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Activity and polymorphisms of butyrylcholinesterase in a Polish population
Chemico-Biological Interactions xxx (2016) 1e8
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Activity and polymorphisms of butyrylcholinesterase in a Polish population _ ca, Monika Zuk, Jacek Jasiecki*, Joanna Jon Anna Szczoczarz, Anna Janaszak-Jasiecka, Krzysztof Lewandowski, Krzysztof Waleron, Bartosz Wasa˛ g sk, Gdan sk, Poland Medical University of Gdan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2015 Received in revised form 5 April 2016 Accepted 18 April 2016 Available online xxx
Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify individuals at risk of prolonged paralysis following the administration of neuromuscular blocking agents, like succinylcholine, pesticides and nerve agents. In this study, the activity of BChE and its sensitivity to inhibition by dibucaine and fluoride was evaluated in 1200 Polish healthy individuals. In addition, molecular analysis of all exons, exon-intron boundaries and the 30 UTR sequence of the BCHE gene was performed in a group of 72 subjects with abnormal BChE activity (<2000 U/L and >5745 U/L) or with DN (Dibucaine Number) or FN (FluorideNumber) values outside the reference range (DN < 78 and FN < lower than wild type). In a studied group, BChE activity range was similar to those observed in other populations. BChE activity screening allowed to detect UA and UF phenotypes in 26 (2.2%) and 15 (1.2%) individuals, respectively. Observed UA or UF phenotypes were confirmed by direct sequencing and heterozygous c.293A > G or c.1253G > T substitutions were identified in all cases. Nine out of 18 (50%) individuals with BChE activity below 2000 U/L had a mutation in 50 UTR (32G/A), intron 2 (c.1518-121T/C) or exon 4 (c.1699G/A; the K variant mutation). Majority of the individuals with BChE activity 6000 U/L were wild type. To summarize, the range of BChE activity in a Polish population is similar to those observed in other countries. We conclude that the BChE phenotyping assay is a reliable method for identification of individuals with the UA and UF genotypes. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: BChE Butyrylcholinesterase BChE activity assay BCHE gene variants Polymorphisms Pseudocholinesterase
1. Introduction Human butyrylcholinesterase (P06276; BChE) is a serine hydrolase widely distributed throughout the body with the highest levels detected in plasma and liver [1,2]. The enzyme hydrolyzes not only acetylcholine but also longer-chain chemicals containing choline ester bonds e.g., succinylcholine, and other non-choline esters, such as aspirin, cocaine and many others [3]. The exact physiological function is unclear although it acts as an endogenous bioscavenger against AChE (P22303) inhibitors. BChE provides protection against administrated or inhaled poisons by hydrolyzing
Abbreviations: AChE, acetylcholinesterase; BChE, butyrylcholinesterase; BMI, body mass index; BTC, S-butyrylthiocholine iodide; DN, dibucaine number; FN, fluoride number; DTNB, 5,50 -dithiobis(2-nitrobenzoic acid). * Corresponding author. E-mail address: [email protected] (J. Jasiecki).
or sequestering the toxic compounds before they reach their targets e synaptic AChE, and cause neurological damage [4]. The BCHE gene is located on the long arm of chromosome 3 at q26.1-q.26.2 (GRCh38/hg38). It consists of 64569 bp spanning 4 exons and 3 introns. The gene encodes a transcript of 2447 nt in size and a protein of 602 amino acids residues which includes 574 residues in the mature protein and 28 residues in the signal peptide. Human BChE (huBChE) is a globular, tetrameric serine esterase with a molecular mass of 340 kDa. Each monomer carries sialylated glycans on Asn residues at positions 17, 57, 106, 241, 256, 341, 455, 481, and 486, which increase the molecular mass of the protein from 66 to 85 kDa [5,6]. To date, more than 100 genetic variants of the BCHE gene have been described. Point mutations, small insertions and deletions have been identified. Some of these genetic alterations are molecular basis of phenotypic variants of BCHE, such as: atypical, fluoride-resistant or silent gene and K, J or H variants. BCHE genetic
http://dx.doi.org/10.1016/j.cbi.2016.04.030 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: J. Jasiecki, et al., Activity and polymorphisms of butyrylcholinesterase in a Polish population, ChemicoBiological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.030
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J. Jasiecki et al. / Chemico-Biological Interactions xxx (2016) 1e8
alterations can affect the function of the enzyme, which leads to the prolonged neuromuscular blockade by succinylcholine [7e10]. Moreover, there is a number of BCHE genetic variants called “silent” which expression result in a production of a protein with reduced or without enzymatic activity [11e17]. The characterization of BChE phenotypes in humans is performed by biochemical methods, which include measurement of the enzyme activity, DN (dibucaine number) and FN (fluoride number) values. DN value is defined as the percent of BChE activity that is inhibited by dibucaine. It is used to differentiate individuals who are resistant to dibucaine inhibition due to alterations of the anionic site of the BChE [18,19]. The DN and the BChE enzyme activity measurement can help to identify subjects at risk for prolonged paralysis following the administration of muscle relaxants such as succinylcholine or mivacurium. Such individuals, with decreased enzyme activity and DN values less than 30 are called Atypical (A) BChE phenotype [20e23]. The fluoride (F) variant of BChE enzyme is resistant to inhibition by 0.050 mM sodium fluoride in in vitro assay. Individuals who are compound fluoride and atypical heterozygous (AF phenotype) have prolonged response to succinylcholine. Therefore, DN, FN and total BChE activity have to be used in a combination to assign a biochemical phenotype of the individuals [21,24]. Many studies have shown that high serum BChE activity is associated with obesity, insulin resistance, metabolic syndrome, hyperlipidemia, coronary artery disease or hypertension [25e27]. It was also reported that butyrylcholinesterase K (BChE-K) is associated with increased risk of developing Alzheimer's disease [28e32]. BChE has been shown to inactivate ghrelin and affects the circulating levels of this peptide hormone, with consequences for weight gain, fat metabolism, and aggression levels [33e35]. Data from the BChE knockout mice, which on a fat diet are obese and significantly heavier than wild types, indicate a role for BChE in fat utilization [25]. Furthermore, SNPs of the human BCHE gene have been associated with body mass index (BMI) [26,36]. The most common assay used for the determination of BChE activity and enzyme phenotyping is based on Ellman's colorimetric method developed in 1961 and universally used until now [40]. It is rapid, simple and cheap method which can be easily adapted for high-throughput analysis. In this study, the BChE activity and phenotype was determined by methods and protocols developed and described by our group previously [41]. 2. Materials and methods S-butyrylthiocholine iodide (BTC, cat. no. 20820), 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent cat. no. D8130), dibucaine hydrochloride (cat. no. D0638), sodium fluoride (cat. no. 201154) were purchased from Sigma-Aldrich (Germany). 0.1 M sodium phosphate buffer (PB, pH 7.4), stock and working solutions of BTC, DTNB, dibucaine and sodium fluoride were prepared as previously described [41]. All solutions were prewarmed to 25 C prior to use. 2.1. Subjects Blood and serum samples from 1200 anonymized healthy individuals (556 males and 644 females, aged from 2 to 94 (mean age 48) years) were obtained from a collection of the Central Clinical Laboratory of the Medical University of Gdansk. The local Ethical Committee approved the study (NKBBN/304/2013) and waived the need for informed consent from donors. The selected individuals enrolled to the study were asymptomatic and designated as a healthy, with complete blood count and serum activity of hepatic enzymes ALT and AST in the reference range. All blood and serum samples were stored in 0.3 mL aliquots at e 80 C prior to analysis.
