Leistner, Sandra; (1996) Molecular genetics of hunter disease and the psuedodeficiency of arylsulphatase a. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Hunter disease (mucopolysaccharidosis type II or MPS II) is an X-linked recessive lysosomal storage disease resulting from a deficiency of L-iduronate 2-sulphate-sulphatase (IDS) (EC 3.1.6.13), which is involved in the catabolism of glycosaminoglycans (GAGs). The gene has been mapped to chromossome Xq28.1 and a 2.3 kb cDNA clone covering the entire coding sequence of human IDS has been isolated and sequenced. The organization of the coding sequences within the IDS gene has also been determined. The presence of a pseudogene which is highly homologous to exons 2 and 3 and introns 2, 3 and 7 in the functional gene is reported in the literature. Mutation analysis has been carried out to identify the primary genetic defect in 34 patients who had been diagnosed clinically and/or enzymically as suffering from Hunter disease. Genomic DNA was screened to detect large deletions in the IDS gene by using PCR reactions specific for the 5' and 3' ends of the gene. Complete deletion of the gene was found in three patients with the severe form of the disease. To characterize the mutations in the remaining 31 patients each exon was amplified by PCR followed by SSCP analysis and sequencing. All 9 exons from controls and each of the patients amplified successfully with one exception in which exon 4 appeared to be deleted. An SSCP change was detected in 28 out of 30 of these patients. Heterozygosity was observed in exon 3 at position +12, in patients and controls, a position at which there is a discrepancy between the 2 published sequences for the gene. This is an indication of the presence of more than one X-chromosome, duplication of part of the gene or a pseudogene. Six point mutations previously described in a number of patients of unrelated origin were identified in 9 of our patients, making these codons possible "hot spots" for mutations in the Hunter gene. 16 novel mutations were found which are predicted to be the disease-causing mutations because they cause frame-shifts, changes in the charge of an amino acid side chain or affect the splicing of mRNA. They are spread all over the gene and occur in patients with mild, intermediate and severe forms of the disease. An attempt was made to correlate genotype and phenotype in these patients. A benign deficiency (pseudodeficiency) of the lysosomal enzyme, aryl sulphatase A (ASA) (EC 3.1.6.8) towards the synthetic substrates, complicates the diagnosis of metachromatic leukodystrophy (MLD). It is due to a single base change in the 3'- untranslated region of the gene (1524+95 A[greater-than]G). This mutation (PD2) has always been reported to occur on a chromosome carrying a second mutation in the ASA gene, PD1, which abolishes an N-glycosylation site (N350S). In the present study, analysis of the two PD mutations in the ASA gene, separately and together in a compound allele, was carried out in a large group of subjects with neurological symptoms and low ASA activity, including close relatives and MLD patients and correlated with ASA activity. A strategy for genetic counselling and prenatal diagnosis in families of individuals with low ASA activity and neurological symptoms carrying a PD allele or both PD and MLD alleles has been proposed based on these results. In cases with low ASA and neurological symptoms but no indication of MLD the PD1 and PD2 mutations are detected separately. If family studies cannot reveal the phase of the 2 mutations for a heterozygous patient, then the PD test (i.e. for the allele containing both PD1 and PD2) is applied. For those families carrying both MLD and PD mutations, testing for PD1 and PD2 and the MLD mutation(s) if known can clarify the basis of the low ASA activity. When the MLD mutation(s) is not known or if it is in the same allele as the PD2 mutation, the sulphatide loading test has to be used to resolve the problem.

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