysis. Patients lacking the muscular isoform of PFK typically present with myopathy and compensated hemolysis (glycogenosis type VII or Tarui's disease). Since 1965 about 30 cases of muscular PFK deficiency have been reported. In most cases family history suggests a recessive inherited trait. We describe a family of Ashkenazi Jewish origin with two members in subsequent generations suffering from muscular PFK deficiency. The propositus, a 19-year-old male patient presented with weakness, myalgias and exercise intolerance since early infancy. His father also had early fatigue on exercise with myalgias; the mother and a 12-year-old brother were asymptomatic. Muscle biopsy of both the propositus and his father showed increased glycogen storage and absent histochemical stain for PFK. Biochemical studies of muscle revealed a markedly decreased PFK activity and DNA analysis of the muscle PFK gene revealed compound heterozygosity in both cases. This is the first description of proven muscle PFK deficiency (glycogenosis type VII) in two subsequent generations.

[Metabolic myopathies]. / Miopatías metabólicas.

AIM: To review the metabolic myopathies manifested only by crisis of myalgias, cramps and rigidity of the muscles with decreased voluntary contractions and normal inter crisis neurologic examination in children and adolescents. DEVELOPMENT: These metabolic myopathies are autosomic recessive inherited enzymatic deficiencies of the carbohydrates and lipids metabolisms. The end result is a reduction of intra muscle adenosine triphosphate, mainly through mitochondrial oxidative phosphorylation, with decrease of available energy for muscle contraction. The one secondary to carbohydrates intra muscle metabolism disorders are triggered by high intensity brief (< 10 min) exercises. Those secondary to fatty acids metabolism disorders are triggered by low intensity prolonged (> 10 min) exercises. The conditions in the first group in order of decreasing frequency are the deficiencies of myophosforilase (GSD V), muscle phosphofructokinase (GSD VII), phosphoglycerate mutase 1 (GSD X) and beta enolase (GSD XIII). The conditions in the second group in order of decreasing frequency are the deficiencies of carnitine palmitoyl transferase II and very long chain acyl CoA dehydrogenase. CONCLUSIONS: The differential characteristics of patients in each group and within each group will allow to make the initial presumptive clinical diagnosis in the majority and then to order only the necessary tests to achieve the final diagnosis. Treatment during the crisis includes hydration, glucose and alkalinization of urine if myoglobin in blood and urine are elevated. Prevention includes avoiding exercise which may induce the crisis and fasting. The prognosis is good with the exception of rare cases of acute renal failure due to hipermyoglobinemia because of severe rabdomyolisis.

TITLE: Miopatias metabolicas.Objetivo. Revisar las miopatias metabolicas manifestadas solamente por crisis de mialgias, calambres y rigidez musculares con dificultad para contraer los musculos afectados y el examen neurologico normal entre las crisis en niños y adolescentes. Desarrollo. Estas miopatias metabolicas se deben a deficits enzimaticos heredados en forma autosomica recesiva del metabolismo de los carbohidratos y lipidos. El resultado final es una reduccion del trifosfato de adenosina principalmente a traves de la fosforilacion oxidativa mitocondrial con disminucion de la energia disponible para la contraccion muscular. Las secundarias a trastornos del metabolismo de los carbohidratos se producen por ejercicios de alta intensidad y breves (< 10 min) y las secundarias a trastornos de los lipidos, por ejercicios de baja intensidad y prolongados (> 10 min). Los deficits enzimaticos en el primer grupo son de miofosforilasa (glucogenosis V), fosfofructocinasa muscular (glucogenosis VII), fosfoglicerato mutasa 1 (glucogenosis X) y beta enolasa (glucogenosis XIII), y en el segundo, de carnitina palmitol transferasa tipo II y de acil-CoA deshidrogenasa de cadena muy larga. Conclusiones. Las caracteristicas diferenciales de los pacientes en cada grupo y dentro de cada grupo permitiran el diagnostico clinico presuntivo inicial en la mayoria y solicitar solamente los examenes necesarios para corroborar el diagnostico. El tratamiento de las crisis consiste en hidratacion, glucosa y alcalinizacion de la orina. Las medidas preventivas son evitar el tipo de ejercicio que induce las crisis y el ayuno. No existe cura o tratamiento especifico. El pronostico es bueno con la excepcion de casos raros de insuficiencia renal aguda debido a la elevacion sanguinea de la mioglobina producto de una rabdomiolisis grave.