2.2. Micro-ellman assay for measuring BChE activity and phenotyping Butyrylcholinesterase activity was determined spectrophotometrically by modified Ellman's method with the use of BTC as a substrate. Briefly, 10 ml of serum was added to 190 ml of PB (100 mM, pH 7.4) to achieve initially 20-fold dilution. 400-fold dilutions were prepared in 96-well microtiter plates in a final reaction volume of 200 ml of PB (100 mM, pH 7.4) with 0.5 mM DTNB and 5 mM BTC. All samples were mixed thoroughly by repetitive pipetting. 10 ml of diluted serum samples were added to the wells of a microtiter plate containing 40 ml of PB (100 mM, pH 7.4). Then, 50 ml of DTNB (2 mM in PB) was added, and incubated (25 C, 10 min) in a microplatereader. For BChE phenotyping, dibucaine hydrochloride or sodium fluoride were also added to final concentrations of 100 mM and 50 mM respectively. The reactions were initiated by addition of 100 ml BTC (10 mM in PB). The absorbance was monitored at 412 nm by a Tecan Infinite M200Pro (1 min intervals for 5 min, 25 C). One unit (U) of enzyme activity is defined as the amount that hydrolyzes 1 mmol of butyrylthiocholine per 1 min. The molar absorption coefficients of TNB at 412 nm is 14150 M(1)cm(1) at 25 C. The pathlength in the microtiter plates used in the assays was estimated experimentally as 0.58 cm. The detailed protocol was described previously [41]. All experiments were performed in triplicates and for measurements and calculations Tecan Magellan V7.0 software was applied. 2.3. DNA extraction, PCR amplification and sanger sequencing Genomic DNA was extracted from 1 mL of peripheral blood leukocytes using Blood Mini kit in accordance with the manufacturer's protocol (A&A Biotechnology, Poland). All four exons, exonintron boundaries and 30 UTR of the BCHE were amplified by PCR. A 25-mL PCR mixture contained 60 ng of extracted DNA, 10 pmoles of each forward and reverse primers (Table 1), dNTPs, buffer, and Marathon Taq DNA polymerase (A&A Biotechnology, Poland). Primer sequences, amplicons size and position within BCHE gene are presented in Table 1 and Fig. 1. Amplification was performed with an initial denaturation at 95 C for 5 min, followed by 35 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s, and extension at 72 C for 30 s with a final extension at 72 C for 10 min. PCR products were purified using Clean Up (A&A Biotechnology, Poland). Bidirectional DNA sequencing of PCR amplification products was performed using BigDye Terminator v.3.1 cycle sequencing kit and 3130 Genetic Analyzer according to the manufacturer's
Table 1 PCR primer sequences and amplicons size for BCHE sequencing analysis. Primer
Exon
Amplicon size (bp)
Sequence 50 -30
M1F M1R M2-1F M2-1R M2-2F M2-2R M2-3F M2-3R M2-4F M2-4R M3F M3R M4-1F M4-1R M4-2F M4-2R
1
344
2
452
2
593
2
532
2
420
3
415
4
381
4
517
F- AGACTACCTGCAATTGTAAAGCA R- TCTCATCCCACAGAATGAGC F- CCTATGTAGGCCTTTACAGAAGC R- TTGATCTATGTTCTGACAGCAAG F- GCCACAGTCTCTGACCAAGTG R- TTCTGTTCCTAGCTTCATAAAGAG F- TGTTCACCAGAGCCATTCTG R- ACAACATCACCCAAGGCCTC F- AGTGAGTTTGGAAAGGAATCC R- AGAGACCAAGCAAAGCTAAGC F- CACTAAGCCCAGTTCACATACG R- CATCACCGTGCCTTGGAG F- TGTACTGTGTAGTTAGAGAAAATGGC R- TACTAAGTTAAAGATGTGAGGAATC F- AGATCAAGGCAAAAATATCAGGAGC R- ATAAGGTGTTTTAAAGTGGCTGAG
NCBI Reference Sequence: NC_000003.12.
Please cite this article in press as: J. Jasiecki, et al., Activity and polymorphisms of butyrylcholinesterase in a Polish population, ChemicoBiological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.030
J. Jasiecki et al. / Chemico-Biological Interactions xxx (2016) 1e8
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Fig. 1. Human BCHE gene structure and position of detected genetic variants. BCHE exons are indicated in red (coding) and black (50 UTR and 30 UTR) blocks. Introns are marked gray. PCR primers used for amplification and sequencing are denoted as horizontal arrows. Green vertical lines show the locations of ten BCHE genetic variants detected in the study. Two types of mutations numbering is presented. Residues numbers denoted in black are for the mature BChE protein and such numbering is present in the PDB database and in most publications about BChE. In contrast, residues numbers in genetics databases (NCBI, denoted in red, NM_000055.2, NP_000046.1) add 28 amino acids for the signal peptide. For example Asp70 in the PDB database is Asp 98 in the NCBI database. NCBI Reference Sequence: NC_000003.12. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
protocol (ThermoFischer Scientific, USA). Sequences were analyzed by Sequencher v.4.10 DNA Software (Gene Codes Corporation, USA) and aligned with BCHE sequence NM_000055.2 (http://www.ncbi. nlm.nih.gov).
3. Results In a studied group of 1200 healthy individuals, BChE activity as well as DN and FN values of serum were measured using 5 mM BTC as a substrate and dibucaine hydrochloride or sodium fluoride at final concentrations of 100 mM and 50 mM, respectively. The results (Fig. 2) showed that in Polish population range of BChE activity is normal with a mean of 3600 U/L. In order to find a new genetic variants of BCHE, the entire coding sequence and 30 UTR of the gene were analyzed in 18 individuals with BChE activity lower than 2000 U/L and 10 with higher than 5745 U/L. In addition, BCHE genotype was determined in 26 individuals with DN < 78 and 15 subjects with FN < lower than wild type. The phenotype and genotype were in a full agreement in all sequenced individuals with aberrant DN and FN. In all 26 and 15 individuals the expected heterozygous c.293A > G/p.D98G (atypical variant) and c.1253G > T/p.G418V (fluoride-2 variant) substitutions were
observed, respectively. The A variant (c.293A > G/p.D98G) was found only in heterozygous (UA) form in 26 (2.1%) individuals of the studied group (Table 2). In 23 out of 26 (88%) UA individuals, the quantitative K variant c.1699G > A/p.A567T (rs1803274) was also detected. K variant is frequently associated with the A variant as described earlier [13]. In addition, K variant was identified in three individuals without A variant. In 13 out of 37 (35%) individuals with K variant, the coincidence of c.-32G > A (rs1126680) and c.1518121T > C (rs55781031) substitutions was found. Interestingly, in 9 out of 18 (50%) subjects with BChE activity below 2000 U/L coincidence of c.-32G > A, (rs1126680), c.1518-121T > C and c.1699G > A was observed (Fig. 2, Table 2). In contrast, most of the individuals with BChE activity 6000 U/L or above were found to be a wild type. Sequencing analysis revealed one specimen with heterozygous c.849G > C/p.E283D (rs16849700) substitution. In this individual the presence of K variant was also detected. Finally, in 28 out of 72 (39%) samples, the genetic variant in 30 UTR of BCHE was identified. Of these, 82% (23/28) were found to carry compound heterozygous or homozygous c.*189G > A (rs3495) substitution and homozygous c.*293dupT (rs3836432) duplication. Additionally, in four of these individuals, the fourth heterozygous alteration was detected downstream of 30 UTR (g.165772869G > T, rs77307863). In one of
Please cite this article in press as: J. Jasiecki, et al., Activity and polymorphisms of butyrylcholinesterase in a Polish population, ChemicoBiological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.030
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J. Jasiecki et al. / Chemico-Biological Interactions xxx (2016) 1e8
