| |
August 1996
Volume 60 |
Number 8
|
| |
|
| The Hereditary
Deficiencies of Serum Cholinesterase: An Update for Anesthesiologists |
Bert N. La Du, Jr., M.D., Ph.D.
Sérgio L. Primo-Parmo, Ph.D.
Pharmacogenetic analysis to find the reason for abnormal, prolonged
responses to succinylcholine began shortly after its introduction
in the 1950s. Werner Kalow, M.D., and colleagues at the University
of Toronto found that patients with a deficiency of serum cholinesterase
-- also known as pseudocholinesterase, the nonspecific cholin-esterase,
or butyrylcholinesterase (BChE) -- reacted as if they had been
given an enormous overdose of the drug; a few of them died. It
was soon realized that if artificial ventilation with adequate
oxygenation was instituted and continued for several hours, the
drug would diffuse away from the myoneural junctions, and complete
recovery would follow.
Laboratory methods were then developed to measure the level of
serum cholinesterase and determine its degree of enzymatic inhibition
with selective compounds such as dibucaine (dibucaine number)
and fluoride (fluoride number). Unusual resistance to dibucaine
inhibition identified the atypical variant, and resistance to
inhibition by fluoride was used to detect both types of fluoride-resistant
variants. These tests made it possible to diagnose several hereditary
types of succinylcholine sensitivity by analyzing a serum sample
rather than directly exposing the person to succinylcholine. Family
studies were carried out using these new serum tests, and it became
apparent that succinylcholine sensitivity was inherited as a simple
recessive, autosomal mendelian trait. Thus, this kind of drug
sensitivity requires that affected persons have inherited abnormal
cholinesterase genes from both parents.1
It is fortunate that serum cholinesterase and the white blood
cells used as a source of genomic DNA are both readily accessible
for clinical investigations. Not only can they be used for diagnostic
tests of the individual, but for detailed family pedigree analyses
as well. Genomic DNA extracted from a patient's blood cells can
be analyzed to compare the entire 1,722-nucleotide sequence of
the region coding for the BChE enzyme. Polymerase chain reaction
(PCR) amplification of genomic DNA from a small blood sample is
enough to allow sequencing of the entire region of the BChE gene
that codes for the BChE amino acid sequence. A genetic mutation
such as a single base substitution can be identified by comparison
of the variant esterase DNA sequence with that of the usual, or
"wild type," BChE. One can then predict how a change
in DNA sequence of the variant gene would alter the amino acid
sequence of the variant enzyme.
For example, a single nucleotide difference, located within the
triplet codon for amino acid 70, is sufficient to change that
residue from aspartate to glycine in the atypical
(dibucaine-resistant) BChE. This slight structural alteration
reduces the binding affinity for choline ester substrates for
the atypical enzyme and makes it almost completely ineffective
as an esterase for hydrolyzing succinylcholine.
We can analyze the entire 1,722-nucleotide coding sequence and
thus deduce the sequence of the 574 amino acids of a person's
cholinesterase within a few days. Structural mutations can be
detected and identified easily by this technique. Of course, there
are some difficulties in using this technique. For example, finding
a new mutation is straightforward, but proving that the observed
mutation causes the altered level of enzyme or abnormal catalytic
behavior is, at times, very difficult. The mutation found might
represent an unimportant structural change but may be in linkage
with another, still-undetected mutation that really causes the
trouble. Transfection and expression of the variant enzyme in
tissue culture cells is one way to be sure that the mutation in
question produces a cholinesterase with the expected variant characteristics.
Not all of the mutations affecting BChE will occur in the DNA
of the coding region; some of the mutations causing low activity,
or even abnormally high esterase activity, may be located in the
gene's regulatory region, which determines the rate of protein
synthesis. Several candidates for future study of such mutations
have been collected based on no findings of abnormalities in the
coding region of the gene.
Current Status of BChE Variants
At present, we know the unique molecular basis responsible for
at least 25 different types of BChE deficiencies [Table
1]. The list of defects causing no or very low levels of cholinesterase
activity (i.e., "silent" variants) has grown to 17,
and this number certainly will continue to grow. At present, about
half of the searches for the molecular basis of a silent BChE
phenotype in a new family will produce an entirely new, previously
unidentified mutation rather than rediscover a previously identified
variant.
The large number of structural variants of BChE in the human population
was not anticipated. Clinically relevant mutations due to amino
acid substitutions occur throughout the length of the enzyme molecule.
No major region of the enzyme is immune from mutational changes
that affect the stability and catalytic properties of the esterase
[Figure 1].
