Human Molecular Genetics, 2000, Vol. 9, No. 9 1273-1281
© 2000 Oxford University Press
A transcription-activating polymorphism in the ACHE promoter associated with acute sensitivity to anti-acetylcholinesterases
1Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, 2 The IVF Unit, Department of Obstetrics and Gynecology, Barzilai Medical Center, Ben-Gurion University of the Negev, Ashkelon 78306, Israel, 3Department of Obstetrics and Gynecology, Sourasky Medical Center, The Sackler School of Medicine, Tel Aviv University, Tel Aviv 64239, Israel and 4The Research Laboratory, Herzog Hospital, PO Box 35300, Jerusalem 81351, Israel
Received 5 January 2000; Revised and Accepted 27 March 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hypersensitivity to acetylcholinesterase inhibitors (anti-AChEs) causes severe nervous system symptoms under low dose exposure. In search of direct genetic origin(s) for this sensitivity, we studied six regions in the extended 22 kb promoter of the ACHE gene in individuals who presented adverse responses to anti-AChEs and in randomly chosen controls. Two contiguous mutations, a T
A substitution, disrupting a putative glucocorticoid response element, and a 4-bp deletion, abolishing one of two adjacent HNF3 binding sites, were identified 17 kb upstream of the transcription start site. Allele frequencies for these mutations were 0.006 and 0.012, respectively, in 333 individuals of various ethnic origins, with a strong linkage between the deletion and the biochemically neutral H322N mutation in the coding region of ACHE. Heterozygous carriers of the deletion included a proband who presented with acute hypersensitivity to the anti-AChE pyridostigmine and another with unexplained excessive vomiting during a fourth pregnancy following three spontaneous abortions. Electromobility shift assays, transfection studies and measurements of AChE levels in immortalized lymphocytes as well as in peripheral blood from both carriers and non-carriers, revealed functional relevance for this mutation both in vitro and in vivo and showed it to increase AChE expression, probably by alleviating competition between the two hepatocyte nuclear factor 3 binding sites. Moreover, AChE-overexpressing transgenic mice, unlike normal FVB/N mice, displayed anti-AChE hypersensitivity and failed to transcriptionally induce AChE production following exposure to anti-AChEs. Our findings point to promoter polymorphism(s) in the ACHE gene as the dominant susceptibility factor(s) for adverse responses to exposure or to treatment with anti-AChEs. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Chemical hypersensitivity to xenobiotics causes adverse responses to normally subacute levels of a specific chemical, or a group of chemicals. Affected individuals may suffer from exaggerated immune response manifested as inflammation of epithelial and mucosal tissues (1,2). Alternatively, they may present altered capacity for scavenging, modifying or degrading a relevant chemical (3). The aberrantly processed chemical may cause toxicological stress in target tissues, with symptoms that vary in nature and timing according to the tissue, type of exposure and the permeability of the chemical. Mutations leading to such aberrant chemical metabolism were identified largely within coding regions, thus affecting detoxifying protein properties. However, impaired transcriptional activation of genes responsible for detoxification, due to mutations in their regulatory sequences, may be an equally important cause of chemical hypersensitivity. For example, the metal-chelating metallothioneins (4) and some members of the cytochrome P450 chemical-modifying enzyme family (5) respond to exposure to xenobiotics by transcriptional activation which increases protection. Impaired transcriptional activation due to promoter polymorphisms in such genes would hence cause chemical hypersensitivity.