Fig. 2. A. BChE activity of 1200 healthy individuals. The enzyme activity of a single person is represented by a single black dot. BChE activity was measured in Ellman's reaction in the presence of 5 mM BTC and 400-fold diluted serum at 25 C. The activity was estimated based on the mean of hydrolysis rate (OD/min) determined from at least 5 spectrophotometer reads. Red dots and blue triangles with corresponding numbering stand for subjects selected for BCHE sequencing. Individuals with gene substitution in 50 UTR, c.32G > A, (rs1126680) are designated as blue triangles B. DN and FN values were calculated in assays performed at a final concentrations of 100 mM and 50 mM dibucaine hydrochloride or sodium fluoride respectively. All individuals below the red threshold line set at DN ¼ 78 have UA phenotype. All assays were performed in triplicate, standard error for all points is less than 3% of the values. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
these individuals the additional c.*349G > A(rs55849903) substitution was found. 4. Discussion BChE is a stoichiometric bioscavenger against poisoning by organophosphate pesticides and nerve agents. The enzyme is also a promising biopharmaceutic to accelerate cocaine breakdown. Native BChE has low catalytic efficiency against the abused cocaine but through a combination of molecular modeling and experimental analysis, mutants of human BChE with considerably improved catalytic efficiency against cocaine were successfully made [42,43]. There are many examples of enzymes found in nature that have been exploited in medicine and biotechnology due to their inherent catalytic properties. The desired biochemical activities are often difficult to achieve using the native form of the enzymes. The rapid progress in the field of genetic engineering have revolutionized the development of commercially available enzymes. A combination of in silico and experimental methods allowed to modify the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specificity and selectivity towards non-natural substrates. High-throughput phenotypical and/(or) genetic population screening is another method for searching proteins with improved biochemical properties. In order to find a new interesting variants of BChE, 1200 healthy individuals were screened for BChE activity by biochemical assay. From the studied group, 28 individuals with BChE activity lower
than 2000 U/L and higher than 5745 were selected for mutational analysis of BCHE gene. In addition, based on phenotypical inhibitor assays with dibucaine and fluoride, 41 samples of the individuals with aberrant DN and FN values (26 UA an 15 UF) were selected for sequencing. It is difficult to differentiate individuals with heterozygous FN phenotypes from a wild type subjects. Differences between FN values in UU and UF phenotypes are minimal. Therefore, in order to find FN phenotype it is recommended to apply DN value, too. This value is slightly lowered in UF subjects but not so much as in UA individuals, where DN < 78 [44]. Moreover, to set appropriate UF phenotypes, samples should be always compared to wild type phenotypes. In all 72 samples selected for mutational analysis, entire coding sequence of BCHE with surrounding intronic regions and 30 UTR fragment were screened. In total, a BCHE genetic variant has been identified in 69 out of 72 sequenced individuals. In 54 (75%) individuals a genetic variant has been found within a coding sequence of the gene. In this study, the most common BCHE alterations were K variants, Atypical and Fluoride with the following 0.031, 0.0216 and 0.0125 frequencies. The heterozygous (n ¼ 28) or homozygous (n ¼ 9) K variant was observed in 37 individuals. Both atypical and fluoride variants were found only in heterozygous form in 26 and 15 individuals, respectively. In Caucasian populations, the allele of the atypical variant is detected in one out of 25 subjects whereas the K variant allele is found in one out of four persons with a homozygous incidence of 1/63 individuals [2]. Therefore, the frequency of atypical variant observed in this study is in agreement with a previous reports. The K allele frequency was much lower than described previously for populations. In the study, only samples with respect to atypical and fluoride phenotype and
Please cite this article in press as: J. Jasiecki, et al., Activity and polymorphisms of butyrylcholinesterase in a Polish population, ChemicoBiological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.030
J. Jasiecki et al. / Chemico-Biological Interactions xxx (2016) 1e8
5
Table 2 Genetic variants of BCHE detected in a studied group. Number
Subject no.
BChE Activity [U/L]
DN
Exon 1
Exon 2.1
Exon2.2
Exon 2.3
Exon 2.4
30 end of intron 2
Exon 3
Exon 4
1564
83
WT
WT
WT
WT
WT
WT
c.293A > G, htz
WT
WT
c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, WT
c.1699G htz c.1699G htz c.1699G hom c.1699G htz c.1699G hom c.1699G htz c.1699G hom c.1699G htz c.1699G hom c.1699G hom c.1699G hom c.1699G hom c.1699G hom c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz
14
28
2821
72
c.-32G > A, htz c.-32G > A, htz c.-32G > A, hom c.-32G > A, htz c.-32G > A, htz c.-32G > A, htz c.-32G > A, htz c.-32G > A, htz c.-32G > A, htz c.-32G > A, hom c.-32G > A, htz c.-32G > A, htz c.-32G > A, htz WT
15
33
2609
70
WT
WT
c.293A > G, htz
WT
WT
WT
WT
16
35
3176
70
WT
WT
c.293A > G, htz
WT
WT
WT
WT
17
236
3543
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
18
246
3565
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
19
248
1663
75
WT
WT
c.293A > G, htz
WT
WT
WT
WT
20
251
3343
75
WT
WT
c.293A > G, htz
WT
WT
WT
WT
21
353
2522
76
WT
WT
c.293A > G, htz
WT
WT
WT
WT
22
358
2611
75
WT
WT
c.293A > G, htz
WT
WT
WT
WT
23
414
2645
72
WT
WT
c.293A > G, htz
WT
WT
WT
WT
24
470
2213
75
WT
WT
c.293A > G, htz
WT
WT
WT
WT
25
475
3001
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
26
547
1623
86
WT
WT
WT
c.849G > C, htz
WT
WT
WT
27
588
2694
72
WT
WT
c.293A > G, htz
WT
WT
WT
WT
28
603
2208
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
29
766
3484
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
30
836
6350
83
WT
WT
WT
WT
WT
WT
WT
31
855
2804
80
WT
WT
WT
WT
WT
WT
32
884
3285
73
WT
WT
c.293A > G, htz
WT
c.1253G > T, htz WT
WT
WT
33
922
2656
72
WT
WT
c.293A > G, htz
WT
WT
WT
WT
34
1057
2823
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
35
1070
2373
76
WT
WT
c.293A > G, htz
WT
WT
WT
WT
1 2 3 4 5 6 7 8 9 10 11 12 13
16 198 252 327 615 708 772 789 861 926 1002 1016 1146
1893 1991 3132 2755 5991 1975 6326 1943 1828 1900 1819 1945
87 86 85 61 85 83 83 85 84 85 67 87
WT WT WT WT WT WT WT WT WT WT WT WT
WT WT WT c.293A > G, htz WT WT WT WT WT WT c.293A > G, htz WT
WT WT WT WT WT WT WT WT WT WT WT WT
WT WT WT WT WT WT WT WT WT WT WT WT
htz WT htz WT hom WT htz WT htz WT htz WT htz WT htz WT htz WT hom WT htz WT htz WT htz WT
c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz
30 UTR
> A, > A,
c.*189G > A, htz; c.*293dupT, hom WT
> A,
WT
> A, > A,
c.*189G > A, htz; c.*293dupT, hom WT
> A,
WT
> A,
c.*293dupT, hom
> A, > A,
c.*189G > A, htz; c.*293dupT, hom WT
> A,
c.*293dupT, hom
> A,
c.*293dupT, hom
> A,
c.*293dupT, hom
> A,
WT
> A,
WT
> A,
WT
> A,
WT
> A,
WT
> A,
WT
> A, > A,
c.*189G > A, htz; c.*293dupT, hom WT
> A,
WT
> A,
WT
> A,
WT
> A,
WT
> A,
WT
> A,
> A,
c.*189G > A, htz; c.*293dupT, hom; c.*349G > A, htz WT
> A,
WT
> A,
WT
> A,
> A,
c.*189G > A, htz; c.*293dupT, hom; c.*513G > T, htz WT
> A,
WT
> A, > A,
c.*189G > A, htz; c.*293dupT, hom WT
> A,
WT (continued on next page)
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J. Jasiecki et al. / Chemico-Biological Interactions xxx (2016) 1e8
Table 2 (continued )
*
Number
Subject no.
