Title: Genetic and immunological analyses of patients with increased serum butyrylcholinesterase activity and its C5 variant form Akizuki S, Ohnishi A, Kotani K, Sudo K Ref: Clinical Chemistry & Laboratory Medicine, 42:991, 2004 : PubMed
Recent evidence has denied genetic abnormality as a mechanism of the C5 variant of butyrylcholinesterase (BChE) and proposed the binding of an unknown protein with the C4 component. The present study aimed to evaluate whether the coding sequences and non-translated sequences of the BChE gene at exons 1 to 4, 3q are structurally different in subjects having elevated BChE with and without the C5 variant phenotype. We also attempted to identify the unknown protein associated with the C5 variant and measured the BChE-specific activity in the C5 variant with an enzyme-linked immunosorbent assay (ELISA) using anti-BChE monoclonal antibody. We investigated five subjects, four of whom had elevated plasma BChE (three C5-positive [C5(+)] and one C5-negative [C5(-)]) and one control with a normal plasma BChE level. Direct DNA sequencing of the BChE gene revealed no relevant genetic mutations and no abnormal migrations in the genes of all five subjects. Precipitation of the patients' sera with anti-human immunoglobulin A (IgA), -IgG, -IgM, anti-human albumin antibodies had no effect on the BChE activity. The measured BChE activity in C5(+) was 30 to 54% higher than the activity calculated from BChE protein content. The present results suggest that the C5(+) phenotype is not associated with any genetic abnormality in the CHE1 locus, and BChE-specific activity is enhanced in the C5(+) variant. However, the exact nature of the unknown protein related to the C5(+) phenotype remains unclear.
Dibucaine number (DN) and fluoride number (FN) of the recombinant 330 I mutant ChE (r330 I) expressed in human kidney cells (293 cell) were compared with recombinant usual ChE (rUU), by several assay kits and substrates. All of them showed lower the values compared with rUU. However, the r330 I/rUU ratios about FN determined by several substrates were higher than that determined by propionyl thiocholoneiodide (PTCI), which was recommended by American Association for Clinical Chemistry. In conclusion, commercially available assay kits may not be suitable for the determination of L330 I.
Title: Butyrylcholinesterase genes in individuals with abnormal inhibition numbers and with trace activity: one common mutation and two novel silent genes Dey DC, Maekawa M, Sudo K, Kanno T Ref: Annals of Clinical Biochemistry, 35:302, 1998 : PubMed
A random population was screened for abnormal dibucaine and fluoride numbers (DN & FN) to find some common mutations in butyrylcholinesterase (BCHE) gene. Of 2375 unrelated individuals, 10 were found to have low DN and FN and were selected for further studies. DNA analysis of these hypocholinesterasemics revealed that seven patients were heterozygous for missense mutation at codon 330 (TTA to ATA; BCHE*330I). The frequency of BCHE*330I mutation was calculated to be at least 0.29% among the Japanese. On the other hand, two novel mutations were found in three families and two individuals including probands whose enzyme activity was very low (silent gene). Polymerase chain reaction and single stranded conformation polymorphism (PCR-SSCP) and restriction fragment length polymorphism (PCR-RFLP) were used for identification of the common and known mutation types such as BCHE*250P (ACT to CCT), BCHE*365R (GGA to CGA), and BCHE*539T (GCA to ACA; K-polymorphism), whereas PCR-SSCP was used in combination with direct DNA sequencing for new mutations like BCHE*446V (TTT to GTT) and BCHE*451X (GAA to TAA).
