Kazuko Sukegawa

Morquio disease: Isolation, characterization and expression of full-length cDNA for human N-acetylgalactosamine-6-sulfate sulfatase

We cloned and sequenced a full-length cDNA of human placental N-acetylgalactosamine-6-sulfate sulfatase, the enzyme deficient in Morquio disease. The 2339-nucleotide sequence contained 1566 nucleotides which encoded a polypeptide of 522 amino acid residues. The deduced amino acid sequence was composed of a 26-amino acid N-terminal signal peptide and a mature polypeptide of 496 amino acid residues including two potential asparagine-linked glycosylation sites. Expression of the cDNA in transfected deficient fibroblasts resulted in higher production of this sulfatase activity than in untransfected deficient fibroblasts. The cDNA clone was hybridized to only a 2.3-kilobase species of RNA in human fibroblasts. The amino acid sequence of N-acetylgalactosamine-6-sulfate sulfatase showed a high degree of homology with those of other sulfatases such as human arylsulfatases A, B or C, glucosamine-6-sulfatase, iduronate-2-sulfatase and sea urchin arylsulfatase.

Treatment of MPS VII (Sly disease) by allogeneic BMT in a female with homozygous A619V mutation

A 12-year-old girl with Sly disease (mucopoly- saccharidosis VII; β-glucuronidase deficiency), who is homozygous for the A619V mutation, had a successful allogeneic BMT, donored by an HLA-identical unrelated female to replace the deficient enzyme. Within 5 months after BMT, the enzyme activity of the recipient’s lymphocytes increased to normal range. No signs of acute or chronic GVHD were observed. For the successive 31 months post-BMT, β-glucuronidase activity in her lymphocytes was maintained at almost normal levels and excretion of glycosaminoglycans in the urine was greatly diminished. Ultrastructural findings demonstrated no abnormal vacuoles and inclusion bodies in the cytoplasm of her rectal mucosal cells. Coincident with the restoration of the enzyme activity, clinical improvement was dramatic. Especially notable were improvements in motor function. The patient was able to walk alone for a long time without aid, and she even became able to ride a bicycle and take a bath. In addition, recurrent infections of the upper respiratory tract and the middle ears decreased in frequency and severity, and dyspnea on exertion, severe snoring and vertigo have substantially improved. Thus, allogeneic BMT in this patient produced a better quality of life and provided a more promising outlook.

BETA -GALACTOSIDASE-DEFICIENT MOUSE AS AN ANIMAL MODEL FOR GM1-GANGLIOSIDOSIS

GM1-gangliosidosis is a progressive neurological disease in humans caused by deficiency of lysosomal acid β-galactosidase, which hydrolyses the terminal β-galactosidic residue from ganglioside GM1 and other glycoconjugates. In this study, we generated a mouse model for GM1-gangliosidosis by gene targeting in embryonic stem cells. The mouse homozygous for the disrupted β-galactosidase gene showed β-galactosidase deficiency, presented with progressive spastic diplegia, and died of emaciation at 7–10 months of age. Pathologically, PAS-positive intracytoplasmic storage was observed in neuronal cells of various areas in the brain. Biochemical analysis revealed a marked accumulation of ganglioside GM1 and asialo GM1 in brain tissue. This animal model will be useful for pathogenetic analysis and therapeutic trial of human GM1-gangliosidosis.

Mucopolysaccharidosis type I: Identification of common mutations that cause Hurler and Scheie syndromes in Japanese populations