Organophosphate and carbamate acetylcholinesterase inhibitors (anti-AChEs) are often implicated in chemical hypersensitivity (6,7). These agents impair neurotransmission (8,9) and interact with both AChE and its serum homolog, butyrylcholinesterase (BuChE). Their toxicity has led recently to limitation of their use in the USA as agricultural or home-use insecticides (7,8,10). However, agricultural anti-AChEs are prolifically used in developing countries. Other anti-AChEs are employed as Alzheimer’s disease drugs (11) or as prophylactic agents under anticipation of chemical warfare (12). Certain cases of anti-AChE hypersensitivity were attributed to decreased scavenging capacity in carriers of the mutant ‘atypical’ (6) or ‘silent’ (13) BuChE variants or to polymorphisms in the paraoxonase gene, PON1 (14,15), encoding an organophosphate-hydrolyzing enzyme. However, many cases appear to have another, yet undefined origin(s) (16). Previous studies have shown that anti-AChEs promote overproduction of the readthrough AChE splice variant (AChE-R) in the mouse brain (17). This induction of AChE production, and the consequent increase in scavenging capacity, confer short-term protection during exposure to such chemicals (18). Conversely, we postulated that an impaired ability for such an induction would be associated with hypersensitivity to anti-AChEs. Impaired transcriptional response to chemical stressors may be due to deficient association with specific transcription factors. Functional polymorphisms affecting chemical hypersensitivity to anti-AChEs are therefore likely to be found close to consensus motifs for stress-associated transcription factors, e.g. the glucocorticoid receptor (GR) (19) or hepatocyte nuclear factor 3 (HNF3) (20).
Here, we describe the identification of two adjacent mutations in a distal upstream enhancer domain of the human (h)ACHE gene. One of the mutations, identified in a woman who presented with acute hypersensitivity to the anti-AChE pyridostigmine, was found to constitutively increase AChE expression by abolishing one of two adjacent HNF3 binding sites; the other impairs a GR binding site. Increased sensitivity and impaired transcriptional response to anti-AChEs were also observed in transgenic mice overexpressing hAChE. Moreover, these mice presented increased expression of HNF3ß in target tissues. Altogether, our findings imply an association of ACHE promoter polymorphism(s) with anti-AChE hypersensitivity by way of a mechanism that probably involves both early modulators such as the HNF3ß transcription factor and the downstream responding ACHE gene.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Genotyping and sequencing of promoter regions in the ACHE locus
Genomic DNA from 103 subjects, including several individuals who suffered cholinergic symptoms under anti-cholinesterase exposure, was subjected to length polymorphism analysis at each of the six regions detailed in Materials and Methods. This analysis identified a 4-bp deletion in region I of the ACHE promoter in three heterozygous carriers, including proband I, her mother and proband II. Region I, rich in consensus binding sequences for various transcription factors, includes two sites, 19 bp apart, for binding HNF3ß or HNF5/HNF3
. The more upstream of these sites partially overlaps a glucocorticoid response element (GRE) half-palindromic site (Fig. 1B, top) and is abolished by the newly identified deletion (Fig. 1B, bottom). Further screening of region I in 230 additional individuals identified a second mutation, a TA substitution, which impairs the GRE (Fig. 1B, bottom), and established the allele frequencies of the deletion and the substitution as 0.012 and 0.006, respectively (Table 1), in conformity with a Hardy–Weinberg distribution. Although one carrier of the deletion was hospitalized for multi-infarcts, no increase was detected in the prevalence of any of these mutations in 100 patients hospitalized for infarcts. Carriers of both mutations, who were of various ethnic origins, were additionally screened for the H322N mutation in ACHE (21). Five of the six screened deletion carriers were found to be heterozygous for this mutation as well (Table 1). This compares with two heterozygous individuals out of 16 screened non-carriers. No linkage was found between the TA substitution and the H322N mutation.
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Of the total 333 individuals, 177 were also screened for the BCHE ‘atypical’ allele. The allele frequency of 0.025 determined for this sample (Table 1) agrees with the reported frequency range of 0.015–0.05 for ‘atypical’ BCHE in different ethnic groups of the Israeli population (21). In the group currently being analyzed, none of the individuals directed to us due to suspected anti-AChE sensitivity, nor the carriers of the ACHE promoter mutations, carried the BCHE ‘atypical’ allele.
