Rapid screening for potentially relevant polymorphisms in the human fatty acid amide hydrolase gene using Pyrosequencing™

Prostaglandins & other Lipid Mediators 84 (2007) 128–137

Rapid screening for potentially relevant polymorphisms in the human fatty acid amide hydrolase gene using PyrosequencingTM Alexandra Doehring, Gerd Geisslinger, J¨orn L¨otsch ∗ pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Johann Wolfgang Goethe-University, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany Received 4 May 2007; received in revised form 15 June 2007; accepted 17 June 2007 Available online 22 June 2007

Abstract Introduction: Fatty acid amides such as the endocannabinoid anandamide serve as mammalian lipid transmitters in various physiological and pathophysiological processes including inflammation. They are rapidly degraded by the fatty acid amide hydrolase (FAAH). Non-functional FAAH resulted in reduced inflammatory and nociceptive responses. Evidence suggests that human genetic FAAH variants modulate pain and addiction but their clinical role is still poorly known. We therefore developed reliable high-throughput screening assays for FAAH polymorphisms to facilitate research of their clinical role. Materials and methods: Six simplex PyrosequencingTM assays were developed for FAAH polymorphisms dbSNP rs932816, rs4141964, rs324420, rs324419, rs2295633 and rs12029329 spanning the whole FAAH gene. They are frequent or have been functionally associated. Assays were established and validated in DNA samples from 350 healthy unrelated Caucasians. Results: In all 350 DNA samples the six FAAH polymorphisms were identified correctly as verified by control samples obtained by conventional sequencing. The observed frequencies of homozygous, heterozygous and non-carriers of the minor alleles were in agreement with the Hardy–Weinberg equilibrium. Minor allelic frequencies were: rs932816G > A = 0.26, rs4141964C > T = 0.37, rs324420C > A = 0.20, rs324419C > T = 0.15, rs2295633G > A = 0.35 and rs12029329G > C = 0.25. SNPs were in high linkage except between rs324419 and rs12029329. One single haploblock was identified, spanning either the whole gene range or excluding rs12029329 in the 3 region, depending on the statistical procedure of haloblock assignment. Conclusion: The presently developed PyrosequencingTM assays allow for quick and reliable detection of FAAH genotypes and may facilitate investigations of FAAH genetic functional associations. © 2007 Elsevier Inc. All rights reserved. Keywords: FAAH; Single nucleotide polymorphism (SNP); PyrosequencingTM assays

1. Introduction Fatty acid amides (FAAs) serve as mammalian lipid transmitters involved in various physiological and pathophysiological processes [1–4]. A prototype is anandamide (N-arachidonoyl ethanolamine) [5], which acts as an endogenous cannabinoid. It binds to cannabinoid receptors, preferentially CB1 [6], and participates at physiological functions related to cognition and memory, nociception, motor coordination, temperature homeostasis, and inflammatory responses [7]. Although its effects are similar to those of Δ9 -tetrahydrocannabinol (THC) [8–10], the major ∗

Corresponding author. Tel.: +49 69 6301 4589; fax: +49 69 6301 7636. E-mail address: [email protected] (J. L¨otsch).

1098-8823/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2007.06.001

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Fig. 1. Schematic diagram showing pathways involved in the synthesis and metabolism of arachidonic acid. FAAH catalyzes the hydrolysis of anandamide into ethanolamine and arachidonic acid, which is further metabolized by different pathways. The metabolism of arachidonic acid into anandamide by the FAAH acting in reverse requires very high substrate concentrations in vitro. Another potential metabolic pathway of anandamide is the biosynthesis of PC-anandamide via a yet unknown pathway. For the involved genes single nucleotide polymorphisms with functional consequences concerning inflammation (*) or other clinical fields (**) have been found. Abbreviations: FAAH: fatty acid amide hydrolase, COX: cyclooxygenase, LOX: lipoxygenase, CYP: cytochrome P, PC: phosphorylcholine, NAE: N-acyl ethanolamine.