Diagnostic Tests Now Being Used
The standard research measurements of 1) level of BChE activity,
spectrophotometrically, with benzoylcholine, 2) dibucaine number
and 3) fluoride number with serum or plasma remain the primary
diagnostic tests to diagnose the main types of BChE deficiency.
Some commercial laboratories use kits and simpler tests with other
substrates, but these are often inadequate to do more than conclude
that the BChE activity is reduced. A few laboratories around the
country are all that are needed to perform these more informative
measurements for all the referrals.
On the basis of the activity and the dibucaine number and fluoride
number values, it can be determined whether the more elaborate
and complicated DNA analyses should be undertaken. Again, very
few worldwide centers are enough to provide this service and stock
all the required PCR amplification primers as well as provide
the amplification and sequencing facilities. Details about the
location of diagnostic laboratories, the clinically relevant BChE
variants and their ethnic and geographic distributions, and additional
basic reference information will be available soon on the World
Wide Web.
Future Research Objectives
Finding additional natural mutations of BChE and their frequencies
in different geographic areas will continue in many laboratories
around the world.
Much remains to be done in relating these natural, structural
mutations and the marked changes in function and stability they
produce. Even with the recent aid of crystallographic data on
acetylcholinesterase (AChE), a very homologous protein, it is
not clear, for example, why the fluoride-resistant BChE variants
have a reduced affinity for succinylcholine or why some of the
amino acid substitution variants produce silent phenotypes. Several
laboratories are currently investigating such changes in the BChE
molecule and are studying the properties of artificial mutants
created by site-directed mutagenesis in BChE and AChE. Comparison
of the human BChE with the serum BChE of other primates also will
be of interest. Chimpanzee BChE, for example, also has 574 amino
acid residues, and it has different residues at only six of these
positions.
Observations about the excellent health of a few people who are
homozygous for a frame-shift mutation that precludes their ever
having any active BChE must be considered by any workers who propose
that this enzyme has a definite physiological role in addition
to its ability to inactivate drug and foreign esters. Since there
is only one gene for BChE, we are obliged to conclude that the
complete absence of BChE causes no metabolic problems related
to endogenous substrates. Furthermore, a complete absence of human
BChE seems to have no detrimental effects during any stage of
development and differentiation. Nonetheless, it might contribute
to some of these events in some important if not essential way.
References:
1. Whittaker M, Beckman L, ed. Cholinesterase.
New York: Karger; 1986.
2. Lockridge O. Genetic variants of human butyrylcholinesterase
influence the metabolism of the muscle relaxant succinylcholine.
In: Kalow W, ed. Pharmacogenetics of Drug Metabolism. Oxford:
Pergamon Press; 1992:15-50.
3. Bartels CF, James K, La Du BN. DNA mutations
associated with the human butyrylcholinesterase J-variant. Am
J Hum Genet. 1992; 50:1104-1114.
4. Jensen FS, Bartels CF, La Du BN. Structural
basis of the butyrylcholinesterase H-variant segregating in two
Danish families. Pharmacogenetics. 1992; 2:234-240.
5. Greenberg CP, Primo-Parmo SL, Pantuck EJ, et
al. Prolonged response to succinylcholine: A new variant of plasma
cholinesterase that is identified as normal by traditional phenotyping
methods. Anesth Analg. 1995; 81:419-421.
6. Muratani K, Hada T, Yamamoto Y, et al. Inactivation
of the cholinesterase gene by Alu insertion: Possible mechanism
for human gene transposition. Proc Natl Acad Sci USA. 1991;
88:11315-11319.
7. Hada T, Muratani K, Ohue T, et al. A variant
serum cholinesterase and a confirmed point mutation at Gly-365
to Arg found in a patient with liver cirrhosis. Internal Medicine.
1992; 31:357-362.
8. Hidaka K, Iuchi I, Yamasaki T, et al. Identification
of two different genetic mutations associated with silent phenotypes
for human serum cholin-esterase in Japanese. Rinsho Byori.
1992; 40:535-540.
9. Maekawa M, Sudo K, Kanno T, et al. Genetic
basis of the silent phenotype of serum butyrylcholinesterase in
three compound heterozygotes. Clin Chim Acta. 1995; 235:41-57.
10. Primo-Parmo SL, Bartels CF, Wiersema B, et
al. Characterization of 12 silent alleles of the human butyrylcholinesterase
(BChE) gene. Am J Hum Genet. 1996; 58:52-64.
11. Primo-Parmo SL, Lightstone H, La Du BN. Characterization
of an unstable variant (BChE115D) of human butyrylcholinesterase.
Pharmacogenetics. 1996. In press.
