Mucopolysaccharidosis IVA: a comparative study of polymorphic DNA haplotypes in the Caucasian and Japanese populations.

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulphate sulphatase (GALNS). The genetic heterogeneity at the GALNS locus was studied in 62 mutant alleles and 376 normal alleles in the Caucasian population and also in 40 mutant and 100 normal alleles in the Japanese population. For this study, six different restriction fragment length polymorphisms (RFLPs) at the GALNS locus were analysed to search for the frequency of each RFLP produced by StyI, SphI, RsaI, HaeIII, StuI and HapII restriction endonucleases. We detected a total of 27 haplotypes in the Caucasian and Japanese population. Of these 27 haplotypes, 18 haplotypes were present in the Caucasian population and the most common of these was haplotype 1 (ABHcde) in both mutant and normal alleles. In contrast, in the Japanese population we found 20 of the 27 haplotypes and the most common in mutant and normal alleles was haplotype 2 (abhcDE). Within these two populations a parent in the MPS IVA family has an average probability of greater than 77% (in the Caucasian population 77.27% and in the Japanese population 78.26%) of being heterozygous, and hence informative for linkage, at one or more GALNS RFLP sites. Our results delineate the molecular heterogeneity of MPS IVA haplotypes, as well as their significant interpopulation variation, and make prenatal diagnosis and carrier detection possible in the majority of families with one affected child.

Mucopolysaccharidosis IVA: submicroscopic deletion of 16q24.3 and a novel R386C mutation of N-acetylgalactosamine-6-sulfate sulfatase gene in a classical Morquio disease.

The N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene, which is responsible for autosomal recessive mucopolysaccharidosis IVA (MPSIVA), has been assigned to the long arm of chromosome 16, subregion 24.3, an area where the adenine phosophoribosyltransferase (APRT) gene and renal dipeptidase (DPEP I) gene are also localized. Molecular genetic studies on a severely affected patient with MPSIVA (Morquio disease), without karyotypic abnormality, revealed a partial submicroscopic deletion of 16q24.3 and a single point mutation on the other allele, with no functional GALNS activity. The patient, his mother, and siblings were hemizygous for GALNS and APRT loci, evidenced by informative RFLP and gene dosage analyses combined with a fluorescence in situ hybridization, utilizing a partial genomic clone of GALNS, but heterozygosity was retained at the DPEP I locus and proximal D16S7. Haplotyping of the family members revealed recombinational events between DPEP I locus and three other polymorphic loci on the paternal chromosome, localizing GALNS gene on the proximal side to DPEP I gene. As estimated from the genetic distance between two flanking markers of proximal D16S7 and distal DPEP I locus, size of the deletion was less than 3Mb. Mother of the boy and two older siblings were asymptomatic, despite this interstitial deletion of the Giemsa-light G band. The remaining paternal allele had no gene rearrangement but GALNS activity was not encoded as Arginine at 386 was replaced with Cysteine (R386C), suggesting this alteration accounts for the severe phenotype. Allelic loss of APRT is frequently observed in cancer tissues, thereby suggesting that the tumor suppressor gene locates near the APRT locus. No family member has evidence of any malignant disease. This study is apparently the first documentation of interstitial deletion of 16q24.3, involving GALNS and APRT genes.

Mucopolysaccharidosis IVA: identification of a common missense mutation I113F in the N-Acetylgalactosamine-6-sulfate sulfatase gene.

Mucopolysaccharidosis IVA is an autosomal recessive lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase. The recent isolation and characterization of cDNA and genomic sequences encoding GALNS has facilitated identification of the molecular lesions that cause MPS IVA. We identified a common missense mutation among Caucasian MPS IVA patients. The mutation was originally detected by SSCP, and successive sequencing revealed an A-->T transversion at nt 393. This substitution altered the isoleucine at position 113 to phenylalanine (I113F) in the 622 amino acid GALNS protein and was associated with a severe phenotype in a homozygote. Compound heterozygotes with one I113F-allele mutation have a wide range of clinical phenotypes. Transfection experiments in GALNS-deficient fibroblasts revealed that the mutation drastically reduces the enzyme activity of GALNS. Allele-specific oligonucleotide or SSCP analysis indicated that this mutation accounted for 22.5% (9/40) of unrelated MPS IVA chromosomes from 23 Caucasian patients, including 6 consanguineous cases. Of interest, the I1e 113-->Phe substitution occurred in only Caucasian MPS IVA patients and in none of the GALNS alleles of 20 Japanese patients. These findings identify a frequent missense mutation among MPS IVA patients of Caucasian ancestry, that results in severe MPS IVA when homoallelic, and will facilitate molecular diagnosis of most such patients and identification of heterozygous carriers. In addition to this common mutation, 10 different point mutations and 2 small deletions were detected, suggesting allelic heterogeneity in GALNS gene.

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 non-carrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.

Mucopolysaccharidosis IVA: screening and identification of mutations of the N-acetylgalactosamine-6-sulfate sulfatase gene.

Mutations causing mucopolysaccharidosis IVA in 15 Japanese and one Caucasian patient were characterized. To screen these mutations, we used a combination of single strand conformation polymorphism analysis and heteroduplex analysis for PCR products of targeted cDNA or genomic DNA. Various small mutations were identified in 23 of 26 alleles, while the other six alleles had large rearrangements. Cycle sequencing of PCR products revealed 15 different mutations, including 12 missense, one nonsense, one frame shift (2 bp deletion) and one splice site mutation, in accord with the broad range of clinical phenotypes. Two alleles have different mutations in the same nucleotide position of exon 3 (R94C, CGC-->TGC; R94G, CGC-->GGC), diagnosed by sequencing and by allelic-specific oligohybridization (ASO). One allele had two amino acid changes, E450V in exon 12 and V488M in exon 13, thereby indicating a double point mutation. All 16 mutations reported were confirmed by restriction enzyme assay or by allelic-specific oligohybridization. Transfection of mutagenized cDNAs into patients' fibroblasts showed that all mutations caused completely deficient or markedly decreased N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity, thereby indicating that these mutations were responsible for the enzyme deficiency.