BChE Activity [U/L]
DN
Exon 1
Exon 2.1
Exon2.2
Exon 2.3
Exon 2.4
30 end of intron 2
Exon 3
Exon 4
30 UTR
36
1095
3170
71
WT
WT
c.293A > G, htz
WT
WT
WT
WT
WT
37
1150
2834
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
38
195
2979
58
WT
WT
WT
WT
WT
WT
39 40 41
481 524 11
3042 2496 2640
72 75 78
WT WT WT
WT WT WT
c.293A > G, htz; c.635C > T, htz c.293A > G, htz c.293A > G, htz WT
c.1699G > A, hom c.1699G > A, htz WT
WT WT WT
WT WT c.1253G > T, htz
WT WT WT
WT WT WT
WT WT WT
42
217
2195
81
WT
WT
WT
WT
>
WT
WT
WT
43
381
3596
82
WT
WT
WT
WT
>
WT
WT
WT
WT
44
404
5092
81
WT
WT
WT
WT
>
WT
WT
WT
WT
45
448
3195
83
WT
WT
WT
WT
>
WT
WT
WT
WT
46
477
3780
81
WT
WT
WT
WT
>
WT
WT
WT
WT
47
504
4497
83
WT
WT
WT
WT
>
WT
WT
WT
WT
48
557
4545
82
WT
WT
WT
WT
>
WT
WT
WT
WT
49
587
2909
79
WT
WT
WT
WT
>
WT
WT
WT
WT
50
686
2555
81
WT
WT
WT
WT
>
WT
WT
WT
WT
51
750
3269
78
WT
WT
WT
WT
>
WT
WT
WT
WT
52
887
3083
82
WT
WT
WT
WT
>
WT
WT
WT
WT
53
1059
3331
82
WT
WT
WT
WT
>
WT
WT
WT
WT
54
1190
2928
80
WT
WT
WT
WT
>
WT
WT
WT
WT
55 56
3 5
1665 4233
84 83
WT WT
WT WT
WT WT
WT WT
c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz WT WT
c.*189G > A, htz; c.*293dupT, hom WT WT c.*189G > A, hom; c.*293dupT, hom; c.*513G > T, hom WT
WT WT
WT WT
WT WT
57
102
5887
84
WT
WT
WT
WT
WT
WT
WT
WT
58
133
2642
86
WT
WT
WT
WT
WT
WT
WT
WT
59
159
1468
85
WT
WT
WT
WT
WT
WT
WT
WT
60
310
1872
86
WT
WT
WT
WT
WT
WT
WT
WT
61
339
5335
87
WT
WT
WT
WT
WT
WT
WT
WT
62 63
392 607
6074 6249
87 85
WT WT
WT WT
WT WT
WT WT
WT WT
WT WT
WT WT
WT WT
64
844
7581
83
WT
WT
WT
WT
WT
WT
WT
WT
65
928
4160
86
WT
WT
WT
WT
WT
WT
WT
WT
66
986
1843
86
WT
WT
WT
WT
WT
WT
WT
WT
67
988
1868
87
WT
WT
WT
WT
WT
WT
WT
WT
68
1076
6497
87
WT
WT
WT
WT
WT
WT
WT
WT
69
1099
1542
87
WT
WT
WT
WT
WT
WT
WT
WT
70
1128
6161
85
WT
WT
WT
WT
WT
WT
WT
WT
71 72
1161 1183
729 5745
86 86
WT WT
WT WT
WT WT
WT WT
WT WT
WT WT
WT WT
WT WT
WT c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom; c.*513G > T, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom; c.*513G > T, htz WT c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom; c.*513G > T, hom c.*189G > A, hom; c.*293dupT, hom WT c.*189G > A, hom; c.*293dupT, hom
WT
cited in the Table 2, is known genetic description of 3' UTR e.g. c. *46T>A denotes a T to A substitution 46 nucleotides 3' of the translation termination codon.
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Fig. 3. Predicted putative secondary structure (DG ¼ 46.7 kcal/mol) of the 200 first nt of the BCHE mRNA. Structure was generated for 37 C using the MFOLD algorithm and visualized by RnaViz 2 [50]. It is hypothesized that the hairpin (4) comprising the AUG codon affects protein synthesis, but in wild type mRNA the hairpin is not formed due to binding of regulatory proteins via stem loop (3). c.-32G > A (rs1126680) substitution (shown in red circle) probably lower the affinity of binding plausible proteins and allow to form a hairpin (comprising Kozak sequence as well as translation start site AUG) resulting in reduction of the protein synthesis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
for the highest and lowest BChE activity were selected for mutational analysis. Therefore, it was not a population study which can explain differences between a current study and previous reports. The fluoride variant allele has been previously showed to be present only in one out of 160 individuals [2,26]. In a studied population of Polish healthy individuals, the fluoride allele frequency was much higher. Explanation of this phenomena may be that in a current study the very sensitive method for FN phenotyping was applied. Previous studies have shown that K variants were found in linkage disequilibrium between atypical variants such that in 89e96% of atypical variant carriers, the K variant had been also detected [13]. Results of our study support this hypothesis. In 23 out of 26 samples with Atypical variant, the K variant has been also found. On the other hand, among K allele carriers linkage disequilibrium between atypical variants or compound mutation: c.-32G > A (rs1126680) and c.1518-121T > C (rs55781031) was observed. All 37 studied individuals with K genotype were found to have other genetic variants, atypical variant (n ¼ 23), compound mutation c.-32G > A and c.1518-121T > C (n ¼ 13). In a single cases coincidence of K genotype with fluoride variant or c.849G > C (p.E283D) or 30 UTR alteration was identified. It is notable that 50 UTR c.-32G > A, (rs1126680) variant was preferentially found in cis with K variant (c.1699G > A, p.A567T) and intron 2 alteration (c.1518-121T > C, rs55781031). Previous studies have shown that c.-32G > A substitution in exon 1 was associated with lower BChE activity, obesity, hypertriglyceridemia and with gestational diabetes mellitus [36e39], but the alteration has been reported earlier as 116A BChE variant since there two types of numbering exist in databases (Fig. 1). Our results support this observation. In 11 out of 13 individuals with c.-32G > A, lower BChE activity has been observed (2 homozygous with one of the lowest BChE activity). Moreover, a c.-32G > A, (rs1126680) was found in 9 out of 18 (50%) subjects with BChE activity below 2000 U/L. It was also discussed that c.-32G > A is responsible for lowering of BChE activity by affecting transcription and/or translation. It is plausible that this 50 UTR genetic variant can directly modulate the protein translation through alterations in mRNA secondary structure. It is accepted that protein translation efficiency is often affected by mRNA secondary structure since selfcomplementary sequences within the 50 UTR can form stable stem-loop structures that may disturb initiation of translation
[45e47]. Theoretical model of the mRNA secondary structure of the 50 UTR region of BCHE presented in Fig. 3 was predicted using mFold [48]. It is possible that the position of substitutions in the loop of the stem could affect binding of the regulatory proteins and disturb translation efficiency. Such a process was previously described for 50 UTR of the ferritin receptor mRNA which contains stem loop that recruits binding proteins and control translation efficiency [49]. The biochemical determination of the BChE phenotype is a helpful method used to identify individuals at risk for prolonged apnea after receiving succinylcholine. As demonstrated in the present study, the biochemical determination of BChE phenotype of individuals is a precise method. In the presented study, in 72 specimens selected for the mutational analysis, the full concordance between phenotype and genotype was observed. We conclude that detection of BChE phenotype based on biochemical assays can be useful in clinical practice. However, further molecular analysis of selected individuals can give new insights into the molecular basis of BChE activity. Acknowledgments The authors are grateful to M. Wa˛ sewicz for blood samples collection and serum preparation. This work was supported by Faculty of Pharmacy with Subfaculty of Laboratory Medicine, Medical University of Gdansk, KNOW Program funded by Ministry of Science and Higher Education, Republic of Poland. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2016.04.030. References [1] G. Johnson, S.W. Moore, Why has butyrylcholinesterase been retained? Structural and functional diversification in a duplicated gene, Neurochem. Int. 61 (5) (2012) 783e797. [2] O. Lockridge, Review of human butyrylcholinesterase structure, function, genetic variants, history of use in the clinic, and potential therapeutic uses, Pharmacol. Ther. 148 (2015) 34e46. Epub 2014/12/03. [3] P. Masson, O. Lockridge, Butyrylcholinesterase for protection from organophosphorus poisons; catalytic complexities and hysteretic behavior, Arch. Biochem. Biophys. 494 (2) (2010) 107.