α‐L‐Iduronidase (IDUA) deficiency (mucopolysaccharidosis type I; MPS‐I) is an inborn error of lysosomal degradation of glycosaminoglycans that results in storage of undegraded glycosaminoglycans in lysosomes. Previous studies in Caucasian populations showed that (1) homozygosity or compound heterozygosity for the W402X and Q70X mutations are the common causes of MPS‐I with a severe form (Hurler syndrome), and (2) the presence of R89Q may lead to a milder phenotype. We studied mutations in the IDUA gene from 19 MPS‐I patients, including two pairs of siblings, with various clinical phenotypes (Hurler, 6 cases; Hurler/Scheie, 7 cases; Scheie, 6 cases). We report the presence of two common mutations that account for 42% of the 38 alleles in these patients. One is a novel 5‐bp insertion between the thymidine at nt 704 and a cytosine at nt 705 (704ins5), which is seen only in the Japanese population. The other is a missense mutation, R89Q, which is also seen in Caucasians, although uncommonly. In the 19 Japanese MPS‐I patients, the 704ins5 mutation accounted for 7 of 38 alleles (18%), while the R89Q accounted for 9 of 38 (24%). No Japanese patient was found to carry the W402X or Q70X alleles, the two most common MPS‐I mutations in Caucasians. Homozygosity for the 704ins5 mutation is associated with a severe phenotype, and for the R89Q mutation with a mild phenotype. Compound heterozygosity for these two mutations produced an intermediate phenotype. Haplotype analysis using polymorphisms linked to the IDUA locus demonstrated that each mutation occurs on a different specific haplotype, suggesting that individuals with each of these common mutations derive from common founders. These data continue to document the molecular heterogeneity and racial differences in mutations in MPS‐I.

Molecular heterogeneity in mucopolysaccharidosis IVA in Australia and Northern Ireland: Nine novel mutations including T312S, a common allele that confers a mild phenotype

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive lysosomal storage disorder caused by a genetic defect in N‐acetylgalactosamine‐6‐sulfate sulfatase (GALNS). Previous studies of patients from a British–Irish population showed that the I113F mutation is the most common single mutation among MPS IVA patients and produces a severe clinical phenotype. We studied mutations in the GALNS gene from 23 additional MPS IVA patients (15 from Australia, 8 from Northern Ireland), with various clinical phenotypes (severe, 16 cases; intermediate, 4 cases; mild, 3 cases). We found two common mutations that together accounted for 32% of the 44 unrelated alleles in these patients. One is the T312S mutation, a novel mutation found exclusively in milder patients. The other is the previously described I113F that produces a severe phenotype. The I113F and T312S mutations accounted for 8 (18%) and 6 (14%) of 44 unrelated alleles, respectively. The relatively high residual GALNS activity seen when the T312S mutant cDNA is overexpressed in mutant cells provides an explanation for the mild phenotype in patients with this mutation. The distribution and relative frequencies of the I113F and T312S mutations in Australia corresponded to those observed in Northern Ireland and are unique to these two populations, suggesting that both mutations were probably introduced to Australia by Irish migrants during the 19th century. Haplotype analysis using 6 RFLPs provides additional data that the I113F mutation originated from a common ancestor. The other 9 novel mutations identified in these 23 patients were each limited to a single family. These data provide further evidence for extensive allelic heterogeneity in MPS IVA in British–Irish patients and provide evidence for their transmission to Australia by British–Irish migrants. Hum Mutat 11:202–208, 1998.

Mutation analysis in the iduronate-2-sulphatase gene in 43 Japanese patients with mucopolysaccharidosis type II (Hunter disease).

Our series of studies on Hunter disease in Japanese patients showed allelic heterogeneity of IDS gene mutations, genotype/phenotype correlation and racial differences in distribution of mutations. Twenty-five different small mutations have been characterized. Small mutations in the Japanese population are widely distributed through the IDS gene, although some mutations were unevenly concentrated on exon 5 (28%) and on exon 9 (24%). Mutations were seen at the same codon 468 in exon 9 in 5 patients. These findings are in good agreement with data on other ethnic groups. Two unique mutations linked to a severe phenotype were apparently associated with aberrant splicings; one was a point mutation within exon 3 (P86L), partially activating a cryptic splice acceptor site at 28 bp downstream from the mutation site within exon 3 and producing a 44-base truncated mRNA, and the other was a point mutation at the consensus sequence of the splice donor site of intron 2, causing exon 2 skipping.

A novel common missense mutation G301C in the N-acetylgalactosamine-6-sulfate sulfatase gene in mucopolysaccharidosis IVA.

