A novel approach to identify
Duchenne muscular dystrophy patients for aminoglycoside antibiotics therapy
Shigemi Kimura, Kaori Ito, Toshihiko
Miyagi, Takashi Hiranuma, Kowasi Yoshioka, Shirou Ozasa, Makoto Matsukura,
Makoto Ikezawa, Masafumi Matsuo,Yasuhiro Takeshima, Teruhisa Miike -
Japan
Abstract
:
Aminoglycoside antibiotics have been found to
suppress nonsense mutations located in the defective dystophin gene in mdx
mice, suggesting a possible treatment for Duchenne muscular dystrophy (DMD).
However, it is very difficult to find patients that are applicable for this
therapy, because: (1) only 5-13% of DMD patients have nonsense mutations in the
dystrophin gene, (2) it is challenging to find nonsense mutations in the gene
because dystrophin cDNA is very long (14 kb), and (3) the efficiency of
aminoglycoside-induced read-through is dependent on the kind of nonsense
mutation. In order to develop a system for identifying candidates that qualify
for aminoglycoside therapy, fibroblasts from nine DMD patients with nonsense
mutation of dystrophin gene were isolated, induced to differentiate to myogenic
lineage by AdMyoD, and exposed with gentamicin. The dystrophin expression in
gentamicin-exposed myotubes was monitored by in vitro dystrophin staining and
western blotting analysis. The results showed that gentamicin was able to
induce dystrophin expression in the differentiated myotubes by the read-through
of the nonsense mutation TGA in the gene; a read-through of the nonsense
mutations TAA and TAG did not occur and consequently did not lead to dystrophin
expression. Therefore, it is speculated that the aminoglycoside treatment is
far more effective for DMD patients that have nonsense mutation TGA than for
patients that have nonsense mutation TAA and TAG. In this study, we introduce
an easy system to identify patients for this therapy and report for the first
time, that dystrophin expression was detected in myotubes of DMD patients using
gentamicin.