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Please cite this article in press as: J. Jasiecki, et al., Activity and polymorphisms of butyrylcholinesterase in a Polish population, ChemicoBiological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.030
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Activity and polymorphisms of butyrylcholinesterase in a Polish population _ ca, Monika Zuk, Jacek Jasiecki*, Joanna Jon Anna Szczoczarz, Anna Janaszak-Jasiecka, Krzysztof Lewandowski, Krzysztof Waleron, Bartosz Wasa˛ g sk, Gdan sk, Poland Medical University of Gdan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2015 Received in revised form 5 April 2016 Accepted 18 April 2016 Available online xxx
Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify individuals at risk of prolonged paralysis following the administration of neuromuscular blocking agents, like succinylcholine, pesticides and nerve agents. In this study, the activity of BChE and its sensitivity to inhibition by dibucaine and fluoride was evaluated in 1200 Polish healthy individuals. In addition, molecular analysis of all exons, exon-intron boundaries and the 30 UTR sequence of the BCHE gene was performed in a group of 72 subjects with abnormal BChE activity (<2000 U/L and >5745 U/L) or with DN (Dibucaine Number) or FN (FluorideNumber) values outside the reference range (DN < 78 and FN < lower than wild type). In a studied group, BChE activity range was similar to those observed in other populations. BChE activity screening allowed to detect UA and UF phenotypes in 26 (2.2%) and 15 (1.2%) individuals, respectively. Observed UA or UF phenotypes were confirmed by direct sequencing and heterozygous c.293A > G or c.1253G > T substitutions were identified in all cases. Nine out of 18 (50%) individuals with BChE activity below 2000 U/L had a mutation in 50 UTR (32G/A), intron 2 (c.1518-121T/C) or exon 4 (c.1699G/A; the K variant mutation). Majority of the individuals with BChE activity 6000 U/L were wild type. To summarize, the range of BChE activity in a Polish population is similar to those observed in other countries. We conclude that the BChE phenotyping assay is a reliable method for identification of individuals with the UA and UF genotypes. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: BChE Butyrylcholinesterase BChE activity assay BCHE gene variants Polymorphisms Pseudocholinesterase
1. Introduction Human butyrylcholinesterase (P06276; BChE) is a serine hydrolase widely distributed throughout the body with the highest levels detected in plasma and liver [1,2]. The enzyme hydrolyzes not only acetylcholine but also longer-chain chemicals containing choline ester bonds e.g., succinylcholine, and other non-choline esters, such as aspirin, cocaine and many others [3]. The exact physiological function is unclear although it acts as an endogenous bioscavenger against AChE (P22303) inhibitors. BChE provides protection against administrated or inhaled poisons by hydrolyzing
Abbreviations: AChE, acetylcholinesterase; BChE, butyrylcholinesterase; BMI, body mass index; BTC, S-butyrylthiocholine iodide; DN, dibucaine number; FN, fluoride number; DTNB, 5,50 -dithiobis(2-nitrobenzoic acid). * Corresponding author. E-mail address: [email protected] (J. Jasiecki).
or sequestering the toxic compounds before they reach their targets e synaptic AChE, and cause neurological damage [4]. The BCHE gene is located on the long arm of chromosome 3 at q26.1-q.26.2 (GRCh38/hg38). It consists of 64569 bp spanning 4 exons and 3 introns. The gene encodes a transcript of 2447 nt in size and a protein of 602 amino acids residues which includes 574 residues in the mature protein and 28 residues in the signal peptide. Human BChE (huBChE) is a globular, tetrameric serine esterase with a molecular mass of 340 kDa. Each monomer carries sialylated glycans on Asn residues at positions 17, 57, 106, 241, 256, 341, 455, 481, and 486, which increase the molecular mass of the protein from 66 to 85 kDa [5,6]. To date, more than 100 genetic variants of the BCHE gene have been described. Point mutations, small insertions and deletions have been identified. Some of these genetic alterations are molecular basis of phenotypic variants of BCHE, such as: atypical, fluoride-resistant or silent gene and K, J or H variants. BCHE genetic
http://dx.doi.org/10.1016/j.cbi.2016.04.030 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: J. Jasiecki, et al., Activity and polymorphisms of butyrylcholinesterase in a Polish population, ChemicoBiological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.030
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J. Jasiecki et al. / Chemico-Biological Interactions xxx (2016) 1e8
alterations can affect the function of the enzyme, which leads to the prolonged neuromuscular blockade by succinylcholine [7e10]. Moreover, there is a number of BCHE genetic variants called “silent” which expression result in a production of a protein with reduced or without enzymatic activity [11e17]. The characterization of BChE phenotypes in humans is performed by biochemical methods, which include measurement of the enzyme activity, DN (dibucaine number) and FN (fluoride number) values. DN value is defined as the percent of BChE activity that is inhibited by dibucaine. It is used to differentiate individuals who are resistant to dibucaine inhibition due to alterations of the anionic site of the BChE [18,19]. The DN and the BChE enzyme activity measurement can help to identify subjects at risk for prolonged paralysis following the administration of muscle relaxants such as succinylcholine or mivacurium. Such individuals, with decreased enzyme activity and DN values less than 30 are called Atypical (A) BChE phenotype [20e23]. The fluoride (F) variant of BChE enzyme is resistant to inhibition by 0.050 mM sodium fluoride in in vitro assay. Individuals who are compound fluoride and atypical heterozygous (AF phenotype) have prolonged response to succinylcholine. Therefore, DN, FN and total BChE activity have to be used in a combination to assign a biochemical phenotype of the individuals [21,24]. Many studies have shown that high serum BChE activity is associated with obesity, insulin resistance, metabolic syndrome, hyperlipidemia, coronary artery disease or hypertension [25e27]. It was also reported that butyrylcholinesterase K (BChE-K) is associated with increased risk of developing Alzheimer's disease [28e32]. BChE has been shown to inactivate ghrelin and affects the circulating levels of this peptide hormone, with consequences for weight gain, fat metabolism, and aggression levels [33e35]. Data from the BChE knockout mice, which on a fat diet are obese and significantly heavier than wild types, indicate a role for BChE in fat utilization [25]. Furthermore, SNPs of the human BCHE gene have been associated with body mass index (BMI) [26,36]. The most common assay used for the determination of BChE activity and enzyme phenotyping is based on Ellman's colorimetric method developed in 1961 and universally used until now [40]. It is rapid, simple and cheap method which can be easily adapted for high-throughput analysis. In this study, the BChE activity and phenotype was determined by methods and protocols developed and described by our group previously [41]. 2. Materials and methods S-butyrylthiocholine iodide (BTC, cat. no. 20820), 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent cat. no. D8130), dibucaine hydrochloride (cat. no. D0638), sodium fluoride (cat. no. 201154) were purchased from Sigma-Aldrich (Germany). 0.1 M sodium phosphate buffer (PB, pH 7.4), stock and working solutions of BTC, DTNB, dibucaine and sodium fluoride were prepared as previously described [41]. All solutions were prewarmed to 25 C prior to use. 2.1. Subjects Blood and serum samples from 1200 anonymized healthy individuals (556 males and 644 females, aged from 2 to 94 (mean age 48) years) were obtained from a collection of the Central Clinical Laboratory of the Medical University of Gdansk. The local Ethical Committee approved the study (NKBBN/304/2013) and waived the need for informed consent from donors. The selected individuals enrolled to the study were asymptomatic and designated as a healthy, with complete blood count and serum activity of hepatic enzymes ALT and AST in the reference range. All blood and serum samples were stored in 0.3 mL aliquots at e 80 C prior to analysis.
