Hisao Fukushima

Molecular cloning and expression of cDNA for murine galactocerebrosidase and mutation analysis of the twitcher mouse, a model of Krabbe's disease

Abstract: The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5′ end. It has a 2,278‐nucleotide sequence including a 2,004‐nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG → TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6‐kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3′‐untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.

Mitochondrial tRNAlle mutation in fatal cardiomyopathy

A patient with mitochondrial encephalomyopathy who died from progressive intractable cardiac failure at the age of 18 is reported. At the age of 4, he presented with short stature, but multiorgan disorders including deafness, focal glomerulosclerosis, epilepsy and dilated cardiomyopathy appeared later in his clinical course. Laboratory tests showed hyperlactatemia and hyperpyruvatemia. Histopathological findings demonstrated mitochondrial myopathy with ragged red fibers and focal cytochrome C oxidase-deficient fibers in skeletal and cardiac muscles. The activity of cytochrome C oxidase was 30% less than the control level in skeletal muscle. Sequencing of the entire mitochondrial tRNA genome revealed a novel point mutation in the tRNA(Ile) region (nt 4269). This A-to-G substitution was found in none of the 30 controls by screening using mispairing PCR and Ssp I digestion methods, suggesting that this new mutation was pathogenic in our case.

Clinical experiences in Sakai City Hospital during the massive outbreak of enterohemorrhagic Escherichia coli O157 infections in Sakai City, 1996.

Abstract In the middle of July 1996, a massive outbreak of hemorrhagic colitis (HC) occurred among elementary schoolchildren in Sakai city. This is the most widespread outbreak of O157 infection ever experienced to our knowledge. Lunch foods supplied in the elementary schools in Sakai were contaminated by Escherichia coli (E. coli) O157. One hundred and twenty‐one cases developed hemolytic uremic syndrome (HUS) from 12 680 symptomatic patients, including putative secondary infections, and three girls died during this outbreak. Sakai City Hospital is one of the core medical facilities in this community; hence, 425 children with HC were treated at the hospital. Antibiotics were used extensively on all patients. Among them, 12 children developed HUS. All 425 children, including the patients with HUS, recovered without significant sequelae. In the present paper, the clinical experiences during this massive outbreak of E. coli O157 infection in Sakai City Hospital are described.

Mitochondrial encephalomyopathies with the mutation of the mitochondrial tRNALeu(UUR) gene

Four families with mitochondrial encephalomyopathy are described. Probands of three families had typical clinical presentations of mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS), but the proband of family 4 lacked strokelike episodes. The mitochondrial DNA mutation of tRNA(Leu(UUR)) (transfer ribonucleic acid specific to leucine (UUR codon)) found in MELAS was examined in muscle DNA obtained from biopsy samples of the probands of four families and the maternal relatives of family 2. The mutation was detected in all muscle samples, and the degree of the mutated DNA was 68% to 84% by Southern blot analysis. However, the clinical patterns of the maternal relatives of family 2 were mild and distinctly different from MELAS. The same mutation was also detected in blood-derived DNA samples of all family members examined, including healthy mothers but not fathers, although the degree of mutation did not correlate with the clinical severity. These results confirmed the maternal inheritance of this disease and suggested that the mitochondrial DNA mutation (tRNA(Leu(UUR))) may cause clinical symptoms other than MELAS. The clinical findings of mitochondrial encephalomyopathy should be reinvestigated in terms of the mitochondrial gene mutation; the polymerase chain reaction method will be useful for screening for this mutation of mitochondrial DNA in blood samples.

Structure and sequence of the human α-l-fucosidase gene and pseudogene

Abstract Fucosidosis is a rare lysosomal storage disease resulting from a nearly complete deficiency of α- l -fucosidase enzyme activity. Previously, cDNA encoding human fucosidase was cloned and sequenced. Here we report the determination of the human fucosidase gene structure and sequence as well as the sequence of the fucosidase pseudogene. The gene encoding fucosidase is composed of eight exons spanning 23 kb of DNA. Analysis of the sequence 5′ of the open reading frame indicates the presence of multiple transcription factor binding sites but no TATA box. Northern blot analysis has confirmed an mRNA size of 2.3 kb in human lymphoblasts, testis, and epithelial cells. We have also sequenced the processed pseudogene of fucosidase. The sequence of the pseudogene is 80% identical to that of fucosidase cDNA but does not contain an open reading frame.

