title: E-box mutations in the RAPSN promoter region in eight cases with congenital myasthenic syndrome.
abstract: 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 -27C-->G 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 -38A-->G 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 -38A-->G 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 -38A-->G gains, and -27C-->G looses, binding affinity for different components of nuclear extracts of C2C12 myotubes. Luciferase reporter assays show that both -38A-->G and -27C-->G attenuate reporter gene expression in C2C12 myotubes, and that -27C-->G additionally attenuates reporter gene expression in MyoD- or myogenin-transfected HEK cells. The -27C-->G 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.
authors.affiliation: Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, MN 55905, USA. ohnok@mayo.edu
authors.forename: Kinji
authors.initials: K
authors.lastname: Ohno
authors.forename: Menachem
authors.initials: M
authors.lastname: Sadeh
authors.forename: Ilan
authors.initials: I
authors.lastname: Blatt
authors.forename: Joan M
authors.initials: JM
authors.lastname: Brengman
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Engel
mesh_terms: D000293:Adolescent; D000328:Adult; D000483:Alleles; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002648:Child; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D019066:Facies; D005260:Female; D017930:Genes, Reporter; D005822:Genetic Vectors; D006239:Haplotypes; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics*; D009132:Muscles / Q000378:metabolism; D009154:Mutation; D020294:Myasthenic Syndromes, Congenital / Q000235:genetics*; D010375:Pedigree; D010641:Phenotype; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D009124:Muscle Proteins; C051609:peripheral membrane protein 43K; D008156:Luciferases