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Defects in fatty acid amide hydrolase 2 in a male with neurologic and psychiatric symptoms Sirrs, Sandra; van Karnebeek, Clara D; Peng, Xiaoxue; Shyr, Casper; Tarailo-Graovac, Maja; Mandal, Rupasri; Testa, Daniel; Dubin, Devin; Carbonetti, Gregory; Glynn, Steven E; Sayson, Bryan; Robinson, Wendy P; Han, Beomsoo; Wishart, David; Ross, Colin J; Wasserman, Wyeth W; Hurwitz, Trevor A; Sinclair, Graham; Kaczocha, Martin Mar 28, 2015

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RESEARCH Open AccessDefects in fatty acid amidwith neurologic and psychg5onJdrand pathological neurological processes. Although activation The endocannabinoids anandamide (AEA) and 2-Sirrs et al. Orphanet Journal of Rare Diseases  (2015) 10:38 DOI 10.1186/s13023-015-0248-3Institute, University of British Columbia, Vancouver, CanadaFull list of author information is available at the end of the articleof the ECS has been proposed to have neuroprotective effectsin a number of disease states, abnormal activation of the ECScan also be neurotoxic [1] and alterations in the ECS have beenimplicated in a number of neurological diseases includingParkinson’s disease [2], Huntington’s disease [3], multiplesclerosis [4] and Fragile X syndrome [5]. Alterations inarachidonoylglycerol (2-AG) are degraded by a series offour hydrolases in man [17]. Alterations in endocannabi-noid activity can occur through altered receptor binding[8], alterations in metabolic enzymes [18] or downstreampathways [13]. Fatty acid amide hydrolase (FAAH1; [MIM606581]) is the principal enzyme that hydrolyzes AEA inmammals [18]. Missense variants in FAAH1 have been as-sociated with schizophrenia in genome-wide associatedstudies [19] and homozygosity for a common polymorph-ism in FAAH1 reduces functional activity of the enzymeand is associated with problem drug use [20] which is a* Correspondence: cvankarnebeek@cw.bc.ca†Equal contributors2Departments of Pediatrics, University of British Columbia, Vancouver, Canada3Centre for Molecular Medicine and Therapeutics, Child and Family Researchnaling system that is implicated in a wide variety of normalin man. Alterations in the endocannabinoid system are associated with a wide variety of neurologic and psychiatricconditions, but the phenotype and biochemical characterization of patients with genetic defects of FAAH2 activityhave not previously been described. We report a male with autistic features with an onset before the age of 2 yearswho subsequently developed additional features including anxiety, pseudoseizures, ataxia, supranuclear gaze palsy, andisolated learning disabilities but was otherwise cognitively intact as an adult.Methods and results: Whole exome sequencing identified a rare missense mutation in FAAH2, hg19: g.57475100G > T(c.1372G > T) resulting in an amino acid change (p.Ala458Ser), which was Sanger confirmed as maternally inherited andabsent in his healthy brother. Alterations in lipid metabolism with abnormalities of the whole blood acyl carnitineprofile were found. Biochemical and molecular modeling studies confirmed that the p.Ala458Ser mutation results inpartial inactivation of FAAH2. Studies in patient derived fibroblasts confirmed a defect in FAAH2 activity resulting inaltered levels of endocannabinoid metabolites.Conclusions: We propose that genetic alterations in FAAH2 activity contribute to neurologic and psychiatric disordersin humans.Keywords: Fatty acid amide hydrolase 2, FAAH1, FAAH2, Anandamide, Endocannabinoids, Intellectual developmentaldisability, Ataxia, Anxiety, Psychiatric diseasesBackgroundThe endocannabinoid system (ECS) is a complex neural sig-endocannabinoid signaling have also been implicated in awidevariety of psychiatric conditions including anxiety, depression,schizophrenia [6-14] and autistic spectrumdisorders [15,16].Sandra Sirrs1†, Clara DM van Karnebeek2,3,4,13*†, Xiaoxue PenRupasri Mandal6, Daniel Testa7, Devin Dubin7, Gregory CarbWendy P Robinson10, Beomsoo Han6, David Wishart6, ColinGraham Sinclair4,12 and Martin Kaczocha5,9AbstractBackground: Fatty acid amide hydrolase 2 (FAAH2) is a hy© 2015 Sirrs et al.; licensee BioMed Central. ThCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.e hydrolase 2 in a maleiatric symptoms†, Casper Shyr3,4,10, Maja Tarailo-Graovac3,4,10,etti8, Steven E Glynn9, Bryan Sayson2,4,Ross2,4,10, Wyeth W Wasserman3,4,10, Trevor A Hurwitz11,olase that mediates the degradation of endocannabinoidsis is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,was shown to harbor a distinct missense variant in theEthical issuesSirrs et al. Orphanet Journal of Rare Diseases  (2015) 10:38 Page 2 of 10This study was initiated as