Protective protein gene mutations in galactosialidosis.

Four different protective protein cDNA mutations, 146A-->G (Q49R), 193T-->C (W65R), 268-269TC-->CT (S90L), and 1184A-->G (Y395C), were identified in six Japanese galactosialidosis patients with various phenotypic manifestations, and another mutation, 746T-->A (Y249N), in a patient of French-German origin with an atypical clinical course. Y395C was a common mutation in four Japanese patients in infancy and childhood; two juvenile patients were compound heterozygotes of Y395C and another common mutation, SpDEx7, and the other two infants were compound heterozygotes of Y395C and mutant alleles other than SpDEx7. We confirmed these mutations in genomic DNA by direct-sequence analysis or restriction-site analysis. The mutant cDNA clones, transiently expressed in a transformed galactosialidosis cell line, did not restore the secondarily deficient beta-galactosidase or alpha-neuraminidase activity except for the Y249N mutation that expressed some carboxypeptidase activity and restored the two lysosomal enzyme activities. Pulse-chase analysis detected a small amount of the mature form, as well as the precursor, in the cells transfected with the Y249N cDNA. Only precursor proteins were detected, mature proteins not appearing for the other mutant cDNAs.

Immunohistochemical characterization of transgenic mice highly expressing human lysosomal alpha-galactosidase.

Human lysosomal alpha-galactosidase predominantly hydrolyzes ceramide trihexoside. A transgenic mouse line, C57BL/6CrSIc-TgN(GLA) 1951 Rin, highly expressing human alpha-galactosidase, has been established and investigated biochemically and immunohistochemically in order to clarify the distribution of the expressed enzyme proteins and to evaluate it as a donor model of organ transplantation therapy for Fabry disease caused by a genetic defect of alpha-galactosidase. In these transgenic mice, about five copies of the transgene were integrated, and alpha-galactosidase activity was expressed in liver, kidney, heart, spleen, small intestine, submaxillary gland, skeletal muscle, cerebrum, cerebellum, bone marrow cells and serum. The enzyme activity was about 22 to 11,080-fold higher than that in non-transgenic mice. In liver, heart and kidney tissues, which are important organs for transplantation studies, sufficient amounts of alpha-galactosidase mRNAs were transcribed, and the expressed enzymes, with molecular weights of 54-60 kDa, are abundant in the liver (enzyme activity: 53,965 nmol h-1 mg-1 protein) and heart (39,906 nmol h-1 mg-1 protein), followed by in the kidney tissue (9177 nmol h-1 mg-1 protein), respectively. An immunohistochemical microscopic study clearly demonstrated the distribution of the expressed enzyme proteins in kidney and liver tissues. Highly expressed alpha-galactosidase was detected in glomerular cells, tubular cells and hepatocytes. These transgenic mice will be useful as a donor model for experimental organ transplantation, and also it will enable recurrent biopsies and long-term observation. The organ transplantation data on mice will provide us with important information.

Generation and characterization of transgenic mice expressing a human mutant alpha-galactosidase with an R301Q substitution causing a variant form of Fabry disease.

Transgenic mice expressing a human mutant alpha-galactosidase with an R301Q substitution, which was found in a patient with a variant form of Fabry disease, were established. The mice transcribed a sufficient amount of alpha-galactosidase mRNA, but the steady-state levels of the enzyme protein were decreased in liver, kidney and heart, only residual activity being detected in these tissues. The mice will be useful for the clarification of the defective regulation of the structurally altered enzyme protein expressed by the mutant gene at the organ or individual level as well as for the evaluation of drugs that stabilize and/or activate the mutant alpha-galactosidase.

GM2 gangliosidosis AB variant: clinical and biochemical studies of a Japanese patient.

OBJECTIVE: To determine the clinical features and biochemical basis of the first Japanese patient with the GM2 gangliosidosis AB variant. METHODS: The clinical manifestations and laboratory findings in the patient were investigated. Cultured fibroblasts from the patient were analyzed by means of immunofluorescence staining with an anti-GM2 ganglioside monoclonal antibody and thin-layer chromatography and immunostaining. GM1 ganglioside catabolism in cultured cells was analyzed by pulse labeling, and the amount of GM2 activator in cells was determined by Western blot analysis. Gene analysis was performed according to standard protocols. RESULTS: The patient showed progressive neurologic manifestations of quite early onset. Muscular weakness and hypotonia became evident by 1 month of age, and the patient then developed a startle reaction, severe psychomotor retardation, and myoclonic seizures. Immunocytochemical analysis clearly revealed the accumulation of GM2 ganglioside in cultured fibroblasts from the patient, and thin-layer chromatography confirmed it. Western blot and metabolic studies showed a complete deficiency of GM2 activator. Gene analysis did not reveal any mutations in the protein coding region of the GM2 activator gene. CONCLUSION: The clinical features and biochemical basis of this Japanese patient with GM2 gangliosidosis AB variant were determined. Immunocytochemical analysis using cultured fibroblasts as samples is available for the diagnosis of this disease.