Increased basal levels of blood AChE in carriers of the 4-bp deletion
Two carriers of the deletion showed symptoms that may be associated with cholinergic excitation (see Materials and Methods). Proband III displayed gastrointestinal distress compatible with peripheral nervous system (PNS) excitation, which could be attributed to pesticide exposure in her home vicinity, a crop-growing area. Proband I suffered characteristic acute anti-AChE intoxication in response to a subacute dose of the carbamate anti-AChE pyridostigmine. Several years after this incident, peripheral blood AChE levels were measured in proband I as well as in her parents and were found to be slightly higher than normal in both the proband and her mother (Fig. 2A). No differences were found in serum BuChE activity levels, in BuChE inhibition by succinylcholine or in the BCHE gene sequence (data not shown), excluding BuChE abnormalities and its potential involvement in the proband’s phenotype. Epstein–Barr virus (EBV)-transformed lymphoblast cell lines were established from the second carrier (proband II) and from his non-carrier brother, both negative for the ‘atypical’ BCHE mutation. These cells showed significantly higher AChE activity levels for the deletion carrier than for his brother (Fig. 2A).
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HNF3ß binding assays
The functionality of the two putative HNF3 binding sites in region I was confirmed by electromobility shift assays (EMSAs). Probes containing either one or both of the normal sites all displayed a mobility shift when incubated with cell extracts from HNF3ß-overexpressing COS cells (Fig. 3A). An additional supershift caused by rat HNF3ß-specific polyclonal antibodies identified the binding activity as HNF3ß. In contrast, the mutated upstream site was unable to bind HNF3ß, or to compete with the normal upstream site probe for HNF3ß binding. Nevertheless, the deletion did not interfere with HNF3ß binding to the intact downstream site in a probe representing the mutant allele (Fig. 3A).
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The 4-bp deletion increases HNF3ß-induced ACHE gene expression
To test whether HNF3 binding to the identified sites can modulate transcription from the hACHE gene, we employed the AC6 AChE promoter–reporter DNA constructs to which we added the normal (wtAC6) or deleted (
AC6) region I sequence. When transfected into COS cells, each of these three constructs directed hACHE expression, with both versions of region I slightly enhancing the minimal promoter’s effect (Fig. 3B). Co-transfection with a construct encoding rat HNF3 increased AChE expression by 50 and 100% for the normal and mutant versions, respectively, compared with the minimal promoter. Rat HNF3ß increased AChE expression by 60% for the mutant, but had no effect on the normal allele. Hence, region I includes a functional HNF3-dependent enhancer and the 4-bp deletion increases its effect on AChE expression.
Inherited increases in AChE basal levels impair its anti-AChE-induced overexpression and increase sensitivity to these inhibitors
To test whether AChE overexpression impairs individual responses to subacute doses of anti-AChEs, we used transgenic mice overexpressing human synaptic AChE (22). Exposure to a subacute dose of pyridostigmine caused severe diarrhea in hAChE-transgenics as compared with mild symptoms in control FVB/N mice. Additionally, average survival time under exposure to a lethal dose of diisopropylfluorophosphate (DFP) was 1.9 ± 0.4 min for hAChE-transgenic mice as compared with 5.5 ± 3.3 min for age- and sex-matched control mice (n = 4 for each group; P < 0.05, Student’s t test). Two hours after exposure to a subacute dose of DFP, intestinal AChE was found to be inhibited to 49 ± 38% and 38 ± 7% of its initial activity in transgenic and normal mice, respectively. In situ hybridization revealed ~5-fold increases in the stress-associated AChE-R mRNA transcripts in the small intestine (known to express HNF3ß) (23) of normal mice. Labeling was localized primarily to the intestinal epithelium, muscularis mucosa and intestinal gland regions where proliferation of epithelial cells takes place (Fig. 4). In contrast to normal mice, transgenics displayed initially higher levels of intestinal AChE-R mRNA, yet showed no significant difference between DFP- and saline-injected groups (Fig. 4).
