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

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 Pyrosequencing™ 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 Pyrosequencing™ 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.