The Pharmacogenomics Journal (2011) 11, 207–213 & 2011 Macmillan Publishers Limited. All rights reserved 1470-269X/11 www.nