The enzyme butyrylcholinesterase (BCHE, 3q26.1–3q26.2, OMIM 177400) hydrolyzes drugs containing ester bonds such as neuromuscular blocking drugs (succinylcholine and mivacurium), local anesthetics (procaine, chloroprocaine, and cocaine), and heroin.1,2 Although mivacurium is no longer commercially available in the United States, it is still widely used in Europe.3 Succinylcholine is rapidly hydrolyzed by BCHE, which accounts for its short duration of action.4 Mutations in the BCHE gene affect the function of this enzyme leading to prolonged neuromuscular blockade.5,6 Besides the normal (usual, U) phenotype, the most frequent variations of BCHE are the atypical (A) and Kalow (K) variants, which describe the nucleotide substitution of A with G at position 209 (A209G) and of G with A at position 1615 (G1615A), respectively. This leads to an amino acid change from aspartic acid to glycine at codon 70 and from alanine to threonine at codon 539, respectively. Both variants have been associated with a prolongation of muscle relaxation after administration of succinylcholine and mivacurium.7–9
In Caucasian populations, the allele frequency of the A-variant is about 0.02, with a homozygous incidence of 3 in 1000 individuals,10 whereas the allele frequency of the K-variant varies between 0.12 and 0.27, with a homozygous incidence of 1 in approximately 63 individuals.6,11–14 Genetic investigations have provided evidence that the K-variant alone causes moderate prolongation of the action of mivacurium; however, the effect is more pronounced in patients carrying both A- and K-variants.15 This result was in agreement with previous findings of linkage disequilibrium between A- and K-variants in which the K-variant was found in 89%–96% of A-variant carriers.6,9,16
An unmistakable distinction among the four genotypic combinations of A- and K-variants has, until now, only been possible by molecular genetic methods but not by means of enzyme activities and dibucaine inhibition.6,17,18 In this regard, several molecular genetic methods have been introduced such as direct sequencing and a number of methods based on polymerase chain reaction (PCR) combined with restriction enzymes19–21 and fluorescent hybridization probes.7,16,22 Genotyping of BCHE by automated sequencing is the most popular method.6,23 However, the relatively high cost and time-consuming nature of this and other methods has led to searches for an alternative genetic method. In the present experiments, we explored the use of denaturing high-performance liquid chromatography (dHPLC), which has been successfully used for detection of nucleotide changes in a wide range of genetic diseases24–27 and has been shown to be one of the most cost-effective methods.28 The dHPLC method has the following clear advantages(i): an unmistakable distinction between normal samples (wild type) and mutation carries,(ii) fully automated analysis, and(iii) no need to use restriction enzymes or labeled oligonucleotides.
In the present study, we established and validated a rapid, highly sensitive, accurate and inexpensive detection method for the nucleotide changes responsible for the A- and K-variants in the BCHE gene. We also discuss the application potential for the fast scanning of A- and K-carriers in clinical studies in which neuromuscular blocking drugs are investigated and these variants play an important role.
Forty-six individuals were included in the study. Sixteen individuals were previously identified as carriers of A- and K-variants (n = 15) or the wild type BCHE sequence (n = 1) by automated sequencing as described below. Their written informed consent for molecular genetic analysis was obtained. The former were either patients referred to our center because of a prolonged duration of action of succinylcholine or relatives of known variant carriers. A random sample of 30 anonymous blood donors from the general population was chosen as test subjects. The study was approved by the regional ethics committee (Ethikkommission beider Basel).