Developmental changes of 6-phosphofructo-1-kinase subunit levels in erythrocytes from normal dogs and dogs affected by glycogen storage disease type VII.

1. The subunit proportions (L:M:C) of the PFK isozymes from normal adult erythrocytes were 2:86:12. Affected adult erythrocyte 6-phosphofructo-1-kinase (PFK) isozymes contained normal L-type (31%) and C-type (61%) subunits as well as a small amount (8%) of truncated M-type subunit. 2. When measured within 24 hr of birth, both normal and affected dog erythrocytes contained high PFK activities due to elevated levels of the L-type subunit. As the dogs matured, PFK activity decreased due to a greater than 99% loss of the L-type subunit. 3. By 2 weeks of age, the M-type and C-type subunits in normal dog PFK isozymes increased several-fold and attained near adult levels. 4. During post-natal development, the L-type subunit from affected dog erythrocytes decreased more rapidly than from normal dog erythrocytes; but it was maintained at a higher level in the affected adult erythrocytes. Also, in the affected dog erythrocytes, truncated M-type subunits were detected; and the initially high levels of the C-type subunit decreased approximately 50% after 4 weeks.

Presence of a truncated M-type subunit and altered kinetic properties of 6-phosphofructo-1-kinase isozymes in the brain of a dog affected by glycogen storage disease type VII.

6-Phosphofructo-1-kinase (PFK) activity in the brain of a dog affected by glycogen storage disease type VII was only 31% of the PFK activity in the normal dog brain. PFK in the normal dog brain was composed of L-type, M-type and C-type subunits with apparent molecular weights of 78,000, 86,000, and 88,000, respectively, and subunit proportions (L:M:C) of 27:49:24. PFK in the affected dog brain was composed of nearly equal levels of the normal L-type and C-type subunits, but a normal M-type subunit was not detected. Using antidog muscle PFK IgG, immunoblots of gels containing partially purified PFK from the affected dog brain revealed a small amount of immunoreactive protein with an apparent molecular weight of 84,000, suggesting the presence of a truncated M-type subunit. Kinetic studies indicated that the PFK isozymes in the affected dog brain exhibited significantly different kinetic regulatory properties when compared to the PFK isozyme pool in the normal dog brain.

Partial block of glycolysis in late-onset phosphofructokinase deficiency myopathy.

A late-onset, myopathic variant of phosphofructokinase (PFK) deficiency has been previously described in two patients of Ashkenazic descent. We report here on a non-Ashkenazic woman with the onset, at the age of 48 years, of a progressive limb girdle myopathy that was not preceded by a history of exercise intolerance. Muscle biopsy findings at the age of 58 years showed deposition of amylopectin-like material in muscle fibers and the absence of histochemical PFK activity. Enzymatic PFK activity in vitro was only 4% of normal. Since the forearm ischemic exercise test induced a sub-normal production of serum lactate, the patient underwent phosphorus magnetic resonance spectroscopy (31P-MRS), a non-invasive method that allows in vivo assessment of the functional status of the glycolytic pathway and mitochondrial oxidative metabolism by measuring the high-energy phosphates and cytosolic pH. In vivo, 31P-MRS disclosed a residual glycolytic flux and a normal rate of ATP production both at rest and during exercise. These results suggest that, in some patients, muscle PFK deficiency may be partial in vivo, and more severe in vitro, possibly due to protein or mRNA instability rather than absence. The presence of these findings in a patient with the late-onset myopathic form is compatible with a distinct pathogenetic mechanism, relying on progressive polysaccharide accumulation, rather than on acute energetic shortage in muscle fibers.

Isozymes of human phosphofructokinase: biochemical and genetic aspects.

