A missense mutation in human fatty acid amide hydrolase associated with problem drug use

Abstract

Problem drug use and dependence are neurobehavioral disorders of complex origin. Although environmental factors contribute to drug abuse and addiction, genetic factors also play a significant role estimated at 40-60% of the total risk. Nonetheless, the precise identities of human genes that confer vulnerability to problem drug use remain mostly unknown. Here, we describe a natural single nucleotide polymorphism in the human gene that encodes the principal endocannabinoid-inactivating enzyme, fatty acid amide hydrolase (FAAH), that in homozygous form is strongly associated with both street drug use and problem drug/alcohol use. This single nucleotide polymorphism results in a missense mutation (385C-->A) that converts a conserved proline residue to threonine (Pro129-->Thr), producing a FAAH variant that displays normal catalytic properties but an enhanced sensitivity to proteolytic degradation. Collectively, these results suggest that genetic mutations in FAAH may constitute important risk factors for problem drug use and support a potential link between functional abnormalities in the endogenous cannabinoid system and drug abuse and dependence.

Figures

Figure 1

Percentage of FAAH 385A/385A homozygous subjects in groups of patients reporting street drug use and/or problem drug or alcohol use, matched controls with no drug/alcohol use, alcohol use but no drug use, and nicotine use but no drug/alcohol use. Corrected P values are shown; Bonferroni corrections are also shown in Table 2. Error bars represent standard errors calculated for a binomial distribution of the indicated homozygote frequencies.

Figure 2

Distribution of odds ratios and 95% CIs for data analyses summarized in Table 3. An odds ratio of 1 indicates that the probability of an individual with the homozygous mutation having the specific user trait indicated is no different from the probability of the genotype appearing in the control population.

Figure 3

Relative structural stability of wild-type and Pro129→Thr human FAAH proteins. (A) Heat-inactivation profiles for wild-type and Pro129→Thr FAAH variants. Membranes from COS-7 cells transfected with human FAAH proteins were preincubated for 30 min at the indicated temperatures and then assayed for oleamide hydrolysis activity. Activities are presented as a percentage of the activity observed for control reactions preincubated at 37°C (n = 3 for each temperature). Control activities for oleamide hydrolysis were 2.36 nmol × mg−1 × min−1 and 1.59 nmol × mg−1 × min−1 for wild-type and Pro129→Thr FAAH variants, respectively. (B) Protease sensitivity profiles of wild-type and Pro129→Thr FAAH variants. Membranes from COS-7 cells transfected with human FAAH proteins were preincubated with trypsin for the time points indicated, after which the levels of remaining FAAH protein were measured by covalent labeling with an active site-directed fluorescent probe, FP-rhodamine. Levels of FAAH protein are presented as a percentage of the levels observed for control samples run in the absence of trypsin (n = 3 for each time point). *, P < 0.025; **, P values < 0.01, Student's t test. (Inset) Levels of FAAH estimated by Western blot analysis after incubation of the enzymes with trypsin for the indicated time points.