Brain- and muscle-type promoters of the dystrophin gene are selected in peripheral lymphocytes and Epstein Barr virus-transformed lymphoblastoid [correction of lymphoplastoid] cells.

Promoter-specific transcripts of the dystrophin gene in peripheral lymphocytes and Epstein Barr virus-transformed lymphoblastoid cells were analysed using reverse transcription-nested polymerase chain reaction. Two DNA fragments, corresponding to the alternative first exons transcribed from either brain- or muscle-type promoters, were both amplified from cDNA prepared from normal lymphocytes and lymphoblastoid cells. The nucleotide sequences of the amplified products were 100% homologous to the 5' termini of the cDNA of brain- and muscle-type dystrophins, respectively. Neither fragment was amplified from the lymphoblastoid cells of a patient with Duchenne muscular dystrophy, who has a partial deletion involving the brain- and muscle-type promoters in the dystrophin gene. These findings showed that the brain-type as well as the muscle-type promoter of the dystrophin gene was active in lymphocytes and lymphoblastoid cells.

Dystrophin immunohistochemistry in a symptomatic carrier of Becker muscular dystrophy.

Immunohistochemical localization of dystrophin was studied in a symptomatic carrier of Becker muscular dystrophy (BMD). Muscle biopsy specimens from a female carrier showed findings compatible with slowly progressive muscular dystrophy by ordinary histochemical examinations. Immunohistochemical study, using an antiserum raised against a synthetic peptide fragment of dystrophin, demonstrated a mixture of staining patterns, including continuous but faint positive fibres, partially disrupted fibres and negative fibres. These findings were identical to those of patients with BMD and appear to differ from previous findings in female carriers of Duchenne muscular dystrophy. This report is the first immunohistochemical study of a symptomatic female proven by molecular genetic analysis to be a carrier of BMD.

Adult Sandhoff's disease: R505Q and I207V substitutions in the HEXB gene of the first Japanese case.

We describe a 31-year-old Japanese man with adult Sandhoff s disease presenting as spinocerebellar degeneration. There was a marked cerebellar atrophy on MRI, and proliferation of abundant PAS-positive foamy macrophages in the rectal mucosa. The activities of total beta-Hex, beta-Hex A, and beta-Hex B in leucocytes of the patient were 14%, 15%, and 6% of control values, respectively. However, oligosacchariduria or ultrastructural storage materials in liver tissue were nil. Direct sequencing of cDNA and genomic DNA, and restriction digestion revealed two different homozygous base substitutions in the HEXB gene: the G1514-->A substitution (R505Q) and the A619-->G substitution (1207V). The parents were consanguineous. His healthy mother, an enzymatic heterozygous carrier, was homozygous for 1207V, but heterozygous for R505Q mutation. Thus, the patient is probably homozygous for both base substitutions and a R505Q mutation may be linked to the phenotype of adult Sandhoff's disease.

Urinary excretion of the vitronectin receptor (integrin alpha V beta 3) in patients with Fabry disease.

A renal disorder is one of the important manifestations of Fabry disease, but the details of the pathogenesis have not been clarified yet. We examined the possibility that the vitronectin receptor (VNR, integrin alpha V beta 3), one of the integrins, is involved in the progression of the renal injury in Fabry disease. We measured the urinary excretion of beta 3 originating from VNR in Fabry patients by immunoblotting analysis and enzyme-linked immunosorbent assay (ELISA). Immunofluorescent microscopic analyses for VNR and globotriaosylceramide were performed on urinary sediments from Fabry patients. Furthermore, beta 3 and vitronectin in kidney tissues were analyzed immunohistochemically. Immunoblotting analysis and ELISA showed that the urinary excretion of beta 3 originating from VNR was significantly increased in the Fabry group compared with both the pathological and healthy control groups. Immunofluorescent microscopy revealed the expression of VNR and accumulation of globotriaosylceramide in urinary sediments from the Fabry patients. Increased expression of beta 3 was observed in glomerular epithelial cells, and in Bowman's capsular epithelial layer and tubular cells, and the amount of vitronectin was moderately increased in the kidney tissues from the Fabry patients. The urinary excretion of VNR was increased, and the expression of VNR was observed in Fabry kidney tissues. The expression of VNR may be involved in the progression of the renal injury in this disease.