DNA Isolation and PCR Amplification
Genomic DNA was isolated from 200 μL EDTA-anticoagulated blood using QIAamp Blood mini kit (Qiagen GmbH, Hilden, Germany). For the PCR amplification, we designed two primer pairs specific for exons 2 and 4, where the nucleotide changes for the A- and K-variants are localized. The primer pair for exon 2 (5′-TCTTTTGCTCTGCATGCTTATTG-3′ and 5′-CTTTCAACCCGAGCCAGAAA-3′) amplified a fragment of 460 base pairs. Whereas the primer pair for exon 4 (5′-CTGTGTAGTTAGAGAAAATGGCTTT-3′ and 5′-AGGAAAGAAAGAAATTGAACCAG-3′) amplified a 500 base pairs fragment. The amplification reactions of 25 μL contained approximately 100 ng DNA, 0.4 μM of each primer, and 1 × Mastermix (Eppendorf AG, Hamburg, Germany). The PCR was initialized with a denaturing step at 95°C for 5 min, followed by 35 cycles of 30 s at 94°C, 30 s at 56°C, and 45 s at 72°C, and finished with a final extension step at 72°C for 5 min.
One subject with a wild type BCHE sequence and two carrying A- and K-variants in the heterozygous state were used to establish the dHPLC method, which was then validated on the other 13 mutation carriers and the 30 test subjects. All tested samples were anonymized and blinded analyses were performed, disclosing sequencing results only after completion of dHPLC analysis.
The PCR fragments were directly loaded without previous enzyme digestion or denaturation and then analyzed by dHPLC on a Wave-MD Advantage System equipped with a DNA separation column and an ultraviolet detector (Transgenomic, Omaha, NE). The column temperature, a key variable for an accurate detection of DNA variations, was predicted using the Wavemaker software version 4.1, which is based on the melting properties of the DNA fragment. Heterozygous fragments with one or more internal mismatches are less thermostable than their corresponding homozygous fragments. This reduced thermostability leads to a shorter column retention time, earlier elution from the separation column and earlier detection. Choosing the appropriate column temperature facilitates the distinction of heterozygous from homozygous fragments. Amplification of samples carrying homozygous sequences, either wild type or homozygous mutated alleles, will only produce homozygous fragments. In contrast, samples carrying heterozygous alleles will produce a mixture of PCR products, which include both homozygous and heterozygous fragments. As a result, samples carrying heterozygous alleles will show additional peaks besides the homozygous peak in their elution profiles. To detect a homozygous mutated sample, the sample must be brought into a heterozygous state. For this purpose, we mixed the homozygous mutant with its corresponding wild type sample before PCR amplification. In this way, the homozygous mutated sample is expected to show a heterozygous elution profile in the dHPLC run. The mixing was performed 1:1 on the basis of concentration. A positive (A- or K-variant carrier) and negative control (wild type) sample was included in every dHPLC run in order to clearly distinguish elution profiles.
Amplified PCR fragments of exons 2 and 4 were purified before they were sequenced in both directions using PCR primers. The sequencing reactions were prepared using BigDye® Terminator Cycle Sequencing kit according to the protocol of the manufacturer (Applied Biosystems, Foster City, CA). The samples were purified and loaded on an ABI Prism 3100 Avant Genetic Analyzer with 16 capillaries (Applied Biosystems). The sequence analysis for nucleotide changes was performed using Staden Package v1.5.3 available through the website http://staden.sourceforge.net/.
Pearson's χ2 test statistic was used to compare categorical data and to calculate 95% confidence intervals. The software R version 2.5.0 (http://www.R-project.org) was used for statistical analysis.
Set-up of dHPLC Conditions
dHPLC protocols were developed and optimized to detect the two most common mutations of BCHE, A209G for the A-variant and G1615A for the K-variant. For each variant, chromatographic profiles of heterozygotes and homozygotes were compared at two different temperatures, 56.4°C and 58.4°C for the A-variant, and 53°C and 55°C for the K-variant. The optimal temperature, which significantly distinguished elution profiles of heterozygous from homozygous samples, was 58.4°C and 55°C for the A- and K-variant, respectively (Figs. 1 and 2). Besides the G1615A mutation, the PCR fragment of exon 4 carried a known nucleotide variation (A1914G) in the untranslated region of the 3′end. At 55°C, the A1914G variation alone or in combination with the K-variant was also clearly detectable and easily differentiated (Figs. 2B–C). As expected, the mixed homozygous/wild type mutant generated chromatographic profiles identical to the natural heterozygous sample (Figs. 1B and 2C).
