Clinical Chemistry 43: 924-929, 1997;
(Clinical Chemistry. 1997;43:924-929.)
© 1997 American Association for Clinical Chemistry, Inc.
Genetic mutations of butyrylcholine esterase identified from phenotypic abnormalities in Japan
Masato Maekawa1,a,
Kayoko Sudo2,
Dilip Chandra Dey3,
Jinko Ishikawa3,
Masakazu Izumi3,
Kazuo Kotani3 and
Takashi Kanno3
1
Clinical Laboratory, National Cancer Center Hospital, Tsukiji 5-chome, Chuo-ku, Tokyo, 104 Japan.
2
Department of Laboratory Medicine, Jikei University
School of Medicine, The Daisan Hospital, 4-11-1 Izumi-honcho, Komae,
201 Japan.
3
Department of Laboratory Medicine, Hamamatsu
University School of Medicine, Handa-cho 3600, Hamamatsu City, 431–31
Japan.
a Author for correspondence. Fax 81-3-3542-3815; e-mail mmaekawa{at}gan2.ncc.go.jp
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Abstract
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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, *100S, *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.
Key Words: indexing terms: PCR • single-strand conformation polymorphism • deletion mutation • missense mutation • nonsense mutation • genotype:phenotype correlation
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Introduction
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Several genetic variants of human butyrylcholine esterase (EC
3.1.1.8; BCHE) have been reported to be associated with prolonged apnea
in patients given the muscle relaxant drug
succinylcholine.1
For >30 years, serum BCHE activity, dibucaine
number (DN), and fluoride number (FN) have provided sufficient
information to identify most of the known BCHE phenotypes associated
with succinylcholine sensitivity (1). However, several
additional BCHE variants have been discovered during the last decade,
which makes phenotyping individuals very complex. The full-length BCHE
cDNA of 2.4 kb has been sequenced
(2)(3); the genomic DNA was found
to be at least 73 kb long and to contain four exons interrupted by
three introns at positions corresponding to nucleotides -93, 1433, and
1600 of the BCHE cDNA (4). Moreover, the molecular basis
of several genetic variants of BCHE have been reported, such as
atypical gene (5), fluoride-resistant gene
(6), silent gene (7), K-variant
(8), J-variant (9), and H-variant
(10). In the first report of the molecular basis of the
BCHE silent gene, a frameshift mutation at Gly-117 (GGT to GGAG) was
identified in two unrelated families (7). We previously
reported the genetic basis of the silent gene in four compound
heterozygotes and two homozygotes in Japan
(11)(12). A decrease in serum BCHE activity is
observed in hepatic disease, carcinomas, and chronic debilitating
diseases (13)(14). However, we sometimes find
reduced activity without the above diseases. In this study, we wanted
to determine the frequency of BCHE variants in individuals with
diminished activity, and the characteristics of their laboratory data.
Moreover, we investigated novel BCHE mutations and genotype:phenotype
correlation and discussed the laboratory significance of serum BCHE
activity around the lower limit of the reference range.
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Materials and Methods
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subjects
Group A.
Data regarding individuals deficient in serum
BCHE activity (near zero) were collected from various laboratories in
Japan. These individuals were definitely homozygous for silent genes as
evidenced by the trace BCHE activity in serum. The laboratories found
them by chance in routine laboratory tests. Secondary
hypocholinesterasemia due to hepatic dysfunction was ruled out from
other biochemical data (serum albumin, total cholesterol, and
2-globulin concentration were all within normal limits)
and ultrasonic imaging of the liver. The effects of organophosphorus
insecticides were also ruled out from each patient's recent history.
Thus, these subjects were definitely homozygous for the silent gene of
BCHE.
Group B.
Data regarding individuals diminished in serum
BCHE activity (below the reference range of each laboratory) were
collected from Seirei Health Care Center and other laboratories in
which BCHE activity in serum is tested routinely. Hepatic dysfunction
was also ruled out in these subjects by the criteria described above.
These subjects were therefore possibly heterozygous for silent or
related alleles reducing BCHE activity.
These studies were performed in accordance with the Helsinki
Declaration of 1975, as revised in 1983.
measurement of serum bche activity, dibucaine number and fluoride
number
Serum BCHE activity was measured by using propionylthiocholine
iodide as a substrate (Aldrich, Milwaukee, WI) (15) on a
Shimadzu CL-7200 biochemistry analyzer (Shimadzu, Kyoto, Japan) and
calculated at 37 °C. The reference range of BCHE activity was
4000–8000 U/L. The DN and FN were also determined by the same means.