psychoactive component of Cannabis sativa, it acts much shorter than THC [10,11]. This owes to its rapid metabolism by the fatty acid amide hydrolase (FAAH) [12,13], an intracellular membrane-bound serine hydrolase expressed in human brain, pancreas, kidney, skeletal muscle, liver and placenta [14] that splits fatty acid amides into their corresponding acids, e.g., anandamide into arachidonic acid and ethanolamine (Fig. 1). FAAH knockout mice [15–17] or rodents treated with a selective FAAH inhibitor [18,19] had elevated brain levels of FAAs and reduced inflammatory and nociceptive responses. Compared to wild type mice, FAAH−/− mice exhibited decreased nociceptive and inflammatory responses in behavioral assays, accompanied by clearly elevated (>10-fold) endogenous brain levels of anandamide. Mice expressing FAAH specifically in the nervous system (FAAH-NS mice) still exhibited an antiinflammatory phenotype, which was discussed to be due to peripherally elevated concentrations of fatty acid amides acting through a cannabinoid receptor-independent mechanism [16]. FAAH may also modulate the actions of the widely used cyclooxygenase inhibitors. The inhibition of cyclooxygenases by non-steroidal antiinflammatory drugs was shown to result in decreased quantities of the pro-inflammatory prostaglandins and, because one of its metabolic pathways was blocked, increased concentrations of the arachidonic acid. The latter may be metabolized into endocannabinoids such as anandamide, which is catalyzed by FAAH acting in reverse, however, requiring very high substrate concentrations [20,21]. This evidence suggests that FAAH regulation has phenotypic consequences for inflammation. Therefore, genetic polymorphisms of FAAH are candidates for individual modulation of inflammation, pain and other endocannabinoidrelated biological functions, with potential impact on personalized therapy or risk estimation. A search for “FAAH Homo sapiens” at the NCBI SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/) found 340 single nucleotide polymorphisms (SNPs). For a few of them, functional associations have been reported. Specifically, SNP c.385C > A (nomenclature according to http://www.hgvs.org/mutnomen [22]; dbSNP rs324420 according to the NCBI SNP database) in exon 3, exchanging proline by threonine at protein position 129, caused decreased FAAH expression and net activity, probably due to altered post-translational steps [23]. This variant was associated with overweight and obesity [24] and was over-represented among substance abusers [25–27]. On the other hand, FAAH variants rs932816G > A (5 untranslated region), rs4141964T > C (intron 1) and rs2295633G > A (intron 8) have been associated with increased cold pain perception [28], which is compatible with decreased endocannabinoid concentrations and suggests increased enzymatic activity although molecular consequences of these SNPs have not yet been analyzed. Considering the already known functional associations of FAAH genetic variants and the central role of FAAH in the regulation of fatty acid amides, FAAH is an important candidate for genetic modulation of many further physiological functions of fatty acid amides, such as the endocannabinoid system. To facilitate further investigations towards the functional impact of FAAH genetic variants we developed rapid and reliable screening methods for FAAH SNPs, which was