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HNF3ß and AChE are co-expressed in hematopoietic progenitors and in the brain
To contribute to anti-AChE responses, HNF3ß would be expected to be expressed in AChE-producing cells, which respond to anti-AChE exposure. To examine whether this basic condition is met, HNF3ß mRNA was searched for in brain and blood cells by reverse transcriptase–polymerase chain reaction (RT–PCR) and in situ hybridization (ISH) analyses. HNF3ß production was identified in cortical, cerebellar and hippocampal neurons in the mouse brain (Fig. 5A and data not shown) as well as in AChE-expressing megakaryocytes, lymphocytes and CD34-positive human blood cell progenitors (Fig. 5B). Involvement of HNF3ß with the impaired transcriptional response to anti-AChE exposure would further predict altered HNF3ß expression in AChE-overproducing mice. In keeping with this expectation, brain HNF3ß expression, which was barely detectable in normal mice, was conspicuously higher in AChE-overexpressing transgenics (Fig. 5A). Impaired responses to anti-AChE exposure in AChE-overexpressing mammals can therefore include HNF3 contribution in brain, blood and intestinal epithelium alike.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have identified a transcription activating deletion in a distal enhancer domain of the human ACHE promoter and demonstrated impairment of the transcriptional activation response to anti-AChE exposure in transgenic mice overproducing AChE. In both mice and humans, AChE overproduction was associated with anti-AChE hypersensitivity. Together, this suggests ACHE promoter polymorphisms as novel susceptibility factors for anti-AChE hypersensitivity.
Previously described polymorphisms in the coding regions of BCHE (6) and PON1 (14,15) genes were reported to predispose homozygous carriers to slowly manifested central nervous system (CNS) symptoms of anti-AChE poisoning (6,15). In contrast, the ACHE promoter deletion is manifested in dominant and rapidly developing PNS symptoms in heterozygous carriers. The allele frequency of 0.012 defines this deletion as a rare polymorphism in the Israeli population. The other mutation in this region of the ACHE promoter, a TA substitution, was found to be even less abundant, with an allele frequency of 0.006. As carriers of both mutations are of diverse ethnic origin, it is conceivable that these mutations have more than one founder. However, the higher prevalence of the H322N mutation in carriers of the promoter deletion, compared with the reported allele frequency range of 0.06–0.19 in different ethnic groups of the Israeli population (21), suggests a strong linkage between the two mutations.
DNA sequencing, EMSAs and transfection experiments demonstrated that the transcription activation conferred by the 4-bp deletion was due to elimination of a functional binding site for transcription factors of the HNF3 family. HNF3ß is known to enhance transcription of several genes through distal enhancer domains (e.g. mouse serum albumin) (24). When included in constructs carrying the normal allele and tranfected into COS cells, the normal HNF3 site hampered transcriptional activation, probably by interfering with HNF3 binding to a second site located 19 bp (~65 Å) downstream. The higher steady-state blood AChE levels in carriers of the mutation and the elevated expression in immortalized lymphoblasts from such a carrier, compared with normal homozygotes, imply that similar activation occurs when the enhancer domain is in its natural context.
That AChE overexpression may hamper anti-AChE responses was shown in transgenic mice, which presented with hypersensitivity to anti-AChEs, accompanied by impairment in the transcriptional activation of AChE production. Such activation was shown previously to occur in the brain, both under inhibition and in response to psychological stress (25). Our current findings suggest that transcriptional AChE overproduction in intestinal endothelium may contribute towards overcoming toxicological stress by offering protection to the peripheral cholinergic systems. While the detailed cause of impairments in the transcriptional activation of AChE remain to be uncovered, one possible factor may be HNF3ß which presents increased transcription in the brains of AChE-overexpressing mice.