part of the Treatable IntellectualDisability Endeavor in British Columbia. Informed consentwas obtained from the individuals involved in this studyand approved by the ethics committees of the University ofBritish Columbia (Vancouver, Canada).Whole exome sequencingGenomic DNA was isolated from the peripheral bloodof the patient, unaffected brother, as well as parentsusing standard techniques. Whole exome sequencingwas performed for all four family members using the IonAmpliSeq™ Exome Kit and Ion Proton™ System from LifeTechnologies (Next Generation Sequencing Services,UBC, Vancouver, Canada). An in-house designed bio-informatics pipeline [28] was used to align the reads tothe human reference genome version hg19 and to iden-tify and assess rare variants for their potential to disruptprotein function. The average coverage was 100X. Rarevariants were identified based on a comparison againstalleleic frequencies from dbSNPv138, Exome VariantServer and an in-house database of more than 260exomes and genomes using minor allele frequency(MAF) as <1% as the threshold. The remaining variantswere subsequently screened under a series of geneticFAAH2 gene. Using a variety of techniques, we provideevidence that this mutation compromises FAAH2 activ-ity and propose that this alteration in endocannabinoidsignaling may be the cause of the phenotype observed inthis patient.Methodsmodel for psychiatric disease. However, recently a secondenzyme (FAAH2 [MIM 300654]), present in man but notrodents, was identified and shown to mediate endocanna-binoid degradation [21,22]. Inhibition of FAAH1 orFAAH2 would be expected to increase levels of endocan-nabinoids available for receptor binding. The ECS is impli-cated in neural development [10] and overactivation ofthe ECS during pregnancy has been associated withgrowth and neurocognitive deficits in human offspring[23-25]. Thus it is conceivable that mutations that affectFAAH2 enzyme activity could result in a neurologic orpsychiatric phenotype.The FAAH2 gene resides on the X chromosome inman and has been identified in recent genome wide as-sociation studies as a possible candidate gene for X-linked intellectual disability [26] and autism spectrumdisorders [27]. Here, we present a novel case where amale patient with neurologic and psychiatric symptomsmodels described in the text. We have submitted theFAAH2 missense variant to the LSDB gene variantdatabase (http://grenada.lumc.nl/LOVD2/MR/variants?action=search_unique&select_db=FAAH2).Cloning and transfectionsHuman FAAH1 (NM_001441) and FAAH2 (NM_174912)cDNAs were subcloned into pcDNA4 expression vectors.A FLAG epitope tag was inserted in the C-terminus ofFAAH2. Site-directed mutagenesis was performed usingQuikchange. All constructs were verified by DNA sequen-cing. Human 293T cells and primary fibroblasts were cul-tured in DMEM supplemented with 10% fetal bovineserum, 100 U/ml penicillin/streptomycin, and 2 mM L-glutamine. Transfections were performed using the GenJetPlus transfection reagent (SignaGen, Rockville, MD) ac-cording to the manufacturer’s instructions.Western blottingWestern blot experiments were performed exactly asdescribed [29]. Blots were probed mouse anti-FAAH1(1:1000, Abcam #Ab54615), mouse anti-GAPDH (1:5000,Abcam #Ab8245), mouse anti-FLAG (1:1000, Sigma #F1804),or rabbit anti-FAAH2 (1:200, Abcam #Ab103724)antibodies. The blots were then incubated with the re-spective goat anti-mouse or goat anti-rabbit IgGHRP-conjugated antibodies (Life Technologies), devel-oped using the Immun-star HRP substrate (Bio-Rad), andscanned using a C-DiGiT scanner (Li-COR).Enzyme assaysEnzyme assays using [14C]AEA or [14C]PEA as substrateswere performed exactly as described [22].Endocannabinoid quantification in fibroblastsEndocannabinoid levels were quantified exactly as de-scribed [30]. Briefly, two million affected and unaffectedfibroblasts were harvested and the lipids extracted byaddition of 2:1:1 chloroform:methanol:Tris containingthe appropriate deuterated standards, followed by tworounds of centrifugation at 3000 g. The chloroform phasewas collected, dried, and the lipids resuspended in 2:1chloroform:methanol and quantified on a Thermo TSQQuantum Access Triple Quadropole mass spectrometer.Structure modeling and analysisA FAAH2 model structure was produced by threadingthe sequence of wild-type human FAAH2 onto a hu-manized rat FAAH1 template structure (PDB ID:2WAP)in Modeller 9.13 [31]. The p.Ala458Ser mutation wasmodeled in Coot [32] with a rotamer selected tominimize steric clashes as determined by Molprobity[33]. Atoms within 3.5 Å and capable of forming hydro-gen bonds with the hydroxyl group of p.Ser458 wereidentified in Coot.Sirrs et al. Orphanet