Abstract Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive lysosomal storage disorder caused by a genetic defect in N-acetylgalactosamine-6-sulfate sulfatase (GALNS). In previous studies, we have found two common mutations in Caucasians and Japanese, respectively. To characterize the mutational spectrum in various ethnic groups, mutations in the GALNS gene in Colombian MPS IVA patients were investigated, and genetic backgrounds were extensively analyzed to identify racial origin, based on mitochondrial DNA (mtDNA) lineages. Three novel missense mutations never identified previously in other populations and found in 16 out of 19 Colombian MPS IVA unrelated alleles account for 84.2% of the alleles in this study. The G301C and S162F mutations account for 68.4% and 10.5% of mutations, respectively, whereas the remaining F69V is limited to a single allele. The skewed prevalence of G301C in only Colombian patients and haplotype analysis by restriction fragment length polymorphisms in the GALNS gene suggest that G301C originated from a common ancestor. Investigation of the genetic background by means of mtDNA lineages indicate that all our patients are probably of native American descent.

Non-existence of a tight association between a 444leucine to proline mutation and phenotypes of Gaucher disease: high frequency of a NciI polymorphism in the non-neuronopathic form

SummaryA 444leucine to proline mutation detected by a NciI polymorphism in the human glucocerebrosidase gene was studied to investigate the correlation of the three clinical phenotypes of Gaucher disease with this mutation in 11 Japanese patients with Gaucher disease (type I, 8 patients; type II, 1 patient; type III, 2 patients) and to determine the feasibility of the use of genomic probe DNA for carrier detection and prenatal diagnosis in 8 Japanese families with Gaucher disease and agreeable to family study (type I, 6 families; type III, 2 families). The homoallelic 444leucine to proline mutation was found only in patients with type I disease. Of the 8 type I patients, 5 had the homoallelic mutation and 2 had one mutant allele. One patient with type II disease did not have this mutant allele. Of the 2 type III patients, one had a single mutant allele whereas the other exhibited no mutation of this kind. These results suggest that the 444leucine to proline mutation is very common in the type I (non-neuronopathic form) disease and is not tightly associated only with neuronopathic types of Gaucher disease in Japanese patients. These findings seem to conflict with others showing that this mutation is partially responsible for the occurrence of neuronopathic Gaucher disease. Thus, the NciI polymorphism will not be useful for the diagnosis of subtypes of Gaucher disease. Carrier detection was feasible in three families with type I disease of the 8 families analyzed by the NciI polymorphism.

Mucopolysaccharidosis type IVA: common double deletion in the N-acetylgalactosamine-6-sulfatase gene (GALNS).

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency in N-acetylgalactosamine-6-sulfatase (GALNS). We found two separate deletions of nearly 8.0 and 6.0 kb in the GALNS gene, including some exons. There are Alu repetitive elements near the breakpoints of the 8.0-kb deletion, and this deletion resulted from an Alu-Alu recombination. The other 6.0-kb deletion involved illegitimate recombinational events between incomplete short direct repeats of 8 bp at deletion breakpoints. The same rearrangement has been observed in a heteroallelic state in four unrelated patients. This is the first documentation of a common double deletion a gene that is not a member of a gene cluster.

Fourteen novel mucopolysaccharidosis IVA producing mutations in GALNS gene

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency of the lysosomal N‐acetylgalactosamine‐6‐sulfate sulfatase. Here, we report our analysis of data on 21 patients of diverse ethnic and geographic origins studied by SSCP and sequencing analysis. Sixteen mutations were detected, including 14 new mutations (11 missense, one premature termination, one splice site alteration, and one cryptic site alteration). The donor splice site mutation (IVS4 + 1G→A) predicts that normal splicing will be abolished and that translation would lead to an immediate premature termination (W141X). Another novel nucleotide change outside the coding sequence is an intronic alteration (IVS9‐42C→T:ggtcggtgcggttggtgc) creating a potential cryptic donor site. The nucleotide sequence surrounding this alteration is highly suggestive of a consensus donor splice site. All 12 missense and nonsense mutations were shown by transient expression to abolish or greatly reduce GALNS activity, thereby providing an explanation as to why they produce MPS IVA. All mutations were readily confirmed by restriction enzyme or by allelic specific oligonucleotide analysis (ASO). These findings, coupled with previously reported mutations, bring the total of different mutations to 41 among independent families with MPS IVA, illustrating the extensive allelic heterogeneity among mutations producing MPS IVA. Hum Mutat 10:368–375, 1997.