Dysferlin mutations in Japanese Miyoshi myopathy: relationship to phenotype.

OBJECTIVE: To study dysferlin gene mutations and genotype-phenotype correlations in Japanese patients with Miyoshi myopathy (MM). BACKGROUND: MM is an autosomal recessive distal muscular dystrophy that arises from mutations in the dysferlin gene. This gene is also mutated in families with limb girdle muscular dystrophy 2B. METHODS: The authors examined 25 Japanese patients with MM. Genomic DNA was extracted from the peripheral lymphocytes of the patients. The PCR products of each of 55 exons were screened by single strand conformation polymorphism or direct sequencing from the PCR fragments. RESULTS: The authors identified 16 different mutations in 20 patients with MM; 10 were novel. Mutations in Japanese patients are distributed along the entire length of the gene. CONCLUSIONS: Four mutations (C1939G, G3370T, 3746delG, and 4870delT) are relatively more prevalent in this population, accounting for 60% of the mutations in this study. This study revealed that the G3370T mutation was associated with milder forms of MM and the G3510A mutation was associated with a more severe form.

Slowly progressive dystonia following central pontine and extrapontine myelinolysis.

A 28-year-old woman was hospitalized with dysarthria and oro-mandibular and upper limb dystonia. Approximately 8 years prior to the current admission, the woman became severely hyponatremic due to traumatic subarachnoid hemorrhage-related SIADH. Brain MRIs showed a signal increase in the central pons, thalamus and striatum on T2 weighted images compatible with central pontine and extrapontine myelinolysis. From a few months after that event, dystonia progressed slowly over the subsequent 8 years. We speculate that the particular damage chiefly to the myelin structures by myelinolytic process may have caused an extremely slow plastic reorganization of the neural structures, giving rise to progressive dystonia.

Heterogeneous mutations in the glucose-6-phosphatase gene in Japanese patients with glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive disorder of glycogen metabolism caused by glucose-6-phosphatase (G6Pase) deficiency. It is characterized by short stature, hepatomegaly, hypoglycemia, hyperuricemia, and lactic acidemia. Various mutations have been reported in the G6Pase gene (G6PC). However, in Japanese patients, a g727t substitution was found to be the major cause of GSD-Ia, accounting for 20 of 22 mutant alleles [Kajihara et al., 1995], and no other mutations have been found in this population. We analyzed four Japanese GSD-Ia patients and identified three other mutations in addition to the g727t. They included two missense mutations (R83H and P257L) and one nonsense mutation (R170X). Each of the three mutations exhibited markedly decreased G6Pase activity when expressed in COS7 cells. A patient homozygous for R170X showed multiple episodes of profound hypoglycemia associated with convulsions, while P257L was associated with a mild clinical phenotype. The presence of R170X in three unrelated families may implicate that it is another important mutation in the etiology of GSD-Ia in Japanese patients. Thus, the detection of non-g727t mutations is also important in establishing the DNA-based diagnosis of GSD-Ia in this population.

Glycogen storage disease type Ia: molecular diagnosis of 51 Japanese patients and characterization of splicing mutations by analysis of ectopically transcribed mRNA from lymphoblastoid cells.

Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive disorder of glycogen metabolism caused by a deficiency of glucose-6-phosphatase (G6Pase) that is expressed in the liver, kidney, and intestinal mucosa. Clinical manifestations include short stature, hepatomegaly, hypoglycemia, hyperuricemia, and lactic acidemia. To elucidate a spectrum of the G6Pase gene mutations and their frequencies, we analyzed mutations in 51 unrelated Japanese patients with GSD-Ia. The most prevalent mutation was g727t, accounting for 88 of 102 mutant alleles examined, followed by R170X mutation, which accounted for 6 mutant alleles, and R83H mutation which was observed in 3 mutant alleles. In addition, 3 different, novel mutations, IVS1-1g

Mutation detection by TaqMan-allele specific amplification: application to molecular diagnosis of glycogen storage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency.

We have devised an allele-specific amplification method with a TaqMan fluorogenic probe (TaqMan-ASA) for the detection of point mutations. Pairwise PCR amplification using two sets of allele-specific primers in the presence of a TaqMan probe was monitored in real time with a fluorescence detector. Difference in amplification efficiency between the two PCR reactions was determined by "threshold" cycles to differentiate mutant and normal alleles without post-PCR processing. The method measured the efficiency of amplification rather than the presence or absence of end-point PCR products, therefore allowing greater flexibility in designing allele-specific primers and an ample technical margin for allelic discrimination. We applied the TaqMan-ASA method to detect a prevalent 727G>T mutation in Japanese patients with glycogen storage disease type Ia and a common 985A>G mutation in Caucasian patients with medium-chain acyl-CoA dehydrogenase deficiency. The method can be automated and may be applicable to the DNA diagnosis of various genetic diseases.

Two pathogenic point mutations exist in the authentic mitochondrial genome, not in the nuclear pseudogene.