Rapid Growth and Spontaneous Metastasis of Human Germinal Tumors Ectopically Transplanted into Scid (Severe Combined Immunodeficiency) and Scid‐nudestreaker Mice

Xenograft acceptance, growth and spontaneous metastasis of ectopically transplanted human germinal tumors were compared among scid mice, athymic nude mice and F2 hybrids constructed from scid and nude mice, in relation to the impairments of T and B cell functions in these mice. In scid mice which are deficient in T and B cell functions, human yolk sac tumor (YST‐2) that originated from the ovary grew to enormous sizes in 100% of the animals after both subcutaneous and intraperitoneal transplantation, while only half (59.1% and 51.9%) of the subcutaneous and none of the intraperitoneal transplants were accepted in usual athymic nude mice (BALB/c‐nu/nu and CDl‐nu/nu). The YST‐2 grew rapidly in scid mice, developing 3 to 10 times larger tumors compared to nude‐streaker (AKR/J‐nustr/nustr) and usual nude mice, respectively. Furthermore, ectopically transplanted tumors spontaneously metastasized to distant organs (mostly to the lung) in scid mice (but less frequently in leaky scid mice), while metastases have never been found in nude mice. Although a xenograft of human classic (typical or pure) seminoma of the testis has never been established in nude mice, it grows slowly in one‐third (36.4%) of scid mice and very rapidly in all of scid‐nu (scid/scid; nustr/nustr) double mutant mice. Spontaneous metastases of xenografted seminomas were also observed in distant organs (lymph node, lung, liver, spleen, and kidney). The metastastic distribution of the two human germinal tumors in scid and scid‐nustr mice mimics that found in human. These results (xenograft acceptance, growth of transplanted tumors and degree of metastatic spread) were compatible with the level of T and B cell impairments indicated by FACS analysis, as well as mitogen responses, serum IgG and morphological features of the thymus.

Mutation analysis of a Japanese patient with fucosidosis

AbstractFucosidosis is a rare autosomal recessive disorder resulting from a deficiency of α-L-fucosidase. Recently, various mutations have been reported in this disease, but it is difficult to elucidate the phenotype from the genetic mutations. We report a patient with chronic infantile type fucosidosis, with a compound heterozygote of a nonsense mutation (W148X, Trp at codon 148 to stop codon) and a large deletion, including all exons. This is the first report of a large deletion demonstrated in fucosidosis. It is interesting that this patient has a relatively mild clinical course despite the absence of the mRNA. This case also indicates the difficulty in determining the phenotype from the genotype in fucosidosis.

Molecular study of duchenne and Becker muscular dystrophies in Japanese

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive myopathy with an incidence of 1 in 3500 male births, while Becker muscular dystrophy (BMD), a clinically milder form of myopathy and alMic with DMD, affects 1 in 30 000 males. The incidence of DMD in Japan is reported to be 1 in 4600 male births (Yasuda and Kondo, 1980). Recently, the full-length DMD/BMD cDNA has been cloned and found to be 14 kb containing at least 65 exons spread over 2300 kb of genomic DNA (Koenig et al., 1987, 1988). The gene product, called dystrophin, was also identified (Hoffman et al., 1987), and its abnormality causes DMD, BMD or atypical muscular dystrophy. To study the frequency and distribution pattern of intragenic deletions in Japanese DMD or BMD patients, the dystrophin gene was examined in 28 patients with DMD, BMD or high serum creatine kinase (CK) value, using the polymerase chain reaction (PCR) and Southern blot analysis.

Allele frequencies of intragenic, and 5′ and 3′ markers of the dystrophin gene in Japanese families afflicted with Duchenne or Becker muscular dystrophy

SummaryUsing the polymerase chain reaction method (PCR), we examined the allele frequencies and heterozygosities of 7 polymorphic sites (pERT87, and CA polymorphisms in the 5′ and 3′ regions) of the dystrophin gene in 20 Japanese Duchenne muscular dystrophy and Becker muscular dystrophy (DMD or BMD) families consisting of 36 males, including 23 cases of DMD and BMD, and 28 females. The allele frequencies of three primer and enzyme sets in the pERT87 locus were well comparable to those in the previously reported Japanese female cases but different from in other countries. The frequencies of 5′ markers of the dystrophin gene in Japanese were different from the reported Caucasian frequencies. As for 5′DYS-I and 5′DYS-II, the numbers of alleles in our cases were less than in Caucasians, and the heterozygosities of all three markers (5′DYS-I, II and III) were lower than in Caucasians. However, the 3′CA polymorphisms showed almost the same frequencies and heterozygosities as in Caucasians. All of our females showed a heterozygous pattern for at least one locus, with the combination of the seven markers. The usefulness of linkage analysis involving PCR methods with these intragenic, and 5′ and 3′ markers of the dystrophin gene in the carrier and prenatal diagnosis of DMD and BMD was confirmed by the successful prenatal diagnoses in 15 fetuses, the exception being one case considered to have a new mutation.

A missense mutation (G197→A) in theα-l-fucosidase gene of fucosidosis patients leads to loss ofα-l-fucosidase

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from the absence of α-l-fucosidase activity. Two natural missense mutations (G197→A) and (A860→G) within the α-l-fucosidase gene have been reported to be homozygous in four patients with fucosidosis. Expression of wild-type and mutated α-l-fucosidase cDNAs in COS-1 cells revealed complete deficiency of α-l-fucosidase for the G197→A transition and a normal level of enzyme for A860→G. We therefore conclude that the change of G197→A is responsible for fucosidosis in the patients while A860→G is a normal polymorphic variant of α-l-fucosidase.