Mutations in the Catalytic Subunit of Acetylcholinesterase do not Appear Responsible for Congenital Myasthenic Syndrome Associated with End-Plate Acetylcholinesterase Deficiency

Abstract

A congenital myasthenic condition was described several years ago that is characterized by a deficiency in end-plate acetylcholinesterase (AChE); the absence of the inflammatory immune responses of myasthenia categorizes the condition as congenital (Engle, et al., 1977). Since that time several additional patients with a similar disorder have been identified. Patient blood samples were drawn for DNA extraction and isolation of lymphocytes which have been immortalized by Epstein-Barr virus transformation. To ascertain the genetic basis of the disease, we have examined the structure of the gene encoding the catalytic subunits of AChE by comparing patients and control individuals. Southern analysis revealed no differences between patient and control DNA, suggesting no major chromosomal rearrangements had occurred. PCR amplification of genomic DNA yielded clones covering exon 4 and the alternatively spliced exons 5 and 6; this region was analyzed by nuclease protection and sequencing. While allelic differences were detected, we found no differences in exonic or intronic areas which might give rise to distinctive splicing pattern differences in patients and control individuals. The ACHE gene was cloned from genomic libraries from one of the patients and a control. Identical patterns of mRNA production and species of expressed AChE were revealed when the cloned genes were transfected into COS and HEK cells. Cotransfection of the genes expressing the catalytic subunits from either patient or control with a gene expressing the tail unit from Torpedo AChE (Krejci et. al., 1991) yielded asymmetric species (18 + 14 S) which require assembly of catalytic subunits and tail unit. Thus, the catalytic subunits of AChE expressed in the congenital myasthenia syndrome appear identical in sequence, arise from similar splicing patterns and will assemble with a tail unit to form a heterologous species. These findings point to the genetic abnormality likely existing in the tail unit or in a protein responsible for assembly of the catalytic subunit with the tail subunit.