Technical advancements in molecular genetics have shown various mitochondrial DNA (mtDNA) abnormalities in patients with mitochondrial myopathies. Recently, it has been revealed that, in these patients, the nuclear DNA carries sequences similar to those of the mtDNA (nuclear pseudogene) and it has several point mutations previously reported to be pathogenic. We verified the existence of the T3250C and T3291C mutations, which we have found in patients with mitochondrial myopathy, in the authentic mitochondrial genome. A long polymerase chain reaction provides a powerful tool for avoiding nuclear pseudogene amplification and for ruling out ambiguity in the detection of the mutation for diagnosis.

[Recurrent episodes of fever of unknown origin as temporal lobe epilepsy].

A 67-year-old woman was admitted to our department for the recurrent fever of unknown origin that occurred once approximately every 1 month for the last 3 years. No clinical and laboratory abnormality were found, except an interictal EEG showing fronto-temporal spike discharges. During hospitalization a characteristic febrile episode was accompanied by automatism, thereby, it became clear that the undetermined periodic febrile episodes were due to temporal lobe epilepsy. In this case, the thermoregulatory center of the hypothalamus might be symptomatic zone of temporal lobe epilepsy.

Reversal of hypopigmentation in phenylketonuria mice by adenovirus-mediated gene transfer.

Phenylketonuria (PKU) is caused by deficiency of phenylalanine hydroxylase (PAH) in the liver. Patients with PKU show increased L-phenylalanine in blood, which leads to mental retardation and hypopigmentation of skin and hair. As a step toward gene therapy for PKU, we constructed a replication-defective, E1/E3-deleted recombinant adenovirus harboring human PAH cDNA under the control of a potent CAG promoter. When a solution containing 1.2 x 10(9) plaque-forming units of the recombinant adenovirus was infused into tail veins of PKU model mice (Pah(enu2)), predominant expression of PAH activity was observed in the liver. The gene transfer normalized the serum phenylalanine level within 24 h. However, it also provoked a profound host immune response against the recombinant virus; as a consequence, the biochemical changes lasted for only 10 d and rechallenge with the virus failed to reduce the serum phenylalanine concentration. Administration of an immunosuppressant, FK506, to mice successfully blocked the host immune response, prolonged the duration of gene expression to more than 35 d, and allowed repeated gene delivery. We noted a change in coat pigmentation from grayish to black after gene delivery. The current study is the first to demonstrate the reversal of hypopigmentation, one of the major clinical phenotypes of PKU in mice as well as in humans, by adenovirus-mediated gene transfer, suggesting the feasibility of gene therapy for PKU.

Two CPT2 mutations in three Japanese patients with carnitine palmitoyltransferase II deficiency: functional analysis and association with polymorphic haplotypes and two clinical phenotypes.

Carnitine palmitoyltransferase II (CPT II) deficiency manifests as two different clinical phenotypes: a muscular form and a hepatic form. We have investigated three nonconsanguineous Japanese patients with CPT II deficiency. Molecular analysis revealed two missense mutations, a glutamate (174)-to-lysine substitution (E174K) and a phenylalanine (383)-to-tyrosine substitution (F383Y) in the CPT II cDNA. Transfection experiments in COS-1 cells demonstrated that the two mutations markedly decreased the catalytic activity of mutant CPT II. Case 1 (hepatic form) was homozygous for the F383Y mutation, whereas case 3 (muscular form) was homozygous for the E174K mutation. Case 2 and her brother, who were compound heterozygotes for E174K and F383Y, exhibited the hepatic phenotype. We also identified a novel polymorphism in the CPT2 gene, a phenylalanine (352)-to-cysteine substitution (F352C), which did not alter CPT II activity in transfected cells. It was present in 21 out of 100 normal alleles in the Japanese population, but absent in Caucasian populations. Genotyping with the F352C polymorphism and the two previously reported polymorphisms, V368I and M647V, allowed normal Japanese alleles to be classified into five haplotypes. In all three families with CPT II deficiency, the E174K mutation resided only on the F1V1M1 allele, whereas the F383Y mutation was observed on the F2V2M1 allele, suggesting a single origin for each mutation.

[Molecular analysis of a patient with carnitine palmitoyltransferase II deficiency].

Carnitine palmitoyltransferase (CPT), one of the key enzymes of beta-oxidation, translocates long-chain fatty acids from the cytosolic compartment into the mitochondrial matrix to undergo beta-oxidation. Recently, the CPT system has been characterized to consist of two distinct mitochondrial membrane-bound enzymes, CPT I, located on the inner side of the outer mitochondrial membrane, and CPT II, located on the inner mitochondrial membrane. We have investigated a Japanese patient with muscular manifestations who was previously reported as CPT deficiency. Enzymatic analysis of her cultured lymphoblasts revealed that CPT II activity was reduced to 5.8%, indicating that the patient suffered from CPT II deficiency. Molecular analysis identified a missense mutation, a glutamate (174)-to-lysine substitution (E174K), in the CPT II cDNA. The patient was homozygous for the mutation. The presence of the mutation was confirmed by PCR-RFLP with a mismatched primer to generate Mbo II recognition sequence at the mutation site. It has been reported that CPT II deficiency manifests as two different clinical phenotypes: a muscular form and a hepatocardiomuscular form. To our knowledge, this is the first case of CPT II deficiency with muscular symptoms to be characterized by molecular analysis in Japan.