Journal of Rare Diseases  (2015) 10:38 Page 3 of 10Lipidomic assessment using combined direct flow injectionand LC-MS/MS compound identification and quantificationWe have applied a targeted quantitative metabolomicsapproach to analyze the serum samples using a combin-ation of direct injection mass spectrometry (Absolute-IDQ™ Kit) with a reverse-phase LC-MS/MS Kit. The Kitis a commercially available assay from BIOCRATES LifeSciences AG (Austria). This kit, in combination with anABI 4000 Q-Trap (Applied Biosystems/MDS Sciex) massspectrometer, can be used for the targeted identificationand quantification of up to 180 different endogenousmetabolites including amino acids, acylcarnitines, bio-genic amines, glycerophospholipids, sphingolipids andsugars. The method used combines the derivatizationand extraction of analytes, and the selective mass-spectrometric detection using multiple reaction moni-toring (MRM) pairs. Isotope-labeled internal standardsand other internal standards are integrated in Kit platefilter for metabolite quantification. The AbsoluteIDQ kitcontains a 96 deep-well plate with a filter plate attachedwith sealing tape, and reagents and solvents used to pre-pare the plate assay. First 14 wells in the Kit were usedfor one blank, three zero samples, seven standards andthree quality control samples provided with each Kit. Allthe serum samples were analyzed with the AbsoluteIDQkit using the protocol described in the AbsoluteIDQ usermanual. Briefly, serum samples were thawed on ice andwere vortexed and centrifuged at 13,000 × g. 10 μL ofeach serum sample was loaded onto the center of the fil-ter on the upper 96-well kit plate and dried in a streamof nitrogen. Subsequently, 20 μL of a 5% solution ofphenyl-isothiocyanate was added for derivatization. Afterincubation, the filter spots were dried again using an evap-orator. Extraction of the metabolites was then achieved byadding 300 μL methanol containing 5 mM ammoniumacetate. The extracts were obtained by centrifugation intothe lower 96-deep well plate, followed by a dilution stepwith kit MS running solvent. Mass spectrometric analysiswas performed on an API4000 Qtrap® tandem mass spec-trometry instrument (Applied Biosystems/MDS AnalyticalTechnologies, Foster City, CA) equipped with a solvent de-livery system. The samples were delivered to the massspectrometer by a LC method followed by a direct injec-tion (DI) method. The Biocrates MetIQ software was usedto control the entire assay workflow, from sample registra-tion to automated calculation of metabolite concentrationsto the export of data into other data analysis programs. Atargeted profiling scheme was used to quantitatively screenfor known small molecule metabolites using multiple reac-tion monitoring, neutral loss and precursor ion scans.ResultsOur patient (Subject II-1 in Figure 1) is a 25 year oldmale who was born after an uncomplicated pregnancyfrom healthy non-consanguinous Caucasian parents. Hehas a healthy brother and extended family history isnegative for similarly affected children or neuropsychi-atric disorders. He presented as a neonate with hypo-tonia, feeding difficulties and central apneic episodesassociated with an abnormal EEG which demonstratedan overabundance of multifocal sharp waves against adiscontinuous asynchronous background that was ab-normal for age. He had delayed motor and languagemilestones and as a child was noted to have ocularapraxia with a moderate to severe dysarthria made worseby a mechanically based limitation in jaw opening.He developed autistic features by the age of 3 yearsand was diagnosed with an anxiety disorder at the age of7 which responded to selective serotonin reuptake inhib-itors. He remained stable until the age of 22 years whenhe presented with recurrent psychogenic seizures char-acterized by episodic atonic collapses heralded by a flut-tering of his eyes and then becoming unable to speak ormove but retaining full awareness of his environment.These episodes would last seconds up to 30 minutes andoccurred once to several times per day. Psychosocialfunctioning until age 22 years had been relatively stable.He completed school with the help of a scribe becauseof problems with hand co-ordination. At the time ofpresentation he was enrolled in university and wasstudying to obtain a joint degree in accounting and fi-nancing with similar assistance.His neurological examination at the age of 25 yearsdemonstrated preserved neurocognitive abilities, severedysarthria, a supranuclear vertical gaze palsy, left facialhypoplasia, limited jaw opening (despite MRI confirmednormal temporomandibular joint anatomy showing onlyvery limited anterior translation of the mandibular headon mouth