2.2. Micro-ellman assay for measuring BChE activity and phenotyping Butyrylcholinesterase activity was determined spectrophotometrically by modified Ellman's method with the use of BTC as a substrate. Briefly, 10 ml of serum was added to 190 ml of PB (100 mM, pH 7.4) to achieve initially 20-fold dilution. 400-fold dilutions were prepared in 96-well microtiter plates in a final reaction volume of 200 ml of PB (100 mM, pH 7.4) with 0.5 mM DTNB and 5 mM BTC. All samples were mixed thoroughly by repetitive pipetting. 10 ml of diluted serum samples were added to the wells of a microtiter plate containing 40 ml of PB (100 mM, pH 7.4). Then, 50 ml of DTNB (2 mM in PB) was added, and incubated (25 C, 10 min) in a microplatereader. For BChE phenotyping, dibucaine hydrochloride or sodium fluoride were also added to final concentrations of 100 mM and 50 mM respectively. The reactions were initiated by addition of 100 ml BTC (10 mM in PB). The absorbance was monitored at 412 nm by a Tecan Infinite M200Pro (1 min intervals for 5 min, 25 C). One unit (U) of enzyme activity is defined as the amount that hydrolyzes 1 mmol of butyrylthiocholine per 1 min. The molar absorption coefficients of TNB at 412 nm is 14150 M(1)cm(1) at 25 C. The pathlength in the microtiter plates used in the assays was estimated experimentally as 0.58 cm. The detailed protocol was described previously [41]. All experiments were performed in triplicates and for measurements and calculations Tecan Magellan V7.0 software was applied. 2.3. DNA extraction, PCR amplification and sanger sequencing Genomic DNA was extracted from 1 mL of peripheral blood leukocytes using Blood Mini kit in accordance with the manufacturer's protocol (A&A Biotechnology, Poland). All four exons, exonintron boundaries and 30 UTR of the BCHE were amplified by PCR. A 25-mL PCR mixture contained 60 ng of extracted DNA, 10 pmoles of each forward and reverse primers (Table 1), dNTPs, buffer, and Marathon Taq DNA polymerase (A&A Biotechnology, Poland). Primer sequences, amplicons size and position within BCHE gene are presented in Table 1 and Fig. 1. Amplification was performed with an initial denaturation at 95 C for 5 min, followed by 35 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s, and extension at 72 C for 30 s with a final extension at 72 C for 10 min. PCR products were purified using Clean Up (A&A Biotechnology, Poland). Bidirectional DNA sequencing of PCR amplification products was performed using BigDye Terminator v.3.1 cycle sequencing kit and 3130 Genetic Analyzer according to the manufacturer's
Table 1 PCR primer sequences and amplicons size for BCHE sequencing analysis. Primer
Exon
Amplicon size (bp)
Sequence 50 -30
M1F M1R M2-1F M2-1R M2-2F M2-2R M2-3F M2-3R M2-4F M2-4R M3F M3R M4-1F M4-1R M4-2F M4-2R
1
344
2
452
2
593
2
532
2
420
3
415
4
381
4
517
F- AGACTACCTGCAATTGTAAAGCA R- TCTCATCCCACAGAATGAGC F- CCTATGTAGGCCTTTACAGAAGC R- TTGATCTATGTTCTGACAGCAAG F- GCCACAGTCTCTGACCAAGTG R- TTCTGTTCCTAGCTTCATAAAGAG F- TGTTCACCAGAGCCATTCTG R- ACAACATCACCCAAGGCCTC F- AGTGAGTTTGGAAAGGAATCC R- AGAGACCAAGCAAAGCTAAGC F- CACTAAGCCCAGTTCACATACG R- CATCACCGTGCCTTGGAG F- TGTACTGTGTAGTTAGAGAAAATGGC R- TACTAAGTTAAAGATGTGAGGAATC F- AGATCAAGGCAAAAATATCAGGAGC R- ATAAGGTGTTTTAAAGTGGCTGAG
NCBI Reference Sequence: NC_000003.12.
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J. Jasiecki et al. / Chemico-Biological Interactions xxx (2016) 1e8
3
Fig. 1. Human BCHE gene structure and position of detected genetic variants. BCHE exons are indicated in red (coding) and black (50 UTR and 30 UTR) blocks. Introns are marked gray. PCR primers used for amplification and sequencing are denoted as horizontal arrows. Green vertical lines show the locations of ten BCHE genetic variants detected in the study. Two types of mutations numbering is presented. Residues numbers denoted in black are for the mature BChE protein and such numbering is present in the PDB database and in most publications about BChE. In contrast, residues numbers in genetics databases (NCBI, denoted in red, NM_000055.2, NP_000046.1) add 28 amino acids for the signal peptide. For example Asp70 in the PDB database is Asp 98 in the NCBI database. NCBI Reference Sequence: NC_000003.12. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
protocol (ThermoFischer Scientific, USA). Sequences were analyzed by Sequencher v.4.10 DNA Software (Gene Codes Corporation, USA) and aligned with BCHE sequence NM_000055.2 (http://www.ncbi. nlm.nih.gov).
3. Results In a studied group of 1200 healthy individuals, BChE activity as well as DN and FN values of serum were measured using 5 mM BTC as a substrate and dibucaine hydrochloride or sodium fluoride at final concentrations of 100 mM and 50 mM, respectively. The results (Fig. 2) showed that in Polish population range of BChE activity is normal with a mean of 3600 U/L. In order to find a new genetic variants of BCHE, the entire coding sequence and 30 UTR of the gene were analyzed in 18 individuals with BChE activity lower than 2000 U/L and 10 with higher than 5745 U/L. In addition, BCHE genotype was determined in 26 individuals with DN < 78 and 15 subjects with FN < lower than wild type. The phenotype and genotype were in a full agreement in all sequenced individuals with aberrant DN and FN. In all 26 and 15 individuals the expected heterozygous c.293A > G/p.D98G (atypical variant) and c.1253G > T/p.G418V (fluoride-2 variant) substitutions were
observed, respectively. The A variant (c.293A > G/p.D98G) was found only in heterozygous (UA) form in 26 (2.1%) individuals of the studied group (Table 2). In 23 out of 26 (88%) UA individuals, the quantitative K variant c.1699G > A/p.A567T (rs1803274) was also detected. K variant is frequently associated with the A variant as described earlier [13]. In addition, K variant was identified in three individuals without A variant. In 13 out of 37 (35%) individuals with K variant, the coincidence of c.-32G > A (rs1126680) and c.1518121T > C (rs55781031) substitutions was found. Interestingly, in 9 out of 18 (50%) subjects with BChE activity below 2000 U/L coincidence of c.-32G > A, (rs1126680), c.1518-121T > C and c.1699G > A was observed (Fig. 2, Table 2). In contrast, most of the individuals with BChE activity 6000 U/L or above were found to be a wild type. Sequencing analysis revealed one specimen with heterozygous c.849G > C/p.E283D (rs16849700) substitution. In this individual the presence of K variant was also detected. Finally, in 28 out of 72 (39%) samples, the genetic variant in 30 UTR of BCHE was identified. Of these, 82% (23/28) were found to carry compound heterozygous or homozygous c.*189G > A (rs3495) substitution and homozygous c.*293dupT (rs3836432) duplication. Additionally, in four of these individuals, the fourth heterozygous alteration was detected downstream of 30 UTR (g.165772869G > T, rs77307863). In one of
Please cite this article in press as: J. Jasiecki, et al., Activity and polymorphisms of butyrylcholinesterase in a Polish population, ChemicoBiological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.030
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J. Jasiecki et al. / Chemico-Biological Interactions xxx (2016) 1e8
