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Mucopolysaccharidosis II is a lysosomal storage disorder caused by mutations in the IDS gene, including exonic alterations associated with aberrant splicing. In the present work, cell-based splicing assays were performed to study the effects of two splicing mutations in exon 3 of IDS , i.e., c.241C > T and c.257C > T, whose presence activates a cryptic splice site in exon 3 and one in exon 8, i.e., c.1122C > T that despite being a synonymous mutation is responsible for the creation of a new splice site in exon 8 leading to a transcript shorter than usual. Mutant minigene analysis and overexpression assays revealed that SRSF2 and hnRNP E1 might be involved in the use and repression of the constitutive 3′ splice site of exon 3 respectively. For the c.1122C > T the use of antisense therapy to correct the splicing defect was explored, but transfection of patient fibroblasts with antisense morpholino oligonucleotides (n = 3) and a locked nucleic acid failed to abolish the abnormal transcript; indeed, it resulted in the appearance of yet another aberrant splicing product. Interestingly, the oligonucleotides transfection in control fibroblasts led to the appearance of the aberrant transcript observed in patients' cells after treatment, which shows that the oligonucleotides are masking an important cis -acting element for 5′ splice site regulation of exon 8. These results highlight the importance of functional studies for understanding the pathogenic consequences of mis-splicing and highlight the difficulty in developing antisense therapies involving gene regions under complex splicing regulation. [ABSTRACT FROM AUTHOR]

Abstract: Background: Hunter syndrome (mucopolysaccharidosis type II) is an X-linked recessive lysosomal storage disease caused by a defect of the iduronate-2-sulfatase (IDS) gene. The result is impaired IDS enzyme function. Methods: To characterize the biochemical and molecular defects in IDS-deficient patients and their families, we measured IDS enzyme activity by fluorimetric enzyme assay and identified the IDS gene mutations in 14 unrelated Taiwanese patients with varying clinical phenotypes. In addition, haplotype analysis was also performed. Results: Three novel (IVS2+1G>C, 1055del12, and G489D) and 7 previously reported (N63K, P228L, K347E, R468Q, R468W, I485R, and 1241delAG) mutations were found. Together R468Q and R468W account for 42.8% mutations found in our patients. Haplotype analysis using IDS flanking markers DXS1113 and DXS1123 revealed that the unrelated R468Q alleles were independent in origin whereas the unrelated R468W alleles are probably of the same origin. The R468Q mutation in patient 1150 and I485R mutation in patient 710 occurred de novo in male meioses. Once the mutation in a family was identified, restriction analysis was also performed for rapid diagnosis of female carriers in 8 families. Leukocyte IDS measurement revealed significantly wide range of IDS activity in normal controls and MPS II carriers (19.2∼70.6 vs. 8.4∼26.6 nmol/h/mg cell protein). The average leukocyte IDS activity of normal controls (n =43) was 43.9±13.3 nmol/h/mg protein, whereas patients with MPS II (n =14) had <5% of mean normal IDS activity (0.9±0.6 nmol/h/mg protein), and carriers (n =13) had a mean activity of 17.5 (±5.7) nmol/h/mg protein. The mean leukocyte IDS activity in female carriers was less than a half of the normal level. Conclusion: Due to a small overlapping range of normal and carriers, the level of enzyme activity cannot be used alone for carrier detection. [Copyright &y& Elsevier]