opening), limited lateral movement of histongue but no associated tongue weakness, bilaterally re-duced fine motor movements, a mild left spastic gaitand a bilateral action tremor. Mental status examinationwas significant for a stereotypy characterized by theplacement of his fisted hands against his head andmostly surfacing during periods of emotional arousal.His mood was initially reported as euthymic but subse-quently he became aware of an underlying depression,which was intermittently associated with suicidal idea-tion and anxiety often preceding his psychogenicseizures.Brain magnetic resonance imaging (MRI) at the age of10 years showed delayed myelination. Repeat MRI at theage of 22 years was unremarkable other than a promin-ent cisterna magna.Over time it became clear that his psychogenic sei-zures represented conversion reactions provoked by se-vere anxiety from a decompensation into a majordepressive illness. As his illness unfolded, his episodesmeriSirrs et al. Orphanet Journal of Rare Diseases  (2015) 10:38 Page 4 of 10became modified by self-punishing behaviors, whichwere manifestations of emotional conflicts. He had apartial response to the combination of sertraline, bupro-pion and quetiapine combined with psychotherapy.Figure 1 Pedigree and Sanger sequencing of a patient with FAAH2results are shown. The pedigree is consistent with X-linked recessive inhBiochemical work up was negative except for an ab-normal whole blood acylcarnitine profile which showedpersistent 10-fold elevations in medium chain specieswith lesser elevations in short and long chain species.Although the patient did not have clinical features tosuggest other disorders which could cause this profile in-cluding multiple acylCoA dehydrogenase deficiency(MADD [MIM 231680]), and defects in riboflavin me-tabolism, these were excluded through single gene se-quencing. The patient did not have a pattern of clinicaldeterioration to suggest Niemann-Pick Disease type C(NPC1 [MIM 257220] and NPC2 [607625]) but, giventhe gaze palsy, defects in NPC1 and NPC2 were ex-cluded through single gene sequencing.Whole exome sequencing was performed as describedin the Methods section. We analyzed the variants pre-dicted to be functional (missense, nonsense and frameshiftchanges, as well as in-frame deletions and splice-site ef-fects) under a series of inheritance models. We only fo-cused on the variants not present in the clinicallyunaffected brother. We identified six hemizygous candi-date genes (FAAH2, KIAA1210, AKAP14, TXLNG,ZMYM3 and NRK), five compound heterozygous candi-date genes (MUC16, CFTR, TTN, KEAP1 and HIVEP2)and one gene (MYO1H) affected by a de novo mutation(see Additional file 1: Table S1). All 12 genes wereevaluated to see if any of the candidates could be associ-ated with the neuropsychiatric phenotype and the abnor-mal acylcarnitine profile, which was present in thispatient. The hemizygous variant located on the X-utation. Pedigree information for our patient and Sanger sequencingtance as the mother is clinically unaffected.chromosome affecting FAAH2 (hg19: g.57475100G > T)was considered a very strong candidate in that it harboreda missense mutation c.1372G > T (p.Ala458Ser), FAAH2is known to play a role in lipid metabolism [21] and al-terations in endocannabinoid metabolism have beendocumented in many different psychiatric disorders[6]. None of the other candidate genes are known toplay a role in lipid metabolism and psychiatric diseases(Additional file 1: Table S1). This variant was con-firmed by Sanger sequencing (Figure 1) as present inthe index case and mother (who had a normal acylcar-nitine profile) and was absent in the father and un-affected brother. The variant is reported in dbSNP(version 138) as heterozygous (rs147173444), nothemi/homozygous, with a low frequency 0.00022, butunreported in NHLBI ESP and our in-house genomedatabase. Among the 61,486 unrelated individuals se-quenced as part of various disease-specific and populationgenetic studies available at the Exome Aggregation Con-sortium (ExAC) website (accessed January 05, 2015), theg.57475100G > T allele was observed in 104 out of 122778allele counts but of those, only 24 are reported as hemi/homozygous. Of note, this dataset includes cohorts of pa-tients with mental illnesses. The variant is predicted dam-aging via all tested prediction tools, including SIFT [34](score 0.041), PROVEAN ([35]; score −2.85) andSirrs et al. Orphanet Journal of Rare Diseases  (2015) 10:38 Page 5 of 10Polyphen2 ([36]; score 0.998) software systems; accordingto the Combined Annotation Dependent Depletion(CADD) [37] scoring system, the variant has a Phred-score of 16.93, considered as likely functionally impactful.X-inactivation