AChE overproduction was manifested under exposure to both the slowly reversible inhibitor pyridostigmine (17) and the irreversible inhibitor DFP (this report). Such feedback response should be crucial for overcoming exposure to irreversible inhibitors, yet is also important during exposure to slowly reversible ones. Thus, de novo synthesis of a new pool of uninhibited enzyme, to replace the non-functioning pool offers a major pathway for down-regulating inhibitor-induced hyper-excitation. The feedback response to anti-AChE exposure preferentially produces the alternatively spliced stress-associated AChE-R variant (ref. 17 and this report). AChE-R displays significantly higher inhibition constants for several anti-AChEs as compared with the normally produced ‘synaptic’ AChE-S variant (A. Salmon and H. Soreq, unpublished data). The increased anti-AChE scavenging capacity of AChE-R supports the notion of its involvement with exposure responses, as it would protect the functionally essential synaptic isoform. However, constitutive AChE overproduction such as that occurring in heterozygous carriers of the upstream deletion may prevent sufficient overproduction of these scavenging AChE molecules (Fig. 6). Under exposure, such carriers would therefore lack a fresh pool of uninhibited enzyme, providing a plausible explanation of their apparent hypersensitivity.
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HNF3 has been reported to participate in the acute-phase response of the liver to trauma or inflammation (20). In addition to its known expression patterns (23), we found HNF3ß to be expressed in hematopoietic cells and hippocampal neurons, both of which are known for their rapid toxicological stress responses (17,26). Our current findings further demonstrate that overexpression of AChE leads to HNF3ß overproduction and creates a predisposition to adverse responses to anti-AChE exposure. The greater sensitivity presented by the hAChE-transgenic mice within minutes of exposure to anti-AChEs, suggests a prior, permanent change in the cholinergic system of these mice which is also attributed to AChE overexpression. This change can be caused by the overexpressed HNF3ß; alternatively, it may involve non-catalytic functions of AChE, such as its previously reported roles in proliferation and differentiation of various cell types (27). Altogether, this predicts the participation of HNF3-regulated AChE in toxicological stress responses in many tissues, and points to HNF3ß as a more general stress-responsive protein than previously realized. AChE regulation by HNF3ß may be further influenced by the ubiquitous stress-related GR (19), known to act with HNF3 either synergistically (28) or competitively (29), plausibly affecting the choice between the two HNF3-binding sites in the ACHE promoter.
We identified the polymorphic region in the hACHE upstream sequence by combined search for regions of sequence homogeneity rich in clustered transcription factor binding motifs. Similar screens may be useful for future identification of polymorphisms, especially in individuals with chemical hypersensitivity and in genes that are subject to transcriptional activation under chemical exposure. The deletion identified in the ACHE promoter appears particularly interesting for screening in individuals suffering from multiple chemical sensitivity (MCS)—a phenomenon still awaiting a clear case definition—which involves multi-organ adverse responses (e.g. gastrointestinal distress and neurological disorders) to normally subacute levels of diverse chemicals (30,31). This syndrome is believed to be caused by neuronal sensitization in specific regions of the CNS limbic system (32). Both stress and anti-AChEs have been shown to elevate AChE expression and to cause cholinergic excitation in the mouse brain (17). As the limbic system is modulated by cholinergic neurons, among others (33), the anti-AChE hypersensitivity presented by proband I suggests a link between increased AChE levels and such MCS-related sensitization.
To conclude, this polymorphism, located 17 kb upstream of the hACHE transcription start site, identifies a new HNF3-binding enhancer domain important for AChE expression. Heterozygosity for the deletion is manifested as constitutive overproduction of AChE; such overproduction, which increases the susceptibility to acute anti-AChE exposures in mice, is likely to be the cause of the hypersensitivity of proband I to pyridostigmine. The proposed link between this mutation and the hypersensitivity points to carriers of this allele as individuals at risk of developing adverse responses under treatment with or exposure to anti-AChEs, which is important in view of the increasing use of anti-AChEs as Alzheimer’s disease drugs (11). Moreover, stress- or anti-AChE-induced increases in AChE levels (17) may cause acquired anti-AChE sensitivity, putting at risk a considerably wider group of individuals (7). This type of chemical hypersensitivity therefore emerges from our study as a complex trait, perhaps involving both early modulators such as transcription factors and downstream responding genes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Subjects
A total of 333 individuals were investigated. Of these, 20 were directed to us for investigation of unexplained symptoms with apparent cholinergic involvement, or were family members of such individuals. The majority were randomly selected, with various medical histories and no reported chemical sensitivity. One hundred were older patients hospitalized due to infarcts. The study was approved by the Institutional Review Board of the Herzog Hospital.
