Molecular studies of mucopolysaccharidosis Type II in ThailandRatana Charoenwattanasatein
( M.Sc. )
Advisor:
M.R. Jisnuson Svasti
Abstract
Mucopolysaccharidosis type II or Hunter syndrome is a rare X-linked recessive lysosomal storage disorder caused by the deficiency or absence of IDS or iduronate-2-sulfatase. The purposes of this study were to investigate patients with MPS II found in Thailand in terms of diagnosis and mutation analysis and to characterize the wild type and mutants of IDS for enzyme activity and protein structure and processing. Three Thai patients with the clinical signs of Hunter syndrome were characterized using IDS activity assay and mutation detection. Two patients had very low levels of IDS activity compared to normal. Mutation analysis was performed by DNA sequencing and Restriction fragment length polymorphism (RFLP) analysis from their genomic DNAs and cDNA. The first patient (P1) had a mutation of c.262C>T (p.R88C), which was the first case of this mutation reported in Thailand. The second patient (P2) had a mutation of c.1403G>A (pR468Q), which was previously reported in Thailand, whereas the last patient (P3) had a mutation of c.1044C>G (p.Y348X), which was a novel mutation. Moreover, recombinant expression vectors for R88C, G140R, Y348X, G224A, R468P, D148V and R468Q were constructed and transfected into COS7 cells. Y348X, G224A, R468P, D148V and R468Q had no IDS activity, whereas R88C and G140R showed the activity of 0.2% and 40% of wild type, respectively. Anti-human IDS antibody was used to study the processing of IDS. The wild type showed both of a precursor form (76 kDa) and a mature form (55 kDa), whereas the full-length mutants showed only the precursor form. Y348X showed a truncated protein at 50 kDa. These results suggested that mutations of the IDS gene had an effect on the protein processing. Using the human arylsulfatase B as a template, the three-dimensional model of IDS was constructed and the positions of the mutant residues were predicted. The changes of IDS protein from R88C, R468Q, R468P and G224A were located near the active site where as G140R and D148V were not. These results suggest that mutations of the IDS gene are diverse. The mutations may lead to altered protein structure and protein processing which result in a decrease in IDS activity and cause Hunter disease. The better understanding in the molecular mechanism may be useful to develop sensitive methods for diagnosis and treatment of disease.