studies showed random X-inactivation inthe mother.We subsequently generated and expressed thep.Ala458Ser FAAH2 mutant in human 293T cells to ex-plore the functional consequences of the p.Ala458Sermutation found in our patient. Despite repeated effortsto enhance its expression, the p.Ala458Ser mutant wasconsistently expressed at a reduced level (~30-40%)compared to the wild type (WT) enzyme (Figure 2A).Enzymatic analysis using two established FAAH2 sub-strates, AEA and palmitoylethanolamide (PEA), re-vealed that substrate hydrolysis by the p.Ala458Sermutant was significantly lower compared with the WTenzyme (Figure 2B). The importance of alanine at p.458is supported by its high conservation among the ami-dase signature family of enzymes and absolute conser-vation among FAAH2 orthologs (Additional file 2:Figures S1 and S2). Alanine at p.458 in FAAH2 corre-sponds to alanine at p.478 in FAAH1. We confirmedthat similar to FAAH2, the p.Ala478Ser FAAH1 mutantpossessed reduced catalytic activity (Figure 2C and D)despite similar levels of expression, indicating that theimportance of this residue is not confined to FAAH2and may extend to many members of the amidase sig-nature family.Alanine at p.458 is situated in a region that is inclose proximity to a loop region that houses p.Ser206,one of the two active site serines in this enzyme class([38]; Figure 3A). Molecular modeling suggests thatsubstitution of this residue with a serine may result inadditional hydrogen bonding interactions with sur-rounding residues (e.g., p.Asn195) (Figure 3B). Toexamine the importance of potential hydrogen bondinginteractions induced by the serine mutation, we mu-tated alanine at p.458 to cysteine or valine, residuesthat possess reduced or no hydrogen bonding poten-tial, respectively. Surprisingly, despite robust expres-sion, the p.Ala458Cys FAAH2 mutant displayedcatalytic activity that was similar in magnitude to thep.Ala458Ser mutation (Figure 2A and B). Furthermore,the p.Ala458Val mutant was expressed at a similar level asthe p.Ala458Ser mutant but possessed only marginallyhigher catalytic activity, suggesting that substitution of ala-nine at p.458 with bulkier amino acids, rather than add-itional hydrogen bonding potential, may account for thedefects in FAAH2 activity in the mutants. Collectively, ourdata establish alanine at p.458 as a residue that is essentialfor substrate hydrolysis by FAAH2 and its importance ishighlighted by its high degree of conservation among ami-dase signature enzymes.To explore effects of the FAAH2 mutation in patienttissues, we examined AEA hydrolysis in cultured fibro-blasts derived from our patient and a corresponding un-affected control. Importantly, human fibroblasts do notexpress FAAH1 (Figure 2E) but robustly express FAAH2(Figure 2F), thus permitting an examination of thisFAAH2 mutation upon endocannabinoid metabolism.AEA hydrolysis by fibroblasts bearing the p.Ala458Sermutation was significantly lower compared to unaffectedcontrol fibroblasts (Figure 2G). Treatment of fibroblastswith URB597, a selective dual FAAH1 and FAAH2 in-hibitor [21,39], completely abolished AEA hydrolysis,thereby confirming that AEA hydrolysis observed in fi-broblasts was mediated by FAAH2 (Figure 2G).To further substantiate the functional consequences ofthe p.Ala458Ser mutation, we profiled the levels of theendocannabinoids, AEA and 2-AG, and related N-acylethanolamines, PEA and oleoylethanolamide (OEA),in fibroblasts from our patient and two unrelated con-trols shown in Additional file 2: Figure S3. Our resultsreveal an elevation of AEA levels in the affected fibro-blasts compared to the controls and provide further sup-port of defective AEA hydrolysis by the FAAH2p.Ala458Ser mutant. Furthermore, detailed lipidomicanalysis on serum from our patient showed perturba-tions in multiple lipid species, with elevations in manylong chain species, most marked with lyso-phosphatidylcholine as shown in Additional file 3: Table S2.DiscussionWe have demonstrated for the first time functional im-pairments in FAAH2 activity in a patient with neurologicand psychiatric symptoms who has a missense mutationin FAAH2 that results in reduced enzyme activity. Sev-eral lines of evidence point to a possible causal relation-ship between defects in FAAH2 activity and thepsychiatric/neurologic phenotype in this patient. Reducedactivity of FAAH1 has been associated with other psychi-atric illnesses [20] so it is reasonable to hypothesize thatalterations in the ECS caused by changes in FAAH2 ac-tivity could also have psychiatric consequences. Ourpatient has a phenotype dominated by anxiety symp-toms and alterations in the ECS have been shown inmany psychiatric