DN is the percent inhibition of activity caused by 0.03 µmol/L
dibucaine and FN is the percent inhibition of activity caused by 4
µmol/L sodium fluoride (15).
pcr–single-strand conformation polymorphism (sscp) analysis and
sequencing
Genomic DNA was extracted from EDTA-treated venous blood with the
procedure of Kunkel et al. (16). PCR-SSCP and direct
sequencing procedures were performed according to our previous paper
(11). Briefly, protein-coding exons of the BCHE gene were
amplified separately as nine fragments by PCR, and each amplified
product was analyzed by SSCP with the Phast System (Pharmacia, Uppsala,
Sweden). Amplified products displaying conformational polymophisms were
directly sequenced mostly on one strand by dideoxy chain termination
with Sequenase (United States Biochemical, Cleveland, OH) or by means
of cycle sequencing (Taq dideoxy terminator cycle sequencing kit; ABI,
Foster City, CA) with an automated DNA sequencer (ABI Model 373A).
simple method for identification of point mutations
To establish a simple method for identification for analysis of
family members or suspected individuals, we developed PCR–restriction
fragment length polymorphism (RFLP) assays to detect mutations
affecting restriction enzyme recognition sites. For mutations that did
not affect convenient restriction enzyme recognition sites, we used
mismatched PCR-RFLP analysis according to our previous paper
(11). Briefly, the amplified products by mismatched PCR
were digested by restriction enzymes (Toyobo, Tsuruga, Japan) for
3 h. The digests were analyzed by electrophoresis through 2%
agarose gels stained with ethidium bromide.
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Results
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genetic mutations found in our subjects
PCR-SSCP and sequencing procedures revealed 12 kinds of genetic
mutations, including 1 deletion, 9 missense, and 2 nonsense mutations
(Table 1
). One of these, designated BCHE539T, was the K-polymorphism
(8). Five of these 12 (BCHEFS4, 24 M, 100S, 267R,
330I) were novel mutations. The formal names of genotypes were
designated as the proposed nomenclature for BCHE genetic variants
(17).
We could easily identify BCHE24M and 100S mutations by PCR-RFLP
analysis because the mutations affected the recognition sites of
NlaIII and HinfI, respectively. BCHE267R and
330I mutations did not affect recognition sites of any restriction
endonucleases, and thus we developed a mismatched PCR-RFLP assay
procedure. We could not design an appropriate method to confirm
BCHEFS4, but SSCP and sequencing confirmed this mutation. Table 2
shows primer sequences for PCR-RFLP with/without mismatched
primer and successful annealing temperature for PCR to identify the
four novel missense mutations.
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Table 2. Primer sequences and other conditions of PCR-RFLP analysis
to identify the four novel missense
mutations.
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group a: individuals homozygous for silent genes
Table 3
shows six new subjects deficient in serum BCHE activity (near
zero) and six individuals published previously
(11)(12). Of the six new subjects, five were
homozygous for BCHE365R and one was homozygous for BCHE250P. Eight of
the 12 subjects were homozygous for the same mutation. BCHE365R was
the most frequent allele in this group (62.5% of total mutant
alleles). K-polymorphism was homozygously observed in 10 of 11 examined
individuals.
group b: individuals diminished in serum bche activity
We analyzed 53 individuals diminished in serum BCHE activity, and
found 10 mutations containing five novel mutations (four missense
mutations and one deletion mutation) in 31 individuals (Table 4
). The K-polymorphism was observed in 13 of the remaining 22
individuals (seven homozygous and six heterozygous). Of the total 53
individuals, 14 were homozygous for the K-polymorphism and 21 were
heterozygous, representing a frequency of 49 of 104 alleles (47% as
allele frequency). Some individuals showed slightly diminished DN and
FN. All of these subjects had the same mutation, TTA(Leu) to ATA(Ile)
at codon 330, as determined by DNA analysis.
bche activity and albumin:bche ratio in individuals heterozygous
for the silent and other related genes
We collected data regarding individuals heterozygous for silent
and other related genes reducing serum BCHE activity. For this purpose,
we excluded the individuals initially found by diminished BCHE
activity. Therefore, the target heterozygous individuals were family
members detected by family analysis of individuals homozygous for
silent genes. Fig. 1
shows BCHE activity and albumin:BCHE ratio. Ten of 23
individuals showed BCHE activity below the lower limit of the reference
range, and 11 individuals had albumin:BCHE ratios higher than the upper
limit of the reference range. BCHE activity in heterozygous individuals
was significantly lower than the normal control (P <0.01),
and the albumin:BCHE ratio was significantly higher (P
<0.01) by Student's t-test.
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Figure 1. BCHE activity and albumin:BCHE ratio in individuals
heterozygous for silent and other related genes reducing serum BCHE
activity.
We collected 23 heterozygous individuals detected by family analysis of
the homozygous individuals for silent genes and plotted BCHE activities
and albumin:BCHE ratios (heterozygote). Dots indicate each
individual's value, and vertical and horizontal
bars are means and ranges of mean ± 2SD. Ranges of normal
controls are shown as ovals (mean ± 2SD) and mean is
indicated as vertical bar.
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Discussion
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We use serum BCHE activity as an indicator of hepatic function.
However, serum BCHE activity is distributed over a wide range even in
healthy control subjects, which might be due to variability of genetic
control of the BCHE gene. Numerous mutations have been identified as
being responsible for silent genes (14)(18).
We analyzed individuals deficient or diminished in serum BCHE activity,
and found 11 kinds of putative silent genes from 62 mutant alleles.