We have identified 12 kinds of genetic mutations of butyrylcholine esterase (BCHE) from phenotypic abnormalities, showing that BCHE activities were deficient or diminished in sera. These genetic mutations, detected by PCR-single-strand conformation polymorphism analysis and direct sequencing, consisted of one deletion (BCHE*FS4), nine missense (BCHE*24 M, *1005, *250P, *267R, *330I, *365R, *418S, *515C, *539T), and two nonsense mutations (BCHE*119STOP, *465STOP). All of the individuals deficient in serum BCHE activity were homozygous for silent genes (6 of 6). Fifty-eight percent of the individuals (31 of 53) with slightly reduced serum BCHE activity were heterozygous for silent genes. They also showed a higher frequency (47% as allele frequency) of the K-variant than the general population (17.5%). Finally, we confirmed low serum BCHE activity in 10 of 23 individuals heterozygous for silent genes.
Title: Human butyrylcholinesterase L330I mutation belongs to a fluoride-resistant gene, by expression in human fetal kidney cells Sudo K, Maekawa M, Akizuki S, Magara T, Ogasawara H, Tanaka T Ref: Biochemical & Biophysical Research Communications, 240:372, 1997 : PubMed
We noticed a Japanese male showed low serum butyrylcholinesterase (BCHE) activity on health examination. The phenotyping analysis revealed a reduced dibucaine number (DN) and an especially low fluoride number (FN), similar to an FS phenotype. A homozygous missense mutation, a T to A transversion at nucleotide 988, was identified in his BCHE gene. This mutation resulted in the replacement of leucine by isoleucine at codon 330 (L330I). DN and FN of recombinant BCHE(L330I) secreted by human fetal kidney cells were compared to recombinant wild-type(usual gene) BCHE and normal serum BCHE. These results showed this amino acid substitution of BCHE, Leu330 to Ile, really caused the abnormal DN and FN. We conclude that the BCHE L330I mutation is a fluoride-resistant gene, a Japanese type fluoride-resistant gene.
Title: Three different point mutations in the butyrylcholinesterase gene of three Japanese subjects with a silent phenotype: possible Japanese type alleles Sudo K, Maekawa M, Kanno T, Akizuki S, Magara T Ref: Clinical Biochemistry, 29:165, 1996 : PubMed
OBJECTIVE:
To investigate genetic mutations in three Japanese subjects homozygous for silent butyrylcholinesterase mutations.
METHODS AND RESULTS:
One of them was compound heterozygous for two mutations; GGA(Gly) to CGA(Arg) at codon 365 (G365R) and CAA(Gln) to TAA(Ter) at codon 119 (Q119X). The other two subjects were homozygous for different missense mutations: CGT(Arg) to TGT(Cys) at codon 515 (R515C) and G365R, respectively. Simple identification methods for all of the mutations were developed and applied for family analysis and to control individuals. Two mutations, G365R and R515C, have been reported in the Japanese population, while the nonsense mutation Q119X was discovered in the present study. Genetic heterogeneity between human populations with regard to the butyrylcholinesterase gene was suggested.
CONCLUSIONS:
Among the three mutations found in this investigation, one was novel, and none of these mutations have been reported outside Japan.
Three Japanese patients showed very low butyrylcholinesterase activity in their sera and appeared to be homozygous for silent genes for butyrylcholinesterase. From DNA analysis, all three patients were compound heterozygotes: GGA(Gly) to CGA(Arg) at codon 365 (G365R) and TTC(Phe) to TCC(Ser) at codon 418 (F418S) in patient 1, G365R and CGT(Arg) to TGT(Cys) at codon 515 (R515C) in patient 2 and ACT(Thr) to CCT(Pro) at codon 250 (T250P) and AGA(Arg) to TGA(Stop) at codon 465 (R465X) in patient 3. The K-variant, GCA(Ala) to ACA(Thr) at codon 539, was also found in patients 1 and 2. Simple identification methods for all the mutations were developed and applied to family analysis and control individuals. The mutant alleles (with silent gene and K-variant) were segregated as predicted by theory in pedigrees of patients 1 and 2. Four of the mutations, F418S, R515C, T250P and R465X, were initially discovered in Japan and genetic heterogeneity among the human population for the butyrylcholinesterase gene was suggested.