conditions including anxiety forwhich a biphasic response (low levels of ECS activationreducing anxiety and higher levels of activation in-creasing anxiety) has been demonstrated [6]. Our pa-tient has some mild specific learning disabilities andFAAH1 knock-out mice have been shown to exhibitlearning deficits that can be reversed with the canna-binoid receptor antagonist rimonabant [40]. Our pa-tient had early onset of autistic features and several linesof evidence link alterations in the ECS to autistic symp-toms [16]. Neuroligin-3 mutations associated with autismFigure 2 Functional analysis of FAAH2 mutations. (A) Expression of FLAG-tagged FAAH2 and FAAH2 mutants in 293T cells. The blots wereprobed with anti-FLAG (Sigma # F1804) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Abcam #ab8245) antibodies. (B) AEA andPEA hydrolysis by cell homogenates expressing FAAH2 and FAAH2 mutants. Results were analyzed using one-way ANOVA followed by Dunnett’spost hoc analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 5–7). (C) Expression of FLAG-tagged FAAH1 and the FAAH1 p.Ala478Ser mutant in293T cells. Blots were probed with an anti-FLAG antibody. (D) AEA hydrolysis by WT and p.Ala478Ser FAAH1 ***, p < 0.001 (n = 4). (E) Humanfibroblasts do not express FAAH1. Fibroblasts from a control patient and the FAAH2 p.Ala458Ser affected patient were probed with anti-FAAH1antibodies (Abcam #ab54615). FAAH1 transfected and untransfected 293T cells served as controls. (F) Expression of FAAH2 in fibroblasts from theaffected p.Ala458Ser patient and an unaffected control. Blots were probed with an anti-FAAH2 antibody (Abcam, ab103724). (G) AEA hydrolysisby control fibroblasts and affected FAAH2 p.Ala458Ser fibroblasts in the absence and presence of URB597. *, p < 0.05; **, p < 0.01 versus unaffectedcontrol. #, p < 0.05 versus affected control (n = 4).Sirrs et al. Orphanet Journal of Rare Diseases  (2015) 10:38 Page 6 of 10Sirrs et al. Orphanet Journal of Rare Diseases  (2015) 10:38 Page 7 of 10disrupt endocannabinoid signaling [15]. FAAH1 knockoutmice also have a behavioral phenotype characterized as re-duced emotionality [41]. It must be acknowledged how-ever that mice lack FAAH2 [21] and that FAAH1 andFAAH2 have different substrate affinities [21], so anycomparison of the human FAAH2 deficiency phenotypeto that of the FAAH1 knock-out mouse must be inter-preted with caution.Our patient had a vertical supranuclear gaze palsywhich we postulate could be related to reduced FAAH2activity within the intracellular lipid droplets that serveas sites of cholesterol ester storage and mobilization[22]. FAAH2 is expressed in a number of human tissues[4]. Although the primary function of FAAH2 in thesetissues remains unclear, lipid droplet localization is re-quired for active hydrolysis of endocannabinoids byFAAH2 [22]. Lipid droplets are an important site of tria-cylglyeride and cholesteryl-ester storage and traffickingand endocannabinoid metabolism by FAAH2 may influ-ence these pathways. Intracellular trafficking of cholesterolFigure 3 Modeled structural environments of the disease-causing mutations in FAAH2. Modeled structure of human FAAH2.The protein backbone is shown as a cartoon (white) with the sidechains of A458 (green) and the catalytic triad residues (K131, S206,S230; orange) shown as sticks. (B) Additional interactions formed bythe p.Ala458Ser mutation. Potential hydrogen bonds are observedbetween the side chain of Ser458 (green sticks) and the side chain ofN195 or the carbonyl oxygens of I202 and F237 (cyan sticks). Distancesin angstroms between interacting atoms are shown in dark red.and some fatty acids occurs through a pathway involvingthe Niemann Pick C1 transporter and modulation ofits function alters the eicosanoid pool in cells [42]. InNiemann Pick C, there is defective intracellular lipidtrafficking and patients with this condition developvertical supranuclear gaze palsy over time [43]. There-fore, we hypothesize that a reduction of FAAH2 activ-ity as a result of the mutation in our patient couldalter the levels of FAAH2 substrates and products andfeedback to alter cholesterol trafficking at the level ofthe lipid droplet and lead to this unusual clinicalfeature.The persistent elevations of medium chain acylcarni-tines in both serum and bloodspot in our patient weresuggestive of a dysregulation of fatty acid oxidation(FAO). The pattern was not suggestive of an enzyme-specific block in beta-oxidation but rather was reminiscentof the pattern seen secondary to medium chain triglycer-ide supplementation, with elevations resulting fromincreased flux through the pathway. The role of endocan-nabinoids in FAO is complex with both agonistic and