Human PFK is under the control of three structural loci that encode muscle-type (M), liver-type (L), and platelet-type (P) subunits. These loci are differentially expressed in various human tissues, resulting in a tissue-specific isozyme distribution patterns. Random tetramerization of these subunits produces various homotetrameric and heterotetrameric isozymes distinguishable by ion-exchange chromatography and subunit-specific mouse monoclonal antibodies. Inherited PFK deficiency is associated with five clinically and biochemically identifiable groups. The largest and best defined of these consists of the patients with glycogenosis type VII (group I). This syndrome results from a total deficiency of the catalytically active M subunit; the molecular pathology of the other four syndromes remains to be elucidated. Subunit- and species-specific hybridoma antibodies to the PFK subunits have permitted not only precise immunochemical analysis of this complex isozyme system, but also chromosomal localization of the PFK loci. In addition, immunochemical homologies among vertebrate PFKs determined using monoclonal antibodies suggest both an ancient duplication of the ancestral PFK gene and the structural conservatism of vertebrate PFK subunits despite this early divergence. Using somatic cell hybrids and subunit-specific antibodies, the PFKM, PFKP, and PFKL loci have been assigned to chromosomes I (region cen leads to q32), 10p and 21, respectively. The localization of PFKL to chromosome 21 and the chromatographic demonstration of a specific increase in the L subunit in red cells from trisomy 21 individuals has thus resolved the controversy about whether the previously observed elevation in PFK activity in Down syndrome represented a gene dosage effect. PFK exhibits both quantitative increases and isozymic shifts secondary to the altered gene expression in neoplasia. Since these alterations are correlated with the rate of growth and not the cell type of origin, PFK appears to be not only a transformation-linked but also a progression-linked discriminant of malignancy.

Identification of three novel mutations in non-Ashkenazi Italian patients with muscle phosphofructokinase deficiency.

We have identified three novel mutations in four non-Ashkenazi Italian patients with muscle phosphofructokinase (PFK-M) deficiency (Tarui disease). Patient 1 was homozygous for an A-to-C substitution at the 3' end of intron 6 of the PFK-M gene, changing the consensus splice-junction sequence AG to CG. The mutation leads to activation of two cryptic splice sites in exon 7, resulting in one 5 bp- and one 12 bp-deleted transcript. An affected brother was also homozygous, and both parents were heterozygous, for the splice-junction mutation. Patient 2 was homozygous for a G-to-C substitution at codon 39, changing an encoded arginine (CGA) to proline (CCA). Patient 3 was heterozygous for an A-to-C substitution at codon 543, changing an encoded aspartate (GAC) to alanine (GCC); the PFK-M gene on the other allele was not expressed, but sequencing of the reported regulatory region of the gene did not reveal any mutation.

Kinetic properties of erythrocyte phosphofructokinase in patients with type VII glycogenosis from two families--close similarity to liver type phosphofructokinase.

The kinetic properties of phosphofructokinases (PFKs) from normal human liver, muscle and erythrocytes, and from erythrocytes of two unrelated patients with type VII glycogenosis (muscle PFK deficiency, McKusick 23280) were analysed in this study. Sensitivity to inhibition by ATP and to inhibition by 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate and citrate were quite different for muscle and liver PFKs. The kinetic characteristics of normal erythrocyte PFK were intermediate between those of muscle and liver PFKs. The kinetic constants of erythrocyte PFK of a patient in one family were indistinguishable from those in the other family. In addition, kinetic behaviour of residual PFK activity in erythrocytes from patients in the two families were quite similar to those of normal liver PFK. These results of kinetic analyses provide convincing evidence for the concept that normal erythrocyte PFK consists of muscle and liver type subunits. Residual erythrocyte PFK activity in type VII glycogenosis is thus concluded to reflect the activity of liver type PFK existing in patient's erythrocytes.

Genetic defect in muscle phosphofructokinase deficiency. Abnormal splicing of the muscle phosphofructokinase gene due to a point mutation at the 5'-splice site.

The genetic defect in muscle phosphofructokinase deficiency (type VII glycogenosis, Tarui disease) was investigated. Six cDNAs for muscle phosphofructokinase, including a full-length clone, were isolated from a non-amplified library of muscle from a patient. By sequence analysis of these clones, a 75-base in-frame deletion was identified. The rest of the sequence was identical to that of the normal cDNA, except for a silent base transition at position 516 (ACT (Thr) to ACC (Thr]. The deletion was located in the 3'-terminal region of exon 13 (numbered with reference to the rabbit muscle phosphofructokinase gene (Lee, C.-P., Kao, M.-C., French, B.A., Putney, S.D., and Chang, S.H. (1987) J. Biol. Chem. 262, 4195-4199]. Genomic DNA of the patient was amplified by polymerase chain reaction. Sequence analysis of the amplified DNA revealed a point mutation from G to T at the 5'-end of intron 13. This mutation changed the normal 5'-splice site of CAG:GTATGG to CAG:TTATGG. A cryptic splice site of ACT:GTGAGG located 75 bases upstream from the normal splice site was recognized and spliced in the patient.

