Human Molecular Genetics, 2003, Vol. 12, No. 7 739-748
DOI: 10.1093/hmg/ddg089
© 2003 Oxford University Press
E-box mutations in the RAPSN promoter region in eight cases with congenital myasthenic syndrome
1Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, MN 55905, USA and 2Department of Neurology, Wolfson Medical Center, Holon, Israel
Received November 12, 2002; Revised January 23, 2003; Accepted February 1, 2003
Myogenic determination factors are basic helix–loop–helix proteins that govern specification and differentiation of muscle cells, and bind to the E-box consensus sequence CANNTG in promoter regions of muscle-specific genes. No E-box mutation has been reported to date. RAPSN encodes rapsyn, a 43 kDa postsynaptic peripheral membrane protein that clusters the nicotinic acetylcholine receptor at the motor endplate. Transcriptional regulation mechanisms of RAPSN have not been studied. We here report two novel E-box mutations in the RAPSN promoter region in eight congenital myasthenic syndrome patients. Patient 1 carries -27C
G that changes an E-box at -27 to -22 from CAGCTG to GAGCTG. An allele harboring -27CG is not transcribed in patient's muscle. Patients 2–8 are of Oriental Jewish stock of Iraqi or Iranian origin with facial malformations, and harbor -38AG that changes another E-box at -40 to -35 from CAACTG to CAGCTG, which does not affect the consensus CANNTG sequence. Haplotype analysis shows that -38AG arises from a common founder. For each mutation, position +1 represents the major transcriptional start site that we determine to be 172 nucleotides upstream of the translational start site. Electrophoretic mobility shift assays reveal that -38AG gains, and -27CG looses, binding affinity for different components of nuclear extracts of C2C12 myotubes. Luciferase reporter assays show that both -38AG and -27CG attenuate reporter gene expression in C2C12 myotubes, and that -27CG additionally attenuates reporter gene expression in MyoD- or myogenin-transfected HEK cells. The -27CG mutation also markedly attenuates the enhancer activity of an E-box on an SV40 promoter. Impaired transcriptional activities of the RAPSN promoter region predict reduced rapsyn expression and endplate acetylcholine receptor deficiency.
* To whom correspondence should be addressed. Tel: +1 5072845102; Fax: +1 5072845831; Email: ohnok{at}mayo.edu
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