Case reports
Proband I, a 30-year-old woman of Ashkenazi Jewish origin and no significant history of adverse drug responses, received a single oral dose of 30 mg pyridostigmine, a dose considered safe, which is given prophylactically under anticipation of chemical warfare (12). Within 1 h, peripheral blood AChE fell to an almost undetectable level, increasingly severe muscle fasciculations developed, accompanied by intense headache, rhinnorea, lacrimation and frequent urination. These acute symptoms continued for 3 days, and resolved into a 5-day period of extreme fatigue, muscle weakness and general malaise.
Proband II, a 72-year-old man, was hospitalized due to a multi-infarct dementia, a condition caused by blood flow deprivation during a multi-focal stroke, which damages several brain regions.
Proband III was a 39-year-old woman of Turkish origin with a history of three spontaneous abortions performed under general anesthesia who suffered from excessive unexplained vomiting during a fourth pregnancy.
Selection of screened promoter regions
Promoter regions prone to transcription-modifying polymorphisms were sought in a cosmid clone (GenBank accession no. AF002993) spanning the hACHE gene and ~22 kb of its upstream sequence. Clusters of putative transcription factor binding elements were identified using the MatInspector 2.0 program (34) (core similarity of 1, matrix similarity of 0.85; Fig. 1A, top). Homogeneous sequence regions rich in nucleotide pairs and susceptible to slippage mutation (35) were identified using the Window statistical program of the University of Wisconsin GCG software package (Fig. 1A, bottom). The combined searches yielded six regions of interest: region I, which spans a cluster of putative binding elements (e.g. glucocorticoid response, hepatic and ubiquitous transcription factors such as AP-1) and is G/T-rich; region II, with high C/A content and a 30-nt G/A-rich domain; regions III–V, containing sequence motifs suspected of forming protein-binding DNA secondary structures (36); and region VI, reported to be important for ACHE transcription (37,38).
Length polymorphism analysis
Screening involved PCR amplification of assigned genomic DNA regions from peripheral blood lymphocytes, using flanking primer pairs with forward primers 5'-labeled with the fluorophore 6-FAM (Applied Biosystems, Foster City, CA). Electrophoresis (ABI377 automated sequencer, Applied Biosystems) included an internal size marker labeled with a second fluorophore, TAMRA (Applied Biosystems). Fragment length was determined by the ABI GeneScan analysis program. Primers spanned nucleotides 5267–5484 (region I), 9173–9606 (II), 18149–18435 (III), 20709–21029 (IV), 21485–21673 (V) and 22259–22534 (VI), numbered as in the AF002996 reverse sequence.
Genetic screening
DNA samples were subjected to length polymorphism analysis as well as sequencing of region I of the ACHE promoter; samples from 177 individuals were also screened for the D70G ‘atypical’ BCHE allele (21) by PCR and subsequent sequencing. All samples positive for mutations in the promoter were also screened for the catalytically neutral H322N polymorphism in the ACHE coding region (21).
Cholinesterase assays
AChE and BuChE activity levels were assessed by measuring rates of acetylthiocholine or butyrylthiocholine hydrolysis, respectively, as described previously (6).
Plasmid constructs
Constructs were engineered using amplified DNA fragments from normal (wt) or mutant () genomic DNA using primers 5267(+) and 5484(–). Ligation upstream of a minimal 600 bp fragment of the hACHE promoter (37) in the AC6 construct yielded wtAC6 and AC6, both encoding human AChE as a reporter.
