title: Congenital end-plate acetylcholinesterase deficiency caused by a nonsense mutation and an A-->G splice-donor-site mutation at position +3 of the collagenlike-tail-subunit gene (COLQ): how does G at position +3 result in aberrant splicing?
abstract: Congenital end-plate acetylcholinesterase (AChE) deficiency (CEAD), the cause of a disabling myasthenic syndrome, arises from defects in the COLQ gene, which encodes the AChE triple-helical collagenlike-tail subunit that anchors catalytic subunits of AChE to the synaptic basal lamina. Here we describe a patient with CEAD with a nonsense mutation (R315X) and a splice-donor-site mutation at position +3 of intron 16 (IVS16+3A-->G) of COLQ. Because both A and G are consensus nucleotides at the +3 position of splice-donor sites, we constructed a minigene that spans exons 15-17 and harbors IVS16+3A-->G for expression in COS cells. We found that the mutation causes skipping of exon 16. The mutant splice-donor site of intron 16 harbors five discordant nucleotides (at -3, -2, +3, +4, and +6) that do not base-pair with U1 small-nuclear RNA (snRNA), the molecule responsible for splice-donor-site recognition. Versions of the minigene harboring, at either +4 or +6, nucleotides complementary to U1 snRNA restore normal splicing. Analysis of 1,801 native splice-donor sites reveals that presence of a G nucleotide at +3 is associated with preferential usage, at positions +4 to +6, of nucleotides concordant to U1 snRNA. Analysis of 11 disease-associated IVS+3A-->G mutations indicates that, on average, two of three nucleotides at positions +4 to +6 fail to base-pair, and that the nucleotide at +4 never base-pairs, with U1 snRNA. We conclude that, with G at +3, normal splicing generally depends on the concordance that residues at +4 to +6 have with U1 snRNA, but other cis-acting elements may also be important in assuring the fidelity of splicing.
authors.affiliation: Department of Neurology, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: K
authors.initials: K
authors.lastname: Ohno
authors.forename: J M
authors.initials: JM
authors.lastname: Brengman
authors.forename: K J
authors.initials: KJ
authors.lastname: Felice
authors.forename: D R
authors.initials: DR
authors.lastname: Cornblath
authors.forename: A G
authors.initials: AG
authors.lastname: Engel
mesh_terms: D000110:Acetylcholinesterase / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D019556:COS Cells; D003094:Collagen; D004252:DNA Mutational Analysis; D005091:Exons / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006801:Humans; D007438:Introns / Q000235:genetics; D008297:Male; D008875:Middle Aged; D009045:Motor Endplate / Q000201:enzymology* / Q000503:physiopathology; D009124:Muscle Proteins; D009154:Mutation; D010375:Pedigree; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012342:RNA, Small Nuclear / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D009124:Muscle Proteins; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D003094:Collagen; D000110:Acetylcholinesterase; C114027:COLQ protein, human