A screening for dystrophin gene deletions in Japanese patients with Duchenne/Becker muscular dystrophy by the multiplex polymerase chain reaction.

A new screening method involving the multiplex polymerase chain reaction was developed to detect dystrophin gene deletions in Japanese patients with Duchenne and Becker muscular dystrophy (DMD/BMD). Eleven exonic regions including deletion "hot spots" were analyzed. Gene deletions were found in 33% of 92 unrelated Japanese patients, mainly in the central portion (exons 43-52) and at the 5' end (exons 1-17). This is a useful laboratory test for the rapid genetic diagnosis of DMD/BMD.

Carrier detection of Duchenne/Becker muscular dystrophy: computer-assisted direct quantitation of gene amplification products.

An improved method by quantitative dystrophin gene deletion analysis was developed for the detection of Duchenne/Becker muscular dystrophy (DMD/BMD) carriers. Exon 52, which had been found to be deleted in DMD probands, was amplified for female family members, together with exon 60 as a reference, at the exponential phase of polymerase chain reaction. The products were separated by electrophoresis, the band intensities on gel photographs were quantitated, and the target/control ratios were calculated. The values for three heterozygous mothers were approximately half those for normal individuals and two definite non-heterozygous mothers. This procedure is easy, rapid and useful for the carrier diagnosis of DMD/BMD.

A case of Duchenne muscular dystrophy with truncated dystrophin. Significance of a cysteine-rich domain for functional expression of dystrophin protein.

A Duchenne muscular dystrophy case showed truncated dystrophin (320 kDa) with an isoelectric point slightly shifted towards a more alkaline pH. From the polymerase chain reaction and immunochemical analysis data, the expressed dystrophin protein was predicted to lack the portion comprising the tail of the rod-like domain, the cysteine-rich domain, and the head of the C-terminal domain. These results indicated the functional importance of the cysteine-rich domain in the dystrophin protein.

Molecular form and subcellular distribution of acid beta-galactosidase in fibroblasts from patients with GM1 gangliosidosis, Morquio B disease and galactosialidosis.

The molecular form and subcellular distribution of acid beta-galactosidase in cultured fibroblasts from patients with beta-galactosidase deficiency (GM1-gangliosidosis, Morquio B disease and galactosialidosis) were studied, using antibodies against three different forms of the human enzyme: a high-molecular-weight multienzymic complex, a recombinant 84-kDa precursor, and a 64-kDa tryptic product of the precursor. The mature enzyme from normal fibroblasts was immunoprecipitated by the anti-complex and anti-64-kDa protein antibodies, but not by the anti-84-kDa precursor one. immunofluorescence staining of normal fibroblasts revealed the granular (lysosomal) distribution with anti-64-kDa protein antibody and the perinuclear reticular distribution with anti-84-kDa precursor antibody, probably representing the Golgi apparatus. Both patterns were demonstrated in Morquio B disease, but the residual enzyme activity was exclusively due to the mature enzyme. In Type 1 galactosialidosis, most of the expressed enzyme was detected as the precursor form with a perinuclear reticular distribution. In type 2 galactosialidosis, more than half of the enzyme activity was due to the mature form with a lysosomal distribution. Fibroblasts from a patient with GM1 gangliosidosis, expressing no beta-galactosidase mRNA, did not react against either anti-64-kDa protein antibody or anti-84-kDa precursor antibody. The combined use of immunoprecipitation and immunostaining was useful for analysing the pathophysiology of the intracellular processing and transport of the mutant beta-galactosidase.

Screening and detection of gene mutations in Japanese patients with Fabry disease by non-radioactive single-stranded conformation polymorphism analysis.

We have applied non-radioactive polymerase chain reaction (PCR)-single-stranded conformation polymorphism (SSCP) to the detection of gene mutations causing Fabry disease. Nineteen of 22 known mutations were detected as electrophoretic mobility shifts on PCR-SSCP analysis. Then, DNA from newly diagnosed Japanese patients with the classical form of Fabry disease was subjected to PCR-SSCP analysis, and 4 novel mutations (1 small deletion, 1 nonsense mutation and 2 missense mutations) and 1 neutral polymorphism were identified. Furthermore, identification of an asymptomatic heterozygote and a hemizygote with moderate clinical manifestations was successfully achieved by application of this method to a family with the variant form of Fabry disease. PCR-SSCP is useful for the gene diagnosis of etiologically heterogeneous Fabry disease.