Cell cultures, transfection and harvesting
COS-1 cells were grown in a humidified chamber in Dulbecco’s modified Eagle’s medium (Biological Industries, Beit Ha’emek, Israel) supplemented with 10% fetal calf serum (FCS) and 2 mM L-glutamine at 37°C, 5% CO2. Lymphocytes transformed with EBV were used to create lymphoblast cell lines (39). These were grown similarly to COS-1 with 16% FCS. Transfections of COS cells with 2 µg plasmid DNA per well were carried out using Lipofectamine (Gibco BRL Life Technologies, Bethesda, MD) according to the manufacturer’s instructions. Cell homogenates, prepared 2 days post-transfection in phosphate-buffered saline (PBS) containing 1% Triton X-100, were assayed for AChE activity, which is not affected by this detergent. For EMSAs, cells were harvested with cold PBS and homogenized in a buffer containing 10 mM NaH2PO4, 400 mM KCl, 10% glycerol, 1 mM dithiothreitol (DTT), 5 µg/ml aprotinin, leupeptin and pepstatin A, and 5 µM NaF. Supernatants, divided into aliquots, were stored at –70°C until use.
Electromobility shift assays
EMSAs were performed using dsDNA probes homologous to restricted parts of region I, essentially as detailed elsewhere (40). Briefly, ~0.5 ng 32P-labeled dsDNA was incubated (2 h on ice) in a total volume of 36 µl of 150 mM KCl, 83 µg/ml poly(dIdC:dIdC), 5 mM DTT, 1 mM EDTA, 5 mM MgCl2, 12% glycerol, 15 mM Tris pH 7.5 and ~20 µg protein from whole cell extracts. Reaction products were electrophoresed in 5% polyacrylamide gels. For competition experiments, a 100-fold molar excess of the corresponding unlabeled probe was used. Pre-incubation of protein extracts (20 min on ice) with anti-HNF3ß polyclonal antibodies (1:1000 dilution) was employed for supershift assays.
RT–PCR
Reactions were performed as described elsewhere (37). Primers designed according to rat HNF3ß (numbered as in accession no. L09647) were 219(+) and 518(–). Primers for hAChE (numbered as in AF002993) were 25587(+) and 26968(–). Annealing temperatures were 55 and 65°C, respectively. Plus and minus denote forward and reverse primers, respectively.
In situ hybridization
ISH was performed as described elsewhere, using a fluorescent product (41) or the Fast-red product (Boehringer-Mannheim GmbH, Germany) (17) for labeling. Biotinylated, 2'-O-methylated cRNA probes were used, complementary to rat HNF3ß mRNA (positions 281–330 in sequence accession no. L09647) or to mouse AChE-R (positions 32–81 in M76540). Fluorescent signal quantification involved one to three sections from separate animals and ISH experiments. Intact villi (excluding the cell-shedding villus tips) were selected using the Adobe Photoshop program. Following determination of signal range, the percentage of labeled areas out of the total selected areas was calculated.
Animal experiments
Mice were injected i.p. with either 1 mg/kg body weight (in ISH experiments) or 7 mg/kg (for survival experiments) of the organophosphate anti-AChE DFP (Sigma, St Louis, MO). Pyridostigmine (0.2 mg/kg; Sigma) served for testing sensitivity to anti-AChEs. Mice were killed 2 h after injection. All experiments were approved by the Committee for Animal Experimentation at the Institute of Life Sciences.
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ACKNOWLEDGEMENTS |
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We thank Dr Lap-Chee Tsui, Toronto, for the AF002993 cosmid; Drs A. Rosenthal and B. Hinzmann, Jena, for their help with the cosmid sequencing; Drs N. Benvenisty and M. Levinson, Jerusalem, for HNF3 cDNA constructs and antibodies; Ms Luba Nemanov for help with experiments; and Drs Alon Friedman, Beer Sheva and David Glick, Jerusalem, for assistance in clinical examination of proband I and for critically reviewing this manuscript. This study was supported by the Israel Science Foundation (590/97), the US Army Medical Research and Development Command (DAMD17-99-1-9547), the Israeli Ministry of Science (9433-1-97) and Ester Neuroscience, Ltd (to H.S.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +972 2 6585109; Fax: +972 2 6520258; Email: soreq@shum.huji.ac.il
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