Cloning and expression of the gene involved in Sanfilippo B syndrome (mucopolysaccharidosis III B) |
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Authors: | Weber B; Blanch L; Clements PR; Scott HS; Hopwood JJ |
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Institution: | Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, Australia. |
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Abstract: | Sanfilippo B syndrome is caused by a deficiency of alpha-N-
acetylglucosaminidase, a lysosomal enzyme involved in the degradation of
heparan sulphate. Accumulation of the substrate in lysosomes results in
degeneration of the central nervous system with progressive dementia often
combined with hyperactivity and aggressive behaviour. In order to clone the
deficient gene, we purified the enzyme from human placenta and obtained
amino acid sequence information. Alignment of one of the CNBr generated
internal peptides to sequence from the database revealed the chromosomal
location of the gene in the 5' upstream flanking region of the gene for
17-beta-hydroxysteroid-dehydrogenase at 17q21.1. The available DNA sequence
was used to clone the cDNA coding for alpha-N- acetylglucosaminidase and
analyse its gene structure. The gene is fully contained in the 5' upstream
flanking region of the gene for 17-beta- hydroxysteroid-dehydrogenase and
interrupted by five introns. The cDNA clone has a length of 2575 bp and
encodes a protein of 743 amino acids. Chinese hamster ovary cells
transfected with the cDNA construct show alpha-N-acetylglucosaminidase
activity about 17-fold over background. This will allow correction studies
with NAG deficient Sanfilippo B cell lines and facilitate the development
of enzyme replacement therapy for these patients.
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