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based on the previously reported clinical relevance. We included variants covering the whole FAAH gene range organized into 15 exons and which have been associated with functional consequences (rs932816G > A, rs4141964T > C, rs324420C > A, and rs2295633G > A) or are frequent with a minor allele frequency > 0,1 (rs324419C > T in exon 7 not causing an amino acid exchange and not yet functionally characterized; c.*620G > C (rs12029329) in the 3 untranslated region). 2. Materials and methods 2.1. DNA source and extraction Blood drawn into ammonium heparin containers was available from 350 unrelated healthy Caucasians who had consented into genotyping (Ethics approval obtained). Genomic DNA was extracted from 200 ␮l blood on a BioRobot EZ1 workstation applying the blood and body fluid spin protocol provided in the EZ1 DNA Blood 200 ␮l Kit (Qiagen, Hilden, Germany). 2.2. Assay development During PyrosequencingTM [29–31], an oligonucleotide (sequencing primer) binds to purified single-stranded DNA close to the mutation site and is elongated by specific dispensing of deoxynucleoside triphosphates (dNTPs). If the dispensed deoxynucleoside triphosphate matches the next nucleotide of the DNA, it is incorporated into the oligonucleotide and pyrophosphate is released. The pyrophosphate together with adenosine-5-phosphosulfate (APS) is converted to ATP, which triggers a luciferase catalyzed luciferin-to-oxyluciferin conversion. The resulting light is shown as a peak with a height proportional to the number of incorporated nucleotides in the so-called pyrograms. PCR primers for amplification of FAAH gene segments and sequencing primers were designed using the PyrosequencingTM Assay Design Software (Version 1.0.6; Biotage AB, Uppsala, Sweden). Specificity of primers was verified by gene alignment (http://www.ncbi.nlm.nih.gov/Blast/). 2.3. Polymerase chain reactions PCR reactions were performed in a 50 ␮l assay volume on a Mastercycler ep gradient S instrument (Eppendorf, Hamburg, Germany), using 5 ␮l genomic DNA (20–30 ␮g/ml), mixed with 0.25 ␮l HotStarTaq plus DNA Polymerase (5 U/␮l) (Qiagen, Hilden, Germany), 5 ␮l 10× PCR buffer, 10 ␮l 5× Q-solution, 1 ␮l of dNTP mix (10 mmol/l each) (Qiagen, Hilden, Germany), 0.1 ␮l of one biotinylated and one non-biotinylated PCR primer (each 100 ␮mol/l) (Table 1), and 28.55 ␮l HPLC-purified water. The PCR was done with an initial denaturation step for 5 min at 95 ◦ C, 40–50 cycles with a 30 s denaturation step at 95 ◦ C, annealing step at primer-specific temperatures (Table 1) for 30 s and elongation step at 72 ◦ C for 30 s, followed by a final elongation step at 72 ◦ C for 5 min. After PCR amplification several samples were evaluated by electrophoresis on ethidium bromide-stained agarose gels; the sizes of the PCR products were 254 bp for rs932816G > A, 344 bp for rs4141964C > T, 138 bp for rs324420C > A, 232 bp for rs324419C > T, 214 bp for rs2295633 G > A and 203 bp for rs12029329G > C. 2.4. PyrosequencingTM analysis Twenty-five microlitres of PCR template (biotinylated and non-biotinylated strands) were pipetted into a well containing 3 ␮l streptavidin-coated sepharose beads (Streptavidin Sepharose High Performance, GE Healthcare BioSciences AB, Uppsala, Sweden), 37 ␮l binding buffer (10 mmol/l Tris(hydroxymethyl)-aminomethan, 2 mol/l NaCl, 1 mmol/l EDTA and 0.1% polyoxyethylenesorbitan monolaureate (Tween 20); pH 7.6) and 15 ␮l HPLC-purified water. This mixture was incubated for 10 min at room temperature (shaker speed 800/min) to form specific complexes between streptavidin-coated sepharose beads and biotinylated strands. The complexes were purified and separated from the non-biotinylated strands on a PyroMark Vacuum Prep Worktable (Biotage, Uppsala, Sweden): by suction, the specific complexes were captured on PrepTool filters, purified into 70% ethanol for 5 s, denatured in 0.2 mol/l NaOH for 5 s, and washed with Tris(hydroxymethyl)-aminomethan (10 mmol/l in water) for 5 s. The complexes were then transferred to a PSQ 96 Plate Low (Biotage, Uppsala, Sweden) prefilled with 0.16 ␮l of 100 ␮mol/l sequenc-

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Table 1 PCR primers, PCR conditions and sequencing primers for genotyping FAAH SNPs using PyrosequencingTM

dbSNP rs932816G > A

dbSNP rs4141964C > T

dbSNP rs324420C > A

dbSNP rs324419C > T

dbSNP rs2295633G > A

dbSNP rs12029329G > C

Primers (each 100 ␮mol/l)

PCR conditions

Forward: 5 -ATCTAACAGCTGGCATGTCTGG-3 , reverse: 5 -biotin-AAGCGCGCCAGAGCCTAG-3 , sequencing: 5 -CTGTGGTGCCCAACC-3 Forward: 5 -ATCCCACTGGCATTGTGGTTC-3 , reverse: 5 -biotin-CGCTCAGCACACATTTTCATTCT-3 , sequencing: 5 -CATGTTCACTGATTGGTC-3 Forward: 5 -ATCCTTTCAATCTGGAACTGACTG-3 , reverse: 5 -biotin-GCATTCTAAAATCAGGGAAAATCA-3 , sequencing: 5 -TGAGACTCAGCTGTCTCA-3 Forward: 5 -biotin-CCCCAGGCTGCTCTAGGTC-3 , reverse: 5 -CAGCGGTCCACATTCATGC-3 , sequencing: 5 -AGCGGAACATGTCCT-3 Forward: 5 -CGGGGCACACGGTATGACT-3 , reverse: 5 -biotin-GCACAGGGCTAAAGTTCTCCA-3 , sequencing: 5 -GTCGGGGTGAACTGT-3 Forward: 5 -CCCTGCTCTGCTGGACACT-3 , reverse: 5 -biotin-CCAATGCCCACCACACAG-3 , sequencing: 5 -CCTTCTCTCTCCAGGA-3