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.

Mutations in the human phosphofructokinase muscle subunit gene (PFKM) are known to cause myopathy classified as glycogenosis type VII (Tarui disease). Previously described molecular defects include base substitutions altering encoded amino acids or resulting in abnormal splicing. We report a mutation resulting in phosphofructokinase deficiency in three patients from an Ashkenazi Jewish family. Using a reverse transcription PCR assay, PFKM subunit transcripts differing by length were detected in skeletal muscle tissue of all three affected subjects. In the longer transcript, an insertion of 252 nucleotides totally homologous to the structure of the 10th intron of the PFKM gene was found separating exon 10 from exon 11. In addition, two single base transitions were identified by direct sequencing: [exon 6; codon 95; CGA (Arg) to TGA (stop)] and [exon 7; codon 172; ACC (Thr) to ACT (Thr)] in either transcript. Single-stranded conformational polymorphism and restriction enzyme analyses confirmed the presence of these point substitutions in genomic DNA and strongly suggested homozygosity for the pathogenic allele. The nonsense mutation at codon 95 appeared solely responsible for the phenotype in these patients, further expanding genetic heterogeneity of Tarui disease. Transcripts with and without intron 10 arising from identical mutant alleles probably resulted from differential pre-mRNA processing and may represent a novel message from the PFKM gene.

Acute renal failure in a patient with phosphofructokinase deficiency.

A 16-year-old Caucasian girl was admitted to hospital with acute renal failure and hemolytic anemia due to rhabdomyolysis following a 3-km walk. (31)P-magnetic resonance spectroscopy provided characteristic spectra of type VII glycogen storage disease (phosphofructokinase deficiency).

A new variant case of muscle phosphofructokinase deficiency, coexisting with gastric ulcer, gouty arthritis, and increased hemolysis.

Muscle phosphofructokinase (PFK) deficiency includes both clinically and genetically heterogeneous conditions. A 22-year-old man with muscle PFK deficiency due to previously unrecognized mutation was admitted because of gastric ulcer. He had noticed mild fatigability on vigorous exercise, but had never experienced painful cramps and myoglobinuria. His history included five time relapses of gastric ulcer and gouty arthritis at ages 19 and 21 years. His laboratory data showing impaired muscle glycolysis, increased hemolysis, and myogenic hyperuricemia had aspects in common with those reported for the classic form of this disease, except that lactate concentrations in his blood increased considerably after exercise. The mutant PFK enzyme of this patient, who was demonstrated to have a missense mutation, could exert some catalytic activity that permitted glycolytic flux in vivo, thus leading to the absence of typical myopathic symptoms. The association of relapsing gastric ulcer with muscle PFK deficiency was detected for the first time. There is a possibility that oxygen radical-induced tissue damage resulting from increased hypoxanthine on exertion plays a role in the pathogenesis of ulceration, since the patient is more tolerant to exercise than reported cases with the classic form of muscle PFK deficiency.

Glycogenosis type VII (Tarui disease) in a Swedish family: two novel mutations in muscle phosphofructokinase gene (PFK-M) resulting in intron retentions.

Phosphofructokinase (PFK) plays a major role in glycolysis. Human PFK is composed of three isoenzyme subunits (muscle [Ml, liver [L], and platelet [P]), which are encoded by different genes. Deficiency of muscle isoenzyme (PFK-M), glycogenosis type VII (Tarui disease), is an autosomal recessive disorder characterized by an exertional myopathy and hemolytic syndrome. Several disease-causing mutations have been identified in the PFK-M gene in Japanese, Ashkenazi Jewish, Italian, French Canadian, and Swiss patients. We describe the genetic defect in a Swedish family with affected individuals in two generations. The patients are compound heterozygotes: two different mutations result in retention of intron 13 or intron 16 sequences into mRNA. A G1127A transition destroys the 5' donor site of intron 13, resulting in a 155-nt retention of the intronic sequence. An a-to-g base change in intron 16 creates a new acceptor splice site, resulting in a 63-nt retention of intronic sequence. Both mutations are predicted to result in premature termination of translation. Some of the transcripts generated from the intron 16 mutated allele also contain intron 10 sequence unspliced.