Evaluation of the dHPLC
Validation of the dHPLC method was performed with 43 samples corresponding to 86 alleles. In these we found 100% concordance between dHPLC and DNA sequencing results leading to an accuracy of 1.0 (95% confidence interval, 0.95–1.0, P < 0.0001) for the A- and K-variants. A summary of the identified A- and K-variants is given in Table 1. In a first step, all 13 samples of individuals with known variants were amplified by PCR and analyzed using the optimized dHPLC conditions, obtaining three and eight heterozygous profiles for A- and K-variants, respectively. After the second dHPLC analysis, we properly recognized all homozygous carriers, which were seven and four for the A- and K-variants, respectively. The A1914G polymorphism in either homozygous or heterozygous form was present in all K-variant carriers. Whereas the homozygous K-variants always carried the polymorphism in homozygous form, the heterozygous K-variants presented the polymorphism in either heterozygous or homozygous forms. Application of the dHPLC protocols was performed on the group of 30 test subjects. These were first analyzed by dHPLC, as described above, and then by automated sequencing. After comparing the dHPLC output with the sequencing results, we correctly distinguished all heterozygous and homozygous samples using the established protocols. In the test group, the allele frequencies of A- and K-variants were 0.017 and 0.25, respectively.
In addition to the A-variant, one patient carried the G344A mutation, which leads to an amino acid change from glycine to aspartate at codon 115 (Gly115Asp, S-variant). This nucleotide change was observed as an aberrant elution profile with two additional peaks, which was easily discriminated from the elution profile of the A-variant and wild type (Fig. 1C). Results from the automated sequencing corroborated this finding.
Knowledge of the genetic status of the two BCHE variants is clinically important, as individuals who are either homozygous for the A-variant or carry a combination of the A- and the K-variants are more sensitive to the neuromuscular blocking drugs succinylcholine and mivacurium.15,17,29 According to our results, the presented dHPLC protocols accurately and rapidly identify the two most frequent variants of the BCHE gene, the A- and K-variant, in their heterozygous and homozygous state. In addition, allele frequencies for A- and K-alleles in the Swiss population, as calculated in this study, were similar to frequencies previously reported.10,12
The high sensitivity, accuracy, and automated analysis of dHPLC favors this method for mutation screening studies.27 The cost of the equipment and the time spent for the development of the dHPLC protocols are the main drawbacks of this approach. However, once protocols are established and validated, a large group of samples can be rapidly and efficiently analyzed for these two common variants. For those samples presenting normal elution profiles, a second PCR amplification and dHPLC run is necessary for the identification of homozygous variants. However, no additional handling steps are needed, diminishing the risk of post-PCR contamination. In the presence of additional nucleotide variations, the analysis of the profiles can be complex. Therefore, a wild type sample and a control sample carrying the heterozygous form of the A- or K-variant should be run together with the unknown samples. This will facilitate the recognition of typical elution profiles and help to differentiate them from other variations. Although the protocol was optimized for the A- and K-variants, other nucleotide variations with similar elution temperatures can be identified including nucleotide changes A1914G and G344A (S-variant). Unique elution profiles were generated by the presence of these additional variations. As the sensitivity of the method is temperature dependent, other unknown rare variants located in the same PCR fragment may not be visible. The inclusion of a control sample is also recommended since the elution profiles vary slightly from run to run. This is most often due to the buffer concentration slightly affecting the conditions of the system. Difficulties in interpreting elution profiles could result from weak detection signals. Thus, an appropriate amount of amplified fragments has to be loaded into dHPLC system. The presented protocols are rapid, as the direct loading of unpurified PCR fragments into the dHPLC system allows for a reduction in handling time. The time needed to run the dHPLC protocol and analyze the samples is very short, averaging <10 min per sample.
