An approach to therapy for ataxia-telangiectasia: Aminoglycoside-induced readthrough of premature termination codons in the ATM gene

Ataxia-Telangiectasia (A-T), an autosomal recessive disease, is characterized by lack of functional Ataxia-Telangiectasia mutated (ATM) protein in cells. A-T patients are disabled due to gradual neurodegeneration. Studies in our laboratory, performed over the past eight years, showed that about 14% of A-T mutations result in primary premature termination codons (PTCs). My research on aminoglycosides was prompted by the observation that A-T patients with some detectable ATM protein (e.g. <10% of normal levels) have a slower and milder progression of A-T symptoms. Furthermore, obligate heterozygotes (i.e. A-T parents) are generally healthy despite having only 40--50% of normal A-T protein levels. Thus, aminoglycoside-induced expression of ATM protein in A-T cells may provide a potential treatment for some A-T patients. This approach may also be applicable toward other genetic diseases that carry PTC mutations (approximately 20% for most genetic diseases).; In recent years we have improved the established protocols for diagnosing A-T and subsequent mutation analysis. Previously whole cell lysates were used to detect ATM protein levels in A-T patients. We improved the sensitivity of ATM western analysis 3-fold by using nuclear extracts for immunoblotting. Following A-T patient diagnosis, we also demonstrated that simple short tandem repeat (STR) haplotyping could identify ancestral mutations.; Aminoglycosides, a group of widely used antibiotics, can induce and promote readthrough of termination codons (UAG, UGA and UAA). I have studied some of the most commonly used aminoglycosides to establish their ability to induce translational readthrough PTC mutations within the ATM gene. A protein truncation test (PTT) was modified in order to improve the sensitivity of ATM protein detection. Plasmids containing different PTCs were constructed from different homozygous A-T patients to provide template for PTT. Aminoglycoside-induced readthrough resulted in the production of relatively large amounts of full-length ATM PTT protein (i.e. up to 800-fold increases over controls).; Subsequently, aminoglycoside-induced ATM expression was assessed in vitro by immunoprecipitation of nuclear extracts from aminoglycoside-treated LCLs from the same patients (i.e. the same PTC mutations). At comparable concentrations, geneticin showed the highest readthrough expression.; We next established that the readthrough ATM protein was functional, using colony survival assay (CSA), radioresistant DNA Synthesis (RDS) and flow cytometry. As controls, beta-actin and SMC1 protein expression levels were tested in A-T and normal cells and found not to be affected by geneticin treatment. In summary, our data showed evidence for geneticin-induced readthrough of ATM expression in A-T patients with PTC mutations, and this ATM protein appears to decrease the cellular phenotypes of A-T cells.