p.R386C Arg386Cys (p.R414C Arg414Cys in primary sequence with 28 amino-acids signal peptide) Found in an homozygous state in a 50 year old woman with almost total BChE deficiency 93U/L
Butyrylcholinesterase (BChE) deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report the characterization of four BCHE mutations associated with prolonged effect of suxamethonium (amino acid numbering based on the matured enzyme): p.20delValPheGlyGlyThrValThr, p.Leu88His, p.Ile140del and p.Arg386Cys. Expression of recombinant BCHE mutants, kinetic analysis and molecular dynamics were undertaken to understand how these mutations induce BChE deficiency. Three of the mutations studied (p.20delValPheGlyGlyThrValThr, p.Ile140del and p.Arg386Cys) lead to a "silent" BChE phenotype. Recombinant BCHE expression studies for these mutants revealed BChE activity levels comparable to untransfected cells. Only the last one (hBChE-L88H) presented BChE activity in the transfected cell culture medium. This BChE mutant (p.Leu88His) is associated with a lower k(cat) value compare to the wild-type enzyme. Molecular dynamics simulations analyses suggest that a destabilization of a structure implicated in enzyme activity (-loop) can explain the modification of the kinetic parameter of the mutated protein.
BACKGROUND: Butyrylcholinesterase (BCHE) deficiency is characterized by prolonged apnea after the use of certain muscle relaxants with the genetic defect lying in the BCHE gene. METHODS: Two Chinese patients with no serum BCHE activity were studied. The BCHE genes were screened for mutations by polymerase chain reaction and direct DNA sequencing. RESULTS: Of the four mutations detected, two novel mutations were identified in the two patients, i.e., F474L, and an insertion of an adenine between nucleotide positions 395 and 396. This information was used to screen the immediate families of the patients for carrier status. CONCLUSIONS: We established the molecular basis of butyrylcholinesterase deficiency in two Chinese patients. The developed mutation detection assay provides a reliable method for identifying mutant BCHE carriers.
BACKGROUND: Measurement of plasma butyrylcholinesterase (BChE) activity and inhibitor-based phenotyping are standard methods for identifying patients who experience post-succinylcholine (SC) apnea attributable to inherited variants of the BChE enzyme. Our aim was to develop PCR-based assays for BCHE mutation detection and implement them for routine diagnostic use at a university teaching hospital. METHODS: Between 1999 and 2002, we genotyped 65 patients referred after prolonged post-SC apnea. Five BCHE gene mutations were analyzed. Competitive oligo-priming (COP)-PCR was used for flu-1, flu-2, and K-variant and direct DNA sequencing analysis for dibucaine and sil-1 mutations. Additional DNA sequencing of BCHE coding regions was provided when the five-mutation screen was negative or mutation findings were inconsistent with enzyme activity. RESULTS: Genotyping identified 52 patients with primary hypocholinesterasemia attributable to BCHE mutations, and in 44 individuals the abnormalities were detected by the five-mutation screen (detection rate, 85%). Additional sequencing studies revealed mutations in eight other patients, including five with novel mutations. The most common genotype abnormality was compound homozygous dibucaine and homozygous K-variant mutations. No simple homozygotes were found. Of the remaining 13 patients, 3 had normal BChE activity and gene, and 10 were diagnosed with hypocholinesterasemia unrelated to BCHE gene abnormalities. CONCLUSION: A five-mutation screen for investigation of post-SC apnea identified BCHE gene abnormalities for 80% of a referral population. Six new BCHE mutations were identified by sequencing studies of 16 additional patients.