Only one of these was a deletion mutation, and the others were all
single-base substitutions. The deletion mutation occurred in the
sequence consisting of two direct repeats; only one copy of the repeat
was retained in the mutant gene. Deletion of short direct repeats could
result from the slippage-mediated frame shifts during DNA replication
synthesis (19). Two of 10 mutations, BCHE24 M and 515C,
occurred at the CpG dinucleotides, well known as mutational hotspots
(20). Six of the 11 single-base substitutions, including
K-polymorphism, BCHE539T, created or interrupted a known restriction
site and could be easily identified by PCR and restriction enzyme
digestion. We developed a mismatched PCR method to detect the remaining
five single-base substitutions that did not alter any known restriction
sites. Therefore, these 11 single-base substitutions can now be easily
identified.
The most frequent mutation, BCHE365R, was found in all of the five
prefectures analyzed (from north to south, Akita, Tokyo, Shizuoka,
Hyogo, and Fukuoka), and four other mutations (BCHE119STOP, 250P,
330I, and 515C) were found in two or more different prefectures. In
Japan, there have been other reports of silent alleles in addition to
our previous reports (11)(12). The silent
genes are BCHE365R (21)(22), an
Alu insertion (23), and a frameshift mutation
at codon 315 (21)(24). In the US, 12 kinds of
silent genes, BCHEFS6 (one base deletion in codon 6), 33C, 37S,
125F, 170E, 198G, 201T, 271STOP, 471R, 500STOP, 518L, and
12E3–8G (altered splicing at the acceptor site of intron 2), have
been reported by La Du's laboratory (18). The 11 silent
genes we found and two other silent genes discovered in Japan are
different from the mutations described by Nogueira et al.
(7) and Primo-Parmo et al. (18), discovered
in the US. Therefore, there appear to be distinct ethnic differences in
genetic mutations of human BCHE genes. It would thus be of interest to
characterize the genetic mutations of BCHE genes from other ethnic
groups. Our PCR-SSCP sequencing procedure and (or) simple
identification method are convenient to screen for genetic mutations
among suspected individuals.
The K-polymorphism was characterized as one of the quantitative
variants (potentially causing low BCHE activity in serum) by Rubinstein
et al. (25). It was associated with a transition from
guanine to adenine at nucleotide 1615, which was associated with an
amino acid change from alanine 539 to threonine (8). Among
the 106 alleles of 53 individuals in group B, the frequency for the
K-polymorphism was 0.47. The frequency was significantly higher than
that in a randomly selected healthy population studied previously
(0.175) (26) (P <0.01 by 
2).
This seems to be because the silent genes are somewhat linked to the
K-polymorphism.
Six individuals homozygous and three heterozygous for BCHE365R (15
alleles) were homozygous for the K-polymorphism. Nine individuals
heterozygous for BCHE365R (nine alleles) were heterozygous for the
K-polymorphism. The BCHE365R, therefore, was strongly linked to
the K-polymorphism (15 alleles were definitely linked and nine were
possibly linked). BCHE119STOP and 418S mutations were also found to
be linked to the K-polymorphism. Our results do not suggest
associations between any other mutations and the K-polymorphism. The
atypical variant mutation has been reported to be linked to the
K-polymorphism in European and American populations
(5)(8)(25). However, in screening
for atypical genes in Japan, the atypical variant has been found to be
very rare (0 of 266 hospital patients, Uchiyama et al.
[27]; 0 of 1096 hospital patients, Tanaka et al.
(28); 1 of 1945 subjects in a population survey, Iuchi
(29)). Therefore, in the development of the BCHE gene, it
seems possible that the K-polymorphism occurs first, and the point
mutation in the common atypical gene, BCHE70G (5), or the
silent gene, BCHE365R, occurs later in each one pedigree possessing
the K-polymorphism.
By accumulation of data regarding individuals heterozygous for the
silent gene, we could determine the effects of this mutation on serum
BCHE activity. In hepatic diseases and other debilitating diseases,
serum BCHE activity decreases, paralleled by reduced concentrations of
serum albumin, because both BCHE and albumin are produced in the liver.
We therefore plotted the albumin:BCHE ratio as well as BCHE activity.
BCHE activity and the ratio were significantly different in individuals
heterozygous for silent genes and healthy controls. About half of the
individuals heterozygous for the silent gene showed low serum BCHE
activity and higher albumin:BCHE ratio in their healthy state. We
should know that the low BCHE activity state originating from genetic
abnormalities is not rare in routine laboratory data, when determining
serum BCHE activity exactly.
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Acknowledgments
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We thank T. Usuda in Seirei Health Care Center for help in
collecting samples, and some doctors for supplying samples from various
regions in Japan. This work was supported in part by Grants-in-Aid for
Scientific Research from the Ministry of Education, Japan (nos.
05454583, 05671920).
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Footnotes
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1 Nonstandard abbreviations: BCHE, butyrylcholine esterase; DN, dibucaine number; FN, fluoride number; SSCP, single-strand conformation polymorphism; and RFLP, restriction fragment length polymorphism. 
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Abstract
Introduction
