Gentamicin-mediated suppression of Hurler syndrome stop mutations restores a low level of alpha-L-iduronidase activity and reduces lysosomal glycosaminoglycan accumulation

Abstract

Hurler syndrome is the most severe form of a lysosomal storage disease caused by loss of the enzyme alpha-L-iduronidase (encoded by the IDUA gene), which participates in the degradation of glycosaminoglycans (GAGs) within the lysosome. In some populations, premature stop mutations represent roughly two-thirds of the mutations that cause Hurler syndrome. In this study we investigated whether the aminoglycoside gentamicin can suppress stop mutations within the IDUA gene. We found that a Hurler syndrome fibroblast cell line heterozygous for the IDUA stop mutations Q70X and W402X showed a significant increase in alpha-L-iduronidase activity when cultured in the presence of gentamicin, resulting in the restoration of 2.8% of normal alpha-L-iduronidase activity. Determination of alpha-L-iduronidase protein levels by an immunoquantification assay indicated that gentamicin treatment produced a similar increase in alpha-L-iduronidase protein in Hurler cells. Both the alpha-L-iduronidase activity and protein level resulting from this treatment have previously been correlated with mild Hurler phenotypes. Although Hurler fibroblasts contain a much higher level of GAGs than normal, we found that gentamicin treatment reduced GAG accumulation in Hurler cells to a normal level. We also found that a reduced GAG level could be sustained for at least 2 days after gentamicin treatment was discontinued. The reduction in the GAG level was also reflected in a marked reduction in lysosomal vacuolation. Taken together, these results suggest that the suppression of premature stop mutations may provide an effective treatment for Hurler syndrome patients with premature stop mutations in the IDUA gene.

Effect of Readthrough Treatment in Fibroblasts of Patients Affected by Lysosomal Diseases Caused by Premature Termination Codons

“…In the last few years, other potential strategies such as pharmacological chaperones [8], or the suppression of pathogenic nonsense mutations through the induction of translational readthrough have emerged [2,[9][10][11][12][13][14][15][16][17][18]. To this effect, aminoglycoside antibiotics such as gentamicin [16,17], and small molecules such as PTC124 [18] and NB84 [19], have been described to induce PTC readthrough, eluding the NMD mechanism and allowing the formation of stable mRNAs encoding for full-length mutant, but probably still quite active, proteins [20,21]. These products reduce proofreading of codon-anticodon recognition in the ribosome, allowing the suppression of PTCs and, as a general rule, the insertion of glutamine or tryptophan at premature UAG/UAA or UGA codons, respectively, occurs [17].…”

Protective Effect of Ethanolic Extract of Hordeum Vulgare Seed on Gentamicin Induced Nephrotoxicity

“…Gentamicin binds with cell wall phospholipids, obstructing the chain reactions of phosphotidyl inositol which impairs cell admired. It results by production of reactive oxygen species (ROS) 5 . The strategies aimed at ameliorating the nephrotoxicity are of clinical interest 6 .…”

Aminoglycoside-Induced Premature Stop Codon Read-Through of Mucopolysaccharidosis Type I Patient Q70X and W402X Mutations in Cultured Cells

“…Gentamicin has been investigated as a read-through agent for MPS I (Keeling et al 2001;Hein et al 2004), and this aminoglycoside has the capacity to cross the bloodbrain barrier. Aminoglycosides are 2-deoxystreptamine derivatives, which can be modified by links at the 4, 5, or 6 positions.…”

“…In a preclinical study using cultured cells, we demonstrated that gentamicin enhanced the read-through of IDUA premature stop codons and induced the synthesis of significant amounts of active a-L-iduronidase (Keeling et al 2001;Hein et al 2004). Aminoglycosides such as gentamicin can induce ribosomal read-through, by binding to the eukaryotic ribosome, causing a conformational change that induces faulty stop codon recognition (Francois et al 2005).…”