Fig. 2. A. BChE activity of 1200 healthy individuals. The enzyme activity of a single person is represented by a single black dot. BChE activity was measured in Ellman's reaction in the presence of 5 mM BTC and 400-fold diluted serum at 25 C. The activity was estimated based on the mean of hydrolysis rate (OD/min) determined from at least 5 spectrophotometer reads. Red dots and blue triangles with corresponding numbering stand for subjects selected for BCHE sequencing. Individuals with gene substitution in 50 UTR, c.32G > A, (rs1126680) are designated as blue triangles B. DN and FN values were calculated in assays performed at a final concentrations of 100 mM and 50 mM dibucaine hydrochloride or sodium fluoride respectively. All individuals below the red threshold line set at DN ¼ 78 have UA phenotype. All assays were performed in triplicate, standard error for all points is less than 3% of the values. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
these individuals the additional c.*349G > A(rs55849903) substitution was found. 4. Discussion BChE is a stoichiometric bioscavenger against poisoning by organophosphate pesticides and nerve agents. The enzyme is also a promising biopharmaceutic to accelerate cocaine breakdown. Native BChE has low catalytic efficiency against the abused cocaine but through a combination of molecular modeling and experimental analysis, mutants of human BChE with considerably improved catalytic efficiency against cocaine were successfully made [42,43]. There are many examples of enzymes found in nature that have been exploited in medicine and biotechnology due to their inherent catalytic properties. The desired biochemical activities are often difficult to achieve using the native form of the enzymes. The rapid progress in the field of genetic engineering have revolutionized the development of commercially available enzymes. A combination of in silico and experimental methods allowed to modify the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specificity and selectivity towards non-natural substrates. High-throughput phenotypical and/(or) genetic population screening is another method for searching proteins with improved biochemical properties. In order to find a new interesting variants of BChE, 1200 healthy individuals were screened for BChE activity by biochemical assay. From the studied group, 28 individuals with BChE activity lower
than 2000 U/L and higher than 5745 were selected for mutational analysis of BCHE gene. In addition, based on phenotypical inhibitor assays with dibucaine and fluoride, 41 samples of the individuals with aberrant DN and FN values (26 UA an 15 UF) were selected for sequencing. It is difficult to differentiate individuals with heterozygous FN phenotypes from a wild type subjects. Differences between FN values in UU and UF phenotypes are minimal. Therefore, in order to find FN phenotype it is recommended to apply DN value, too. This value is slightly lowered in UF subjects but not so much as in UA individuals, where DN < 78 [44]. Moreover, to set appropriate UF phenotypes, samples should be always compared to wild type phenotypes. In all 72 samples selected for mutational analysis, entire coding sequence of BCHE with surrounding intronic regions and 30 UTR fragment were screened. In total, a BCHE genetic variant has been identified in 69 out of 72 sequenced individuals. In 54 (75%) individuals a genetic variant has been found within a coding sequence of the gene. In this study, the most common BCHE alterations were K variants, Atypical and Fluoride with the following 0.031, 0.0216 and 0.0125 frequencies. The heterozygous (n ¼ 28) or homozygous (n ¼ 9) K variant was observed in 37 individuals. Both atypical and fluoride variants were found only in heterozygous form in 26 and 15 individuals, respectively. In Caucasian populations, the allele of the atypical variant is detected in one out of 25 subjects whereas the K variant allele is found in one out of four persons with a homozygous incidence of 1/63 individuals [2]. Therefore, the frequency of atypical variant observed in this study is in agreement with a previous reports. The K allele frequency was much lower than described previously for populations. In the study, only samples with respect to atypical and fluoride phenotype and
Please cite this article in press as: J. Jasiecki, et al., Activity and polymorphisms of butyrylcholinesterase in a Polish population, ChemicoBiological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.030
J. Jasiecki et al. / Chemico-Biological Interactions xxx (2016) 1e8
5
Table 2 Genetic variants of BCHE detected in a studied group. Number
Subject no.
BChE Activity [U/L]
DN
Exon 1
Exon 2.1
Exon2.2
Exon 2.3
Exon 2.4
30 end of intron 2
Exon 3
Exon 4
1564
83
WT
WT
WT
WT
WT
WT
c.293A > G, htz
WT
WT
c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, c.1518121T > C, WT
c.1699G htz c.1699G htz c.1699G hom c.1699G htz c.1699G hom c.1699G htz c.1699G hom c.1699G htz c.1699G hom c.1699G hom c.1699G hom c.1699G hom c.1699G hom c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz
14
28
2821
72
c.-32G > A, htz c.-32G > A, htz c.-32G > A, hom c.-32G > A, htz c.-32G > A, htz c.-32G > A, htz c.-32G > A, htz c.-32G > A, htz c.-32G > A, htz c.-32G > A, hom c.-32G > A, htz c.-32G > A, htz c.-32G > A, htz WT
15
33
2609
70
WT
WT
c.293A > G, htz
WT
WT
WT
WT
16
35
3176
70
WT
WT
c.293A > G, htz
WT
WT
WT
WT
17
236
3543
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
18
246
3565
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
19
248
1663
75
WT
WT
c.293A > G, htz
WT
WT
WT
WT
20
251
3343
75
WT
WT
c.293A > G, htz
WT
WT
WT
WT
21
353
2522
76
WT
WT
c.293A > G, htz
WT
WT
WT
WT
22
358
2611
75
WT
WT
c.293A > G, htz
WT
WT
WT
WT
23
414
2645
72
WT
WT
c.293A > G, htz
WT
WT
WT
WT
24
470
2213
75
WT
WT
c.293A > G, htz
WT
WT
WT
WT
25
475
3001
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
26
547
1623
86
WT
WT
WT
c.849G > C, htz
WT
WT
WT
27
588
2694
72
WT
WT
c.293A > G, htz
WT
WT
WT
WT
28
603
2208
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
29
766
3484
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
30
836
6350
83
WT
WT
WT
WT
WT
WT
WT
31
855
2804
80
WT
WT
WT
WT
WT
WT
32
884
3285
73
WT
WT
c.293A > G, htz
WT
c.1253G > T, htz WT
WT
WT
33
922
2656
72
WT
WT
c.293A > G, htz
WT
WT
WT
WT
34
1057
2823
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
35
1070
2373
76
WT
WT
c.293A > G, htz
WT
WT
WT
WT
1 2 3 4 5 6 7 8 9 10 11 12 13
16 198 252 327 615 708 772 789 861 926 1002 1016 1146
1893 1991 3132 2755 5991 1975 6326 1943 1828 1900 1819 1945
87 86 85 61 85 83 83 85 84 85 67 87
WT WT WT WT WT WT WT WT WT WT WT WT
WT WT WT c.293A > G, htz WT WT WT WT WT WT c.293A > G, htz WT
WT WT WT WT WT WT WT WT WT WT WT WT
WT WT WT WT WT WT WT WT WT WT WT WT
htz WT htz WT hom WT htz WT htz WT htz WT htz WT htz WT htz WT hom WT htz WT htz WT htz WT
c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz c.1699G htz
30 UTR
> A, > A,
c.*189G > A, htz; c.*293dupT, hom WT
> A,
WT
> A, > A,
c.*189G > A, htz; c.*293dupT, hom WT
> A,
WT
> A,
c.*293dupT, hom
> A, > A,
c.*189G > A, htz; c.*293dupT, hom WT
> A,
c.*293dupT, hom
> A,
c.*293dupT, hom
> A,
c.*293dupT, hom
> A,
WT
> A,
WT
> A,
WT
> A,
WT
> A,
WT
> A,
WT
> A, > A,
c.*189G > A, htz; c.*293dupT, hom WT
> A,
WT
> A,
WT
> A,
WT
> A,
WT
> A,
WT
> A,
> A,
c.*189G > A, htz; c.*293dupT, hom; c.*349G > A, htz WT
> A,
WT
> A,
WT
> A,
> A,
c.*189G > A, htz; c.*293dupT, hom; c.*513G > T, htz WT
> A,
WT
> A, > A,
c.*189G > A, htz; c.*293dupT, hom WT
> A,
WT (continued on next page)
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6
J. Jasiecki et al. / Chemico-Biological Interactions xxx (2016) 1e8
Table 2 (continued )
*
Number
Subject no.