[Identification of missense mutations and haplotyping of carnitine palmitoyltransferase II gene].

Carnitine palmitoyltransferase II(CPTII) deficiency manifests as two different clinical phenotypes: an adult form associated with muscular symptoms and an infantile form presenting with hepatocardiomuscular manifestations. We have investigated three Japanese patients with CPT II deficiency. Molecular analysis revealed two novel missense mutations, a glutamate (174)-to-lyine substitution (E174K) and a phenylalanine (383)-to-tyrosine substitution (F383Y) in the CPTII cDNA. Transfection experiments demonstrated that the two mutations reduced CPTII catalytic activity. We also identified a novel polymorphism in the CPTII gene, a phenylalanine (352)-to-cysteine substitution (F352C). According to an expression analysis this mutation did not alter CPTII activity. It was present in 21 out of 100 normal alleles in the Japanese population, but was not observed among Caucasians. Genotyping with the F352C polymorphism and the previously reported polymorphisms V368I and M647V allowed normal alleles to be classified into five haplotypes. In all three families, the E174K mutation resided only on F1V1M1 allele, while the F383Y mutation was observed on F2V2M1 allele, suggesting a single origin of each mutation.

[Chorea with prominent spasticity associated with an expansion of the CAG trinucleotide repeat in the IT15 gene: a case report].

A 60-year-old female, who had been noted to have slowly progressive mental changes since the age of 40, subsequently developed irregular, abrupt, jerking movements of her limbs, and coordination and walking difficulty after 15 years. Her father, aunt and sister had also been suffering from similar involuntary movements and changes of personality. Neurological examination revealed slight dementia, choreic movements and the evidence of pyramidal tract signs; i.e., she showed general hyperreflexia of extremities, bilateral ankle clonus, and extensor plantar responses of lower extremities. laboratory data were all within normal ranges except iron deficiency anemia. Axial MRI showed moderate cortical atrophy, however, without dilatation of the frontal hours of lateral ventricles and the caudate head appeared normal. Our initial impression was dentatorubropallidoluysian atrophy (DRPLA). However, the genetic analysis of her peripheral leukocytes disclosed an expansion of the CAG repeats (43 copies) in the IT15 gene in chromosome 4p, indicating the diagnosis of Huntington's disease (HD). CAG repeats in chromosome 12, known to link with DRPLA, was not expanded. Thus, this case is unique in that the classical symptomatologies of HD was betrayed by the presence of prominent pyramidal tract signs. This patient may be of importance in demonstrating the diversity of neurological symptoms and neuropathologies caused by CAG trinucleotide repeats in HD.

[Acute bacterial meningitis and foramen magnum syndrome as a presentation of odontoid osteomyelitis. A case report].

A 77-year-old male was hospitalized with a 6-day history of severe posterior cervical pain and headache. An initial lumbar puncture revealed polymorphonuclear pleocytosis indicating pyogenic meningitis. The blood and throat cultures on admission were positive for Staphylococcus aureus. A combination of ceftriaxone and ABPC, both of which were sensitive to the pathogen, were intravenously administered for one month, by which he responded well as to the meningitis. However, he was found to have persistent neck pain, and wasting and weakness of the shoulder girdles, which were associated with polyneuropathy-like sensory loss. Neuroimaging studies disclosed a partial destruction of the odontoid process, subluxation of the atlantoaxial joint, MRI evidence of an inflammatory pseudomass formation dorsal to the odontoid process and an increased uptake of radionuclide on bone scanning, all of which were indicative of osteomyelitic processes at the region of the axis; i.e., the meningitis was thought to be secondary to osteomyelitis of the odontoid process and hence the foramen magnum syndrome characteristic to this particular case was explained by the compression of the cervicomedullary junction due to the pseudomass. To our knowledge, this is the first case of primary odontoid osteomyelitis to be reported in Japan.

[Histochemical and biochemical investigations of monoamine oxidase activity level in the brain of the brindled mouse].

In order to make clear the degree of brainstem affection in Menkes disease, it is important to investigate morphological and enzymatic changes of monoamine oxidase (MAO)-containing neurons in the brain in this disease. For this purpose, MAO activity levels in brain tissue from normal and brindled mice were examined histochemically and biochemically. A coupled peroxidatic oxidation method for the histochemistry and a rapid microfluorimetric method for the biochemical assay were adopted. Animals aged 3 days, 8 days, 13 days, 3 months and 12 months were used for the histochemical study. Three-, 7-, and 12-day-old mice were used for the biochemical study. Histochemical examinations showed no significant differences in the stainability, morphology and distribution of MAO positive neurons in the brain between normal and brindled mice at the same age. Biochemical assays revealed that MAO activity levels in the brain of the postnatal brindled mice rose with age as highly as those in the normal control mice. There were no significant differences in them between normal and brindled mice at the same age. The results indicate that in Menkes disease in mice the brainstem affection, if there is, is not so severe as to influence the morphology and enzyme activity of MAO-containing neurons.