an-tagonistic effects depending on the receptor system andtissue involved. In muscle cells, agonists of the type 2 can-nabinoid receptor have been shown to promote FAOthrough transcriptional upregulation of carnitine palmi-toyltransferase I [44]. Such an effect would be consistentwith the acylcarnitine pattern seen in our patient (in-creased flux).We are aware of 2 other citations where FAAH2 muta-tions have been identified in human patients with neuro-logic diseases. In a study of humans with autismspectrum disorder, 2 males with validated nonsense mu-tations in FAAH2 are listed in a supplementary appendix[27]. In a study of patients with X-linked intellectual dis-ability [26], two brothers were identified with a deletioninvolving FAAH2. Clinical and biochemical details of thepatients were not provided in either citation (and effortsto obtain such information were not successful); further-more, the impact of the mutations on enzymatic activitywere not quantified. The p.Ala458Ser mutation wasfound in 104 alleles in the Exome Aggregation Consor-tium website (accessed January 5 2015) but, as the pedi-gree of our patient suggests an X-linked recessivepattern of inheritance, only the 24 male alleles carryingthis mutation are relevant to our case. The fact that thismutation has been identified in other patients in this largedata set is not surprising because the Exome AggregationConsortium includes patients with mental illnesses. IfFAAH2 mutations are indeed related to common condi-tions like autism spectrum disorder and mental illness,then it could be expected that similar mutations could beidentified in multiple patients. Further research howeveris required to prove a causal relationship between FAAH2mutations and these common conditions.Sirrs et al. Orphanet Journal of Rare Diseases  (2015) 10:38 Page 8 of 10Our hypothesis that the functional impairments inFAAH2 activity could contribute to neurological symp-toms in humans has limitations. Information is conflict-ing on the distribution of FAAH2 in human brain. Somereports (www.brainspan.org) but not others [21] havebeen able to identify FAAH2 transcripts in human brain.Much of the information supporting this hypothesis hasbeen derived from FAAH1 knockout mice and, as micelack FAAH2 [21] and FAAH1 and FAAH2 have differentsubstrate affinities [21], the effects of the single geneknockout in mice may not be applicable to humans.Clinical details from other patients with mutations inFAAH2 are needed to see if the unusual clinical featuresseen in our patient (supranuclear gaze palsy, abnormalacylcarnitine profile) are a consistent feature of thephenotype.ConclusionsThis is the first comprehensive clinical and biochemicalreport of FAAH2 deficiency in humans. We propose thatthe neuropsychiatric features may result from FAAH2deficiency and identification and characterization ofmore cases are required to delineate the full spectrumand to further confirm the demonstrated relation be-tween symptom severity and the degree of enzymaticimpairment. Areas deserving further research includeFAAH2 distribution and ECS actions in human brain(www.brainspan.org) as well as the precise functions ofFAAH2 substrates –both known and unknown. Finally,FAAH2 mutations should be considered in the differen-tial diagnosis of patients with undiagnosed neuropsychi-atric impairment.Additional filesAdditional file 1: Table S1. Variants identified on whole exomesequencing in our male patient with neurologic and psychiatric features.Additional file 2: Figure S1. Sequence comparison of selected amidasesignature family members. The sequences of Bacillus subtilis Glu-tRNAGlnamidotransferase subunit A, Pseudomonas putida mandelamide hydrolase,Homo sapiens FAAH2, Homo sapiens FAAH1, and Talaromyces marneffeiPM1 Acetamidase are shown. Asterisk denotes the location of p.Ala458 inFAAH2. The sequences were aligned with Clustal omega and depicted withboxshade. Figure S2. Sequence alignment of FAAH2 orthologs. Shown arethe sequences of FAAH2 from Bactrocera dorsalis, Manacus vitellinus,Chinchilla lanigera, Callithrix jacchus, Macaca mulatta, Homo sapiens,Pongo abelii, Pteropus Alecto, and Myotis lucifugus. Asterisk denotesthe location of p.Ala458. Figure S3. Endocannabinoid and N-acylethanolamine levels in fibroblasts. PEA, OEA, AEA, and 2-AG levelswere quantified in fibroblasts derived from the FAAH2 p.Ala458Serpatient and two unaffected controls. Quantification was performed asdescribed previously (41). AEA – anandamide, 2-AG - 2-arachidonoylglycerol(2-AG), PEA - palmitoylethanolamide, OEA - oleoylethanolamide.Additional file 3: Table S2. Lipidomic studies from serum of our patient.AbbreviationsECS: Endocannabinoid system; AEA: Anandamide; FAAH: Fatty Acid AmideHydrolase; MAF: Minor allele frequency; MADD: Multiple acylCoAdehydrogenase deficiency; MRM: Multiple reaction monitoring;CADD: Combined Annotation Dependent Depletion; WT: Wild Type;FAO: Fatty Acid Oxidation.