Amplification for 45 cycles, annealing temperature 50 ◦ C Amplification for 45 cycles, annealing temperature 55 ◦ C Amplification for 45 cycles, annealing temperature 41 ◦ C Amplification for 50 cycles, annealing temperature 50 ◦ C Amplification for 40 cycles, annealing temperature 61 ◦ C Amplification for 45 cycles, annealing temperature 60 ◦ C

ing primer and 39.84 ␮l annealing buffer (20 mmol/l Tris(hydroxymethyl)-aminomethan and 2 mmol/l magnesium acetate tetrahydrate; pH 7,6). Afterwards, the plate was heated at 80 ◦ C for exactly 2 min in a PSQ 96 Sample Prep Thermoplate Low (Biotage, Uppsala, Sweden) and cooled down to room temperature. Sequencing analysis took place on a Biotage PSQ 96 MA System with Biotage enzyme mix, substrate mix and nucleotides (Pyro Gold Reagents reagent set for SNP genotyping and mutation analysis). For all assays, five samples of each genotype were selected, sequenced by conventional means (AGOWA GmbH, Berlin, Germany) and used as positive controls during PyrosequencingTM . 2.5. Statistics The correspondence between the observed number of homozygous and heterozygous individuals and the numbers statistically expected on the basis of the Hardy–Weinberg equilibrium [32] was assessed by χ2 -tests. Binominal 95% Table 2 Summary of detected allelic and haplotype frequencies of FAAH variants in 350 Caucasians

FAAH SNPs rs932816G > A rs4141964C > T rs324420C > A rs324419C > T rs2295633G > A rs12029329G > C

Allelic frequency of variant allele

95% confidence interval

0.26 0.37 0.20 0.15 0.35 0.25

0.23–0.29 0.34–0.41 0.17–0.23 0.12–0.18 0.31–0.38 0.22–0.28

rs932816 rs4141964 rs324420 rs324419 rs2295633 rs12029329

Allelic frequency of haplotype

95% confidence interval

FAAH haplotypes GCCCGG ACCCGG GTACAC GTCTAG GTCTAC GTCCGG

0.37 0.25 0.19 0.1 0.04 0.02

0.33–0.41 0.22–0.29 0.16–0.22 0.08–0.12 0.03–0.06 0.01–0.04

sum

0.97

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confidence intervals (CI) of allelic frequencies of SNPs and haplotypes are indicated in Table 2. FAAH haploblock organization and linkage disequilibrium (LD) between SNPs with parameters D and r2 [33,34] were analyzed using Haploview [35], which employs an accelerated EM algorithm similar to the partition/ligation method [36]. For haploblock assignment, we used both, the confidence interval based procedure [37] and the “solid spine of LD” method [35] implemented in Haploview. 3. Results The PyrosequencingTM assays correctly identified the FAAH genotypes of the DNA samples as indicated by agreement with the implemented positive controls (Fig. 2). Distributions of homozygous, heterozygous and non-carriers of the minor alleles agreed with the expectations from the Hardy–Weinberg law (χ2 -tests: p > 0.27). High linkage was observed between all SNPs (D > 0.8) except between rs324419 and rs12029329 (D = 0.13, r2 = 0.01; Fig. 3). One single haploblock was obtained for the FAAH gene, which, when obtained with the solid spine of LD method spanned all six SNPs from the 5 to the 3 regions, and when obtained with the confidence interval based method reached from the 5 untranslated region to intron 8 (rs2295633), thus excluding rs12029329 in the 3 untranslated region. Depending on the haploblock size, six or five haplotypes were identified, with the most frequent haplotype (37%) composed of major alleles of each SNP (Table 2). 4. Discussion The presented PyrosequencingTM assays provide a reliable and quick genotyping method for six human FAAH polymorphisms. Our SNP selection includes those variants for which a functional association has been reported, i.e., rs324420C > A linked with problem drug use and with obesity [24–27], and rs932816, rs4141964 and rs2295633 that had been associated with increased cold pain perception [28]. A molecular consequence of FAAH SNPs is so far known only for the coding SNP rs324420C > A. It causes reduced expression and net enzymatic activity of FAAH [23]. Molecular consequences of the other SNPs are yet unknown. The SNPs proposed to modify a pain phenotype [28] are located in non-coding intronic regions of the FAAH gene. By their location, a role in mRNA processing, RNA stability, splicing or changes of transcription rates is conceivable. DbSNP rs932816G > A is located in the 5 -untranslated region and according to a PROSCAN run (Version 1.7; http://www-bimas.cit.nih.gov/molbio/proscan/), it is part of the FAAH promoter. However, a subsequent search for transcription factor binding motives using AliBaba 2.1 (http://darwin.nmsu.edu/∼molb470/fall2003/Projects/solorz/aliBaba 2 1.htm) was negative. No molecular consequence of dbSNP rs12029329 in the 3 untranslated region is known. Its present selection to span the complete FAAH gene range was successful when identifying haploblocks using the solid spine of LD method [35] but unsuccessful when using the confidence interval based procedure [37]. Considering the high linkage disequilibrium of rs12029329 with the other SNPs except for rs324419 and in light of the absence of comparative analyses of the predictive clinical relevance of the two haploblock identification methods, the present SNP selection along the whole gene appears to be justified. Therefore, haplotypes obtained from the analyzed SNPs may serve as a basis for association studies addressing various FAAH involving physiological systems or pathophysiological conditions in Caucasians, whereas for other ethnicities haploblock organization is probably different (for details, see HapMap project at http://www.hapmap.org/). FAAH genotyping may enhance individualized approaches to treatment of inflammation and pain. This is suggested by studies using FAAH−/− mice and selective FAAH inhibitors [15–19]. However, given the complex nature of