Clinical and molecular heterogeneity in hereditary beta-galactosidase deficiency.

Results of a molecular analysis of GM1-gangliosidosis and galactosialidosis in our laboratory are briefly reviewed. A common single base substitution was found in adult/chronic form of GM1-gangliosidosis among heterogeneous beta-galactosidase gene mutations, and restriction site analysis was successfully performed for diagnosis of homozygotes and heterozygotes. All adult galactosialidosis patients had a common mutation at a splice junction which caused skipping of an exon of the protective protein/carboxypeptidase gene. An artificial restriction site was introduced in this case and applied to diagnosis of this disease. The heterogeneous gene mutations were compared and correlated with phenotypic manifestations in these two diseases.

Human beta-galactosidase gene mutations in GM1-gangliosidosis: a common mutation among Japanese adult/chronic cases.

Molecular analysis of the human beta-galactosidase gene revealed six different mutations in 10 of 11 Japanese GM1-gangliosidosis patients. They were the only abnormalities in each allele examined in this study. A 165-nucleotide duplication (positions 1103-1267) was found in two infantile patients, producing an abnormally large mRNA; one patient was probably a homozygote, and the other was a heterozygote of this mutation. The other two infantile patients had different mutations; a 123 Gly(GGG)----Arg(AGG) mutation in one patient and a 316 Tyr(TAT)----Cys(TGT) mutation in the other. A 201 Arg(CGC)----Cys(TGC) mutation, eliminating a BspMI site, was detected in a late-infantile/juvenile patient; the restriction-site analysis of amplified genomic DNA confirmed his heterozygosity for this mutation. A 51 Ile(ATC)----Thr(ACC) mutation was found in all five adult/chronic patients examined in this study. It created a SauI site, and restriction-site analysis confirmed that four patients were homozygous mutants. The other was a compound heterozygote for this mutation and another 457 Arg(CGA)----Gln(CAA) mutation. These mutant genes expressed markedly decreased or completely deficient enzyme activities in beta-galactosidase-deficient human fibroblasts transformed by adenovirus-SV40 recombinants. We conclude that gene mutations are heterogeneous in GM1-gangliosidosis but that the 51 Ile(ATC)----Thr(ACC) mutation is common among the Japanese adult/chronic cases. Genotype-phenotype correlations in GM1-gangliosidosis are briefly discussed.

Human beta-galactosidase gene mutations in morquio B disease.

Three different beta-galactosidase gene mutations--a 273Trp----Leu (mutation F) in both families, 482Arg----His (mutation G) in one family, and 509Trp----Cys (mutation H) in the other family--were identified in three patients with Morquio B disease who were from two unrelated families. Restriction-site analysis using StuI, Nsp(7524)I or RsaI confirmed these mutations. In human fibroblasts, mutation F expressed as much as 8% of the normal allele's enzyme activity, but the other mutations expressed no detectable enzyme activity. We conclude that the unique clinical manifestations are specifically associated with mutation F, a common two-base substitution, in this disease.

Fabry disease: detection of 13-bp deletion in alpha-galactosidase A gene and its application to gene diagnosis of heterozygotes.

Polymerase chain reaction amplification of reverse-transcribed messenger RNA from a patient with Fabry disease revealed a 13-base pair deletion in the 5' region (exon 1) of alpha-galactosidase A complementary DNA. This gene rearrangement was not detected by Southern or Northern analysis. Short direct repeats were present around the breakpoints, and considered to be of pathogenetic significance. Gene diagnosis of the mother and a female cousin was successfully achieved by polymerase chain reaction amplification of genomic DNA; the former as a Fabry disease heterozygote and the latter as a normal homozygote.

Protective protein as an endogenous endothelin degradation enzyme in human tissues.

An enzyme hydrolyzing the carboxyl terminus of endothelin-1 was detected in control human tissues but was deficient in tissues from a patient with galactosialidosis, a metabolic disease caused by the protective protein gene mutation. It was proportional to the amount of immunologically estimated mature protective protein. An antibody against the lysosomal protective protein/beta-galactosidase complex precipitated the enzyme activity almost completely. Transfection of the human cDNA for protective protein resulted in high expression of the enzyme activity in transformed fibroblasts from a galactosialidosis patient. These results indicated that the mature protective protein is a major soluble endogenous endothelin degradation enzyme in human tissues.