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsSS recruited and described the family, coordinated investigations, wrote the1st draft of the manuscript, and coordinated revisions. CvK designed thestudy, led the gene identification process, coordinated investigations andexperiments, and contributed to the 1st draft and revisions of themanuscript. XP performed cloning and mutagenesis, conducted biochemicalanalyses of FAAH2 expression and activity in cells and primary fibroblasts. CSperformed the bio-informatics analysis, contributed to the WES section ofthe manuscript and provided edits.MTG performed the bio-informatics analysis,contributed to the WES section of the manuscript and provided edits. RMcoordinated the lipidomics analyses and contributed to the this section of themanuscript and provided edits. DT performed western blotting of FAAH1 andFAAH2 expression in cells and primary fibroblasts. DD performed westernblotting of FAAH1 and FAAH2 expression in cells and primary fibroblasts. GCconducted biochemical analyses of FAAH2 expression and activity in cells. SEGconducted molecular modeling of the FAAH2 mutant. BS consented the familyfor this study as well as sample collection, provided edits to the manuscriptwith subsequent formatting and preparation for submission. WPR performedand interpreted the X-inactivation studies and provided edits to the manuscripts.BM contributed to lipidomics analyses and manuscript edits. DW led thelipidomics analysis, and contributed to the manuscript 1st draft and edits.CJR performed the confirmatory Sanger sequencing, prepared figures andprovided edits to the manuscripts. WWW led the bio-informatics analyses,contributed to the manuscript 1st draft and provided edits. TAH performedthe psychiatric testing and contributed to the case description as well asmanuscript edits. GS performed and interpreted the acylcarnitine analysis,contributed to gene identification process as well as manuscript 1st draftand edits. MK conceived of the study, carried out assays of FAAH2 activityin cells, performed lipidomic analysis of endocannabinoid levels in fibroblasts,and helped draft the manuscript. All authors read and approved the finalmanuscript.Authors’ informationThese authors Sandra Sirrs, Clara DM van Karnebeek and Xiaoxue Peng arejoint first author.AcknowledgementsWe are indebted to the patient and his family for participation in this study,Margaret O’Riley, RN for her work with the patient, Dr. Maria Penaherrera andMs. Ruby Jiang for X-inactivation studies; Mrs. X. Han for Sanger sequencing;Dr. M. Thomas for consenting; Mrs. M. Higginson for DNA extraction andsample handling; Mr. D. Arenillas and Mr. M. Hatas for systems support, andMrs. D. Pak for research management support (University of BritishColumbia). We are grateful for the scientific input of Dr. Ron Wevers andDr. Leo Kluijtmans (Radboud University Medical Centre, Nijmegen, TheNetherlands). The authors would like to thank the Exome AggregationConsortium (ExAC) and the groups that provided exome variant data forcomparison. A full list of contributing groups can be found at http://exac.-broadinstitute.org/about. This work was funded by the B.C. Children’s HospitalFoundation as “1st Collaborative Area of Innovation” (www.tidebc.org);Genome BC (SOF-195 grant); BC Clinical Genomics Network (#00032 grant);the Canadian Institutes of Health Research (#301221 grant); and by theNational Institute on Drug Abuse grants DA035923 and DA035949; Informaticsinfrastructure supported by Genome BC and Genome Canada (ABC4DE Project).Dr. van Karnebeek is recipient of the Michael Smith Foundation for HealthResearch Scholar Award.Web resourcesThe URLs for data presented herein are as follows:ANNOVAR http://www.openbioinformatics.org/annovar/Bowtie 2 http://bowtie-bio.sourceforge.net/bowtie2/index.shtmlBrainspan Atlas of Developing Brain, www.brainspan.orgCombined Annotation Dependent Depletion, http://cadd.gs.washington.edu/Received: 22 January 2015 Accepted: 3 March 2015Sirrs et al. Orphanet Journal of Rare Diseases  (2015) 10:38 Page 9 of 10References1. Fowler CJ, Rojo ML, Rodriguez-Gaztelumendi A. 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BiochemBiophys Res Commun. 2013;436:377–81.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSirrs et al. Orphanet Journal of Rare Diseases  (2015) 10:38 Page 10 of 10Submit your manuscript at www.biomedcentral.com/submit


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