BChE Activity [U/L]
DN
Exon 1
Exon 2.1
Exon2.2
Exon 2.3
Exon 2.4
30 end of intron 2
Exon 3
Exon 4
30 UTR
36
1095
3170
71
WT
WT
c.293A > G, htz
WT
WT
WT
WT
WT
37
1150
2834
74
WT
WT
c.293A > G, htz
WT
WT
WT
WT
38
195
2979
58
WT
WT
WT
WT
WT
WT
39 40 41
481 524 11
3042 2496 2640
72 75 78
WT WT WT
WT WT WT
c.293A > G, htz; c.635C > T, htz c.293A > G, htz c.293A > G, htz WT
c.1699G > A, hom c.1699G > A, htz WT
WT WT WT
WT WT c.1253G > T, htz
WT WT WT
WT WT WT
WT WT WT
42
217
2195
81
WT
WT
WT
WT
>
WT
WT
WT
43
381
3596
82
WT
WT
WT
WT
>
WT
WT
WT
WT
44
404
5092
81
WT
WT
WT
WT
>
WT
WT
WT
WT
45
448
3195
83
WT
WT
WT
WT
>
WT
WT
WT
WT
46
477
3780
81
WT
WT
WT
WT
>
WT
WT
WT
WT
47
504
4497
83
WT
WT
WT
WT
>
WT
WT
WT
WT
48
557
4545
82
WT
WT
WT
WT
>
WT
WT
WT
WT
49
587
2909
79
WT
WT
WT
WT
>
WT
WT
WT
WT
50
686
2555
81
WT
WT
WT
WT
>
WT
WT
WT
WT
51
750
3269
78
WT
WT
WT
WT
>
WT
WT
WT
WT
52
887
3083
82
WT
WT
WT
WT
>
WT
WT
WT
WT
53
1059
3331
82
WT
WT
WT
WT
>
WT
WT
WT
WT
54
1190
2928
80
WT
WT
WT
WT
>
WT
WT
WT
WT
55 56
3 5
1665 4233
84 83
WT WT
WT WT
WT WT
WT WT
c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz c.1253G T, htz WT WT
c.*189G > A, htz; c.*293dupT, hom WT WT c.*189G > A, hom; c.*293dupT, hom; c.*513G > T, hom WT
WT WT
WT WT
WT WT
57
102
5887
84
WT
WT
WT
WT
WT
WT
WT
WT
58
133
2642
86
WT
WT
WT
WT
WT
WT
WT
WT
59
159
1468
85
WT
WT
WT
WT
WT
WT
WT
WT
60
310
1872
86
WT
WT
WT
WT
WT
WT
WT
WT
61
339
5335
87
WT
WT
WT
WT
WT
WT
WT
WT
62 63
392 607
6074 6249
87 85
WT WT
WT WT
WT WT
WT WT
WT WT
WT WT
WT WT
WT WT
64
844
7581
83
WT
WT
WT
WT
WT
WT
WT
WT
65
928
4160
86
WT
WT
WT
WT
WT
WT
WT
WT
66
986
1843
86
WT
WT
WT
WT
WT
WT
WT
WT
67
988
1868
87
WT
WT
WT
WT
WT
WT
WT
WT
68
1076
6497
87
WT
WT
WT
WT
WT
WT
WT
WT
69
1099
1542
87
WT
WT
WT
WT
WT
WT
WT
WT
70
1128
6161
85
WT
WT
WT
WT
WT
WT
WT
WT
71 72
1161 1183
729 5745
86 86
WT WT
WT WT
WT WT
WT WT
WT WT
WT WT
WT WT
WT WT
WT c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom; c.*513G > T, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom; c.*513G > T, htz WT c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom c.*189G > A, hom; c.*293dupT, hom; c.*513G > T, hom c.*189G > A, hom; c.*293dupT, hom WT c.*189G > A, hom; c.*293dupT, hom
WT
cited in the Table 2, is known genetic description of 3' UTR e.g. c. *46T>A denotes a T to A substitution 46 nucleotides 3' of the translation termination codon.
Please cite this article in press as: J. Jasiecki, et al., Activity and polymorphisms of butyrylcholinesterase in a Polish population, ChemicoBiological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.030
J. Jasiecki et al. / Chemico-Biological Interactions xxx (2016) 1e8
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Fig. 3. Predicted putative secondary structure (DG ¼ 46.7 kcal/mol) of the 200 first nt of the BCHE mRNA. Structure was generated for 37 C using the MFOLD algorithm and visualized by RnaViz 2 [50]. It is hypothesized that the hairpin (4) comprising the AUG codon affects protein synthesis, but in wild type mRNA the hairpin is not formed due to binding of regulatory proteins via stem loop (3). c.-32G > A (rs1126680) substitution (shown in red circle) probably lower the affinity of binding plausible proteins and allow to form a hairpin (comprising Kozak sequence as well as translation start site AUG) resulting in reduction of the protein synthesis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
for the highest and lowest BChE activity were selected for mutational analysis. Therefore, it was not a population study which can explain differences between a current study and previous reports. The fluoride variant allele has been previously showed to be present only in one out of 160 individuals [2,26]. In a studied population of Polish healthy individuals, the fluoride allele frequency was much higher. Explanation of this phenomena may be that in a current study the very sensitive method for FN phenotyping was applied. Previous studies have shown that K variants were found in linkage disequilibrium between atypical variants such that in 89e96% of atypical variant carriers, the K variant had been also detected [13]. Results of our study support this hypothesis. In 23 out of 26 samples with Atypical variant, the K variant has been also found. On the other hand, among K allele carriers linkage disequilibrium between atypical variants or compound mutation: c.-32G > A (rs1126680) and c.1518-121T > C (rs55781031) was observed. All 37 studied individuals with K genotype were found to have other genetic variants, atypical variant (n ¼ 23), compound mutation c.-32G > A and c.1518-121T > C (n ¼ 13). In a single cases coincidence of K genotype with fluoride variant or c.849G > C (p.E283D) or 30 UTR alteration was identified. It is notable that 50 UTR c.-32G > A, (rs1126680) variant was preferentially found in cis with K variant (c.1699G > A, p.A567T) and intron 2 alteration (c.1518-121T > C, rs55781031). Previous studies have shown that c.-32G > A substitution in exon 1 was associated with lower BChE activity, obesity, hypertriglyceridemia and with gestational diabetes mellitus [36e39], but the alteration has been reported earlier as 116A BChE variant since there two types of numbering exist in databases (Fig. 1). Our results support this observation. In 11 out of 13 individuals with c.-32G > A, lower BChE activity has been observed (2 homozygous with one of the lowest BChE activity). Moreover, a c.-32G > A, (rs1126680) was found in 9 out of 18 (50%) subjects with BChE activity below 2000 U/L. It was also discussed that c.-32G > A is responsible for lowering of BChE activity by affecting transcription and/or translation. It is plausible that this 50 UTR genetic variant can directly modulate the protein translation through alterations in mRNA secondary structure. It is accepted that protein translation efficiency is often affected by mRNA secondary structure since selfcomplementary sequences within the 50 UTR can form stable stem-loop structures that may disturb initiation of translation
[45e47]. Theoretical model of the mRNA secondary structure of the 50 UTR region of BCHE presented in Fig. 3 was predicted using mFold [48]. It is possible that the position of substitutions in the loop of the stem could affect binding of the regulatory proteins and disturb translation efficiency. Such a process was previously described for 50 UTR of the ferritin receptor mRNA which contains stem loop that recruits binding proteins and control translation efficiency [49]. The biochemical determination of the BChE phenotype is a helpful method used to identify individuals at risk for prolonged apnea after receiving succinylcholine. As demonstrated in the present study, the biochemical determination of BChE phenotype of individuals is a precise method. In the presented study, in 72 specimens selected for the mutational analysis, the full concordance between phenotype and genotype was observed. We conclude that detection of BChE phenotype based on biochemical assays can be useful in clinical practice. However, further molecular analysis of selected individuals can give new insights into the molecular basis of BChE activity. Acknowledgments The authors are grateful to M. Wa˛ sewicz for blood samples collection and serum preparation. This work was supported by Faculty of Pharmacy with Subfaculty of Laboratory Medicine, Medical University of Gdansk, KNOW Program funded by Ministry of Science and Higher Education, Republic of Poland. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2016.04.030. References [1] G. Johnson, S.W. Moore, Why has butyrylcholinesterase been retained? Structural and functional diversification in a duplicated gene, Neurochem. Int. 61 (5) (2012) 783e797. [2] O. Lockridge, Review of human butyrylcholinesterase structure, function, genetic variants, history of use in the clinic, and potential therapeutic uses, Pharmacol. Ther. 148 (2015) 34e46. Epub 2014/12/03. [3] P. Masson, O. Lockridge, Butyrylcholinesterase for protection from organophosphorus poisons; catalytic complexities and hysteretic behavior, Arch. Biochem. Biophys. 494 (2) (2010) 107.
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Please cite this article in press as: J. Jasiecki, et al., Activity and polymorphisms of butyrylcholinesterase in a Polish population, ChemicoBiological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.030