Background: In Taiwan, DNA-based newborn screening showed a surprisingly high incidence (1/875 in males and 1/399 in females) of a cardiac fabry mutation (IVS4 + 919G >A). The common cardiac variant fabry mutation, IVS4+919G >A, affects the splicing of GLA RNA by introducing a 57-nucleotide insertion between exons 4 and 5 that contains a stop codon and leads to a truncated protein and inactive enzyme. And this mutation affected males have up to 10% residual enzyme activity and present clinically with late-onset hypertrophic cardiomyopathy. Due to the high cost of enzyme replacement therapy and the large number of patients with this mutation, the development of alternative therapies is essential. Several low-molecular-mass compounds, such as histone deacetylase inhibitors or kinase/phosphatase inhibitors, have been identified as modulators of alternative splicing. It may offer a potential alternative to enzyme replacement therapy. We expect to find out a more economic and effective drug by the detailed study of the mechanism of the small molecule modulators on the IVS4+919G >A mutation for the greater benefits of patients with this mutation.
Methods: In this study, we used to generate Epstein-Barr virus-transformed lymphoblast cell lines and incubated with different concentrations of three HDIs (sodium butyrate, valproic acid, and trichostatin A) and Amiloride hydrochloride (Amiloride HCl). To identify the respond of these compound, we were monitored the relative amounts of normal and aberrant splice forms by quantitative real-time polymerase chain reaction, the relative amounts of the normal and truncated α-Gal A protein products were analyzed by Western blotting and enzyme activities.
Results: Western blotting revealed those females heterozygous for the IVS4+919G >A mutation had approximately 50% of the normal level of α-Gal A protein, whereas hemizygous males had approximately 10% of the normal level. The three HDIs were all found to rescue the aberrant RNA splicing and to increase the amount of normal α-Gal A protein. And amiloride HCl increased the splicing ratio (2.5 fold) and enzyme activity (2.2 fold) of α-Gal A in the lymphoblasts with IVS4+919G >A mutation.
Conclusions: Our results provide proof-of-concept that aberrant RNA splicing caused by the cardiac variant fabry mutation, IVS4+919G >A, can be rescued by HDIs. The use of HDIs may become a viable therapeutic strategy for patients with this highly prevalent mutation in the Han population.