Biochemical assays are not able to unequivocally identify heterozygous and homozygous A- and K-variants alone or in combination,18,23 and DNA sequencing of the entire coding region is still the most accurate method to genotype the BCHE of an individual patient. Although DNA sequencing is time consuming for projects enrolling many samples, the presented dHPLC protocols are much faster and are equally accurate in identifying the A- and K-variants. The rapid identification of these common variants is important in clinical studies in which the pharmacokinetics of drugs metabolized by BCHE, such as succinylcholine and mivacurium, are being investigated. In addition, the detection of the K-variant using the presented protocol could be useful in Alzheimer's disease research, as the K-variant has been associated with progression of this disease.30,31
In summary, we established dHPLC protocols to rapidly and accurately detect the two most common variants of the BCHE gene. The presented method is a highly sensitive, accurate, and automated approach for the detection of A- and K-variants. It could be useful as a screening tool in clinical studies of drugs metabolized by BCHE.
1. Girard T, Kindler CH. Pharmacogenetics and anaesthesiology. Curr Pharmacogenetics 2004;2:119–35
2. Kalow W. Atypical plasma cholinesterase. A personal discovery story: a tale of three cities. Can J Anaesth 2004;51:206–11
3. Fink H, Geldner G, Fuchs-Buder T, Hofmockel R, Ulm K, Wallek B, Blobner M. Muscle relaxants in Germany 2005: a comparison of application customs in hospitals and private practices. Anaesthesist 2006;55:668–78
4. Li Wan Po A, Girard T. Succinylcholine: still beautiful and mysterious after all these years. J Clin Pharm Ther 2005;30:497–501
5. McGuire MC, Nogueira CP, Bartels CF, Lightstone H, Hajra A, Van der Spek AF, Lockridge O, La Du BN. Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase. Proc Natl Acad Sci USA 1989;86:953–7
6. Bartels CF, Jensen FS, Lockridge O, van der Spek AF, Rubinstein HM, Lubrano T, La Du BN. DNA mutation associated with the human butyrylcholinesterase K-variant and its linkage to the atypical variant mutation and other polymorphic sites. Am J Hum Genet 1992;50:1086–103
7. Gatke MR, Viby-Mogensen J, Bundgaard JR. Rapid simultaneous genotyping of the frequent butyrylcholinesterase variants Asp70Gly and Ala539Thr with fluorescent hybridization probes. Scand J Clin Lab Invest 2002;62:375–83
8. Cerf C, Mesguish M, Gabriel I, Amselem S, Duvaldestin P. Screening patients with prolonged neuromuscular blockade after succinylcholine and mivacurium. Anesth Analg 2002;94:461–6
9. Levano S, Ginz H, Siegemund M, Filipovic M, Voronkov E, Urwyler A, Girard T. Genotyping the butyrylcholinesterase in patients with prolonged neuromuscular block after succinylcholine. Anesthesiology 2005;102:531–5
10. Whittaker M. Cholinesterases. In: L Beckman, ed. Monograph in human genetics. 1st ed. Basel: Karger, 1986:45–64
11. Evans RT, Wardell J. On the identification and frequency of the J and K cholinesterase phenotypes in a Caucasian population. J Med Genet 1984;21:99–102
12. Gaffney D, Campbell RA. A PCR based method to determine the Kalow allele of the cholinesterase gene: the E1k allele frequency and its significance in the normal population. J Med Genet 1994;31:248–50
13. Jensen FS, Nielsen LR, Schwartz M. Detection of the plasma cholinesterase K variant by PCR using an amplification-created restriction site. Hum Hered 1996;46:26–31
14. Babaoglu MO, Ocal T, Bayar B, Kayaalp SO, Bozkurt A. Frequency and enzyme activity of the butyrylcholinesterase K-variant in a Turkish population. Eur J Clin Pharmacol 2004;59:875–7