Fig. 2. Overview of designed forward and reverse simplex assays with expected and observed pyrograms for detecting FAAH polymorphisms. The relevant positions for genotype identification are framed. The DNA sequence on the reverse DNA strand following the sequencing primer accords to the sequence to be analyzed and determines the nucleotide dispensation order. The pyrograms denote the three possible genotypes for each DNA position. Note that the initial part in the pyrograms corresponding to the dispensation of enzyme and substrate mixes has been omitted. From conventional FAAH sequencing only the fragments relevant for identifying the SNPs (marked with frames) are displayed (different style lines code for the nucleotides). Note that in the National Center for Biotechnology Information SNP database (http://www.ncbi.nlm.nih.gov/SNP/) the sequence of some SNPs are given in the reverse strand, according to the sequence from GeneBank/EMBL accession number NT 032977.

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Fig. 2. (Continued ).

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Fig. 3. Linkage disequilibrium of the analyzed FAAH variants in 350 Caucasians using the solid spine of LD method in Haploview. The numbers in the squares indicate the value of D between two SNPs, empty boxes mean D = 1.

inflammation or of the other systems where FAAH plays a role, FAAH genetics are unlikely to provide the only cause for interindividual variability. For inflammation, a few additional genetic modulators are already known. This includes polymorphisms associated with interleukin alpha, beta or the endogenous IL-1 receptor antagonist, which were found to be associated with the occurrence of low back pain [38]. To this add potentially functional COX-2 polymorphisms modulating the effects of coxibs [39] although a functional consequence was disputed [40], or COX-1 polymorphisms modulating the effects of acetyl salicylic acid [41]. In addition to the here reported FAAH gene, other modulators might contribute to the effects of anandamide. Recently, the existence of a second mammalian fatty acid hydrolase (FAAH-2) was revealed, being expressed in primates, but not in mice or rats [42]. However, concerning the metabolism of anandamide the original FAAH enzyme displayed a much greater hydrolytic activity than FAAH-2. Therefore, FAAH-2, also being only weakly expressed in the human brain [42], seems to play a minor role regarding pain and inflammation. Beyond that, about alternative metabolism routes for N-acyl ethanolamines (NAEs) such as anandamide little is known. Besides in vitro studies suggesting the involvement of different enzymes [43–45], in vivo studies using FAAH−/− mice showed an increase in O-phosphorylcholine-NAEs, a class of endogenous molecules in the mammalian central nervous system, with the corresponding pathway remaining unclear so far [46] (Fig. 1). In conclusion, clinical data regarding the functional role of single nucleotide polymorphisms in the FAAH gene is scarce and has not yet entered clinical practice. Therefore, further research is required for which we developed fast and reliable PyrosequencingTM assays to facilitate detection and clinical evaluation of relevant FAAH genotypes. Acknowledgements The investigations were supported by the European Research Training Group “Roles of eicosanoids in biology and medicine” (GRK 757) at the Johann Wolfgang Goethe University and the Karolinska Institute in Stockholm. References [1] McKinney MK, Cravatt BF. Structure and function of fatty acid amide hydrolase. Annu Rev Biochem 2005;74:411–32. [2] Di Marzo V. Endocannabinoids in the new millennium. Prostag Leukot Essent Fatty Acids 2002;66:91–2.

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