Q&A
A few general comments or questions and responses follow, which
should be of interest to anesthesiologists:
These cholinesterase deficiency conditions are very rare, so
I'll probably never see them.
Although qualitative BChE variants are not frequent, at least
one quantitative variant is very common. About 22 percent
of our general population are carriers of the K-variant, which has
a point mutation that reduces the level of BChE activity by about
one-third. In contrast, about 4 percent of the population are carriers
of the atypical BChE gene, which affects the qualitative properties
of the enzyme. AU (atypical-usual) heterozygous individuals do not
usually show succinylcholine sensitivity, but AK people often do.
The AA frequency is only 1:2,500 (0.04 percent), but the AK frequency
is 0.0088 (0.88 percent); so nearly 1 percent of the population
are AK individuals. There are about 20 times as many AK people
as there are AA homozygotes.
If a person's BChE activity level, dibucaine number and fluoride
number are normal, must he or she have the usual kind of BChE?
Probably, but not necessarily. The standard measurements of cholinesterase
activity -- dibucaine number and fluoride number -- miss detecting
some of the BChE variants that cause sensitivity to succinylcholine.
One such variant requires that the testing be done with succinylcholine
itself rather than the surrogate substrate, benzoylcholine. The
point mutation causing this variant seems to have a decreased affinity
for succinylcholine but not for benzoylcholine.
Another variant difficult to detect by the standard methods is a
thermally unstable cholinesterase that can be diagnosed by freezing
and thawing the serum a couple of times or by incubating the serum
at elevated temperatures. Stressing the enzyme by either of these
conditions shows it to have a faster rate of inactivation than normal
BChE. In all, we probably are unable to identify a genetic basis
for nearly half of the reported exaggerated responses to succinylcholine.
Some of these can probably be explained by nongenetic causes such
as drug interactions. However, if the clinical observations have
been made and confirmed by monitoring the time required for the
twitch response to return to normal, it is likely that the patient
has a new variant undetected by the present methods.
Isn't it impractical to phenotype every patient who might receive
succinylcholine before surgery?
Yes. The cost of phenotyping the serum of every patient coming to
surgery would be greater than any projected benefit of having this
information. However, there is at least one exception -- those patients
who will be given succinylcholine on several occasions as a component
with repeated electroshock treatments. This becomes more important
if the treatments are to be given in locations where prolonged or
exaggerated muscular relaxation might be misinterpreted or might
be difficult to treat. The level and type of BChE are relatively
constant for each individual over many years. For these patients,
it is practical to know the type and the level of serum cholinesterase
they have so that the succinylcholine dose can be optimal for each
patient.
It is also worthwhile to phenotype the first-degree relatives of
those individuals found to have a deficiency of serum BChE. We recommend
that close relatives (i.e., parents, siblings and children) also
be tested.
Do the distinctive BChE phenotypes (i.e., atypical or fluoride
resistance) indicate which genetic mutation is responsible?
Not necessarily. All the atypical (dibucaine-resistant) cholinesterase
mutations around the world so far have been exactly the same point
mutation. Perhaps these will all be traced back to some common ancestor
that was the founder of this mutation.
However, there are at least two different point mutations that produce
fluoride resistance (Fluoride-1 and Fluoride-2). Special tests can
be used to distinguish between the two fluoride-resistant phenotypes,
but these two variants are very similar and would be confused in
most diagnostic laboratories.
Why bother to keep track of the people with succinylcholine sensitivity?
The importance of BChE in the metabolism of other drugs is not as
well-established, except for mivacurium, which was designed to be
inactivated by this enzyme. Some other drugs hydrolyzed by BChE
are aspirin, several local anesthetics, heroin, cocaine and bambuterol.2
However, the importance of BChE in the metabolic inactivation of
these and other new drug substrates depends on how unique the BChE
enzyme is when compared with the contributions from all the other
esterases, the rates of renal excretion, disposition into the tissues
and fat, protein and tissue binding, and many other determinants
of a drug's metabolic pattern and elimination.
Anesthesiologists are in a strategic position to identify and follow
up with investigations on the people with this enzymatic deficiency.
There may be good reasons for knowing who they are and getting back
to them in the future.
Bert N. La Du, Jr., M.D., Ph.D., is Emeritus
Professor of Pharmacology and Director of Research, Department of
Anesthesiology, University of Michigan Medical School, Ann Arbor,
Michigan.
E-mail the author.
Sérgio L. Primo-Parmo, Ph.D.,
is a Visiting Research Fellow, Department of Anesthesiology, University
of Michigan Medical School, Ann Arbor, Michigan.
E-mail the author.
return to top
Home >Newsletters
>August 1996Home >Test
|