15. Gatke MR, Viby-Mogensen J, Ostergaard D, Bundgaard JR. Response to mivacurium in patients carrying the k variant in the butyrylcholinesterase gene. Anesthesiology 2005;102:503–8
16. Yen T, Nightingale BN, Burns JC, Sullivan DR, Stewart PM. Butyrylcholinesterase (BCHE) genotyping for post-succinylcholine apnea in an Australian population. Clin Chem 2003;49:1297–308
17. Jensen FS, Viby-Mogensen J. Plasma cholinesterase and abnormal reaction to succinylcholine: twenty years' experience with the Danish Cholinesterase Research Unit. Acta Anaesthesiol Scand 1995;39:150–6
18. Jensen FS, Skovgaard LT, Viby-Mogensen J. Identification of human plasma cholinesterase variants in 6,688 individuals using biochemical analysis. Acta Anaesthesiol Scand 1995;39:157–62
19. Ceppa F, Gidenne S, Benois A, Fontan E, Burnat P. Rapid identification of atypical variant of plasma butyrylcholinesterase by PCR. Clin Chem Lab Med 2002;40:799–801
20. Lando G, Mosca A, Bonora R, Azzario F, Penco S, Marocchi A, Panteghini M, Patrosso MC. Frequency of butyrylcholinesterase gene mutations in individuals with abnormal inhibition numbers: an Italian-population study. Pharmacogenetics 2003; 13:265–70
21. Shibuta K, Abe M, Suzuki T. A new detection method for the K variant of butyrylcholinesterase based on PCR primer introduced restriction analysis (PCR-PIRA). J Med Genet 1994; 31:576–9
22. La Du BN, Bartels CF, Nogueira CP, Hajra A, Lightstone H, Van der Spek A, Lockridge O. Phenotypic and molecular biological analysis of human butyrylcholinesterase variants. Clin Biochem 1990;23:423–31
23. Barta C, Sasvari-Szekely M, Devai A, Kovacs E, Staub M, Enyedi P. Analysis of mutations in the plasma cholinesterase gene of patients with a history of prolonged neuromuscular block during anesthesia. Mol Genet Metab 2001;74:484–8
24. Tammaro A, Bracco A, Cozzolino S, Esposito M, Di Martino A, Savoia G, Zeuli L, Piluso G, Aurino S, Nigro V. Scanning for mutations of the ryanodine receptor (RYR1) gene by denaturing HPLC: detection of three novel malignant hyperthermia alleles. Clin Chem 2003;49:761–8
25. Bagattin A, Veronese C, Bauce B, Wuyts W, Settimo L, Nava A, Rampazzo A, Danieli GA. Denaturing HPLC-based approach for detecting RYR2 mutations involved in malignant arrhythmias. Clin Chem 2004;50:1148–55
26. Yu B, Sawyer NA, Caramins M, Yuan ZG, Saunderson RB, Pamphlett R, Richmond DR, Jeremy RW, Trent RJ. Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease. J Clin Pathol 2005;58:479–85
27. Fackenthal DL, Chen PX, Das S. Denaturing high-performance liquid chromatography for mutation detection and genotyping. Methods Mol Biol 2005;311:73–96
28. Sevilla C, Moatti JP, Julian-Reynier C, Eisinger F, Stoppa-Lyonnet D, Bressac-de Paillerets B, Sobol H. Testing for BRCA1 mutations: a cost-effectiveness analysis. Eur J Hum Genet 2002;10:599–606
29. Rodriguez-Gonzalez MM, Arribas-Carrion C, Torre-Aznar C, Gonzalez-Miranda F. Prolonged neuromuscular block with mivacurium. Rev Esp Anestesiol Reanim 1997;44:328–9
30. Holmes C, Ballard C, Lehmann D, David Smith A, Beaumont H, Day IN, Nadeem Khan M, Lovestone S, McCulley M, Morris CM, Munoz DG, O'Brien K, Russ C, Del Ser T, Warden D. Rate of progression of cognitive decline in Alzheimer's disease: effect of butyrylcholinesterase K gene variation. J Neurol Neurosurg Psychiatry 2005;76:640–3
31. Combarros O, Riancho JA, Infante J, Sanudo C, Llorca J, Zarrabeitia MT, Berciano J. Interaction between CYP19 aromatase and butyrylcholinesterase genes increases Alzheimer's disease risk. Dement Geriatr Cogn Disord 2005;20:153–7