Toshinori Hori

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.

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.

Intravenous Immunoglobulins Suppress Immunoglobulin Productions by Suppressing Ca2+ ‐Dependent Signal Transduction Through Fc γ Receptors in B Lymphocytes

A high dose intravenous immunoglobulin (IVIG) therapy is used in the treatment of a wide range of autoimmune disorders. However, the mechanisms of the action of IVIGs remain poorly understood. To analyse the mechanisms of effects of IVIGs on immunoglobulin (Ig) production of B cells, the effects of IVIGs on B lymphoblastoid cell lines transformed by Epstein‐Barr virus (LCLs) were investigated. The productions of IgG or IgM of LCLs were dose‐dependently suppressed by polyethylene glycol (PEG)‐treated IVIG or pH 4‐treated 1VIG though the productions were not or only slightly suppressed by pepsin‐treated IVIG. The suppression by IVIGs was blocked by anti‐human IgG Fc or anti‐Fc γ RII. C μ gene expression and μ s C terminal gene expression of LCLs were suppressed by PEG‐treated IVIG, whereas neither C μ gene expression nor μ s C terminal gene expression of LCLs were suppressed by pepsin‐treated IVIG. Although the increase in intracellular calcium concentration in LCLs was not suppressed by pepsin‐treated IVIG, the increase was suppressed by PEG‐treated IVIG. This suppressing effect of PEG‐treated IVIG on intracellular calcium concentration of LCLs was blocked by anti‐human IgG Fc or anti‐ Fc γ RII. Our results suggest that IVIGs suppressed the Ca2+ ‐dependent signal transduction through Fc γ R on B‐cell membrane, consequently, the transcription of C γ mRNA, especially secreted γ mRNA was suppressed in the B cells.

Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population

Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or noncarrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.

Polymerase chain reaction detection of two novel human N-acetylgalactosamine-6-sulfate sulfatase gene polymorphisms by single-strand conformation polymorphism analysis or by StyI and StuI cleavages

We describe two common single-base polymorphisms of the N-acetylgalactosamine-6-sulfate sulfatase gene after StyI or StuI restriction sites. These polymorphisms were readily detected by single-strand conformation polymorphism (SSCP), using the polymerase chain reaction (PCR).

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences

Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetylgalactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the fulllength cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5′end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.

Molecular analysis by Southern blot for theN-acetylgalactosamine-6-sulphate sulphatase gene causing mucopolysaccharidosis IVA in the Japanese population

Mucopolysaccharidosis IVA (MPS IVA; McKusick 253000) is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulphate sulphatase (GALNS: EC 3.1.6.4). Diagnosis is based on the lack of GALNS activity. Classical Morquio disease (a severe phenotype of MPS IVA) holds an important position as a prototype of chondro-osteodystrophy, characterized by specific spondyloepiphyseal dysplasia, short-trunk dwarfism, coxa valga, odontoid hypoplasia and corneal opacities, preserving normal intelligence. Studies on the molecular basis of MPS IVA have been facilitated following purification of GALNS (Masue et al 1991), cloning of the fulMength cDNA (Tomatsu et al 1991), identification of the first two exonic mutations (Fukuda et al 1992), chromosomal assignment to 16q24 (Tomatsu et al 1992; Masuno et al 1993) and cloning of the genomic gene (Nakashima et al 1994). The availability of polymorphic markers makes it feasible to perform an indirect genotype analysis and a more definite estimation of carrier risk. Using GALNS fulllength cDNA, one intragenic restriction fragment length polymorphism (RFLP) has been detected that now provides a high probability genotype diagnosis and carrier detection in informative families. We report here the results of our analysis using the GALNS cDNA probe in 18 Japanese MPS IVA patients and one family. Southern blot analysis of GALNS gene due to the cDNA probe revealed the existence of a highly heterogeneous BamHI polymorphism and some gene alterations in four Japanese patients (5 out of 36 alleles; 13.9% in frequency). In one of five patients we could follow the defective GALNS allele in the family analysis. Northern blot analysis for multiple tissues revealed high expression in adult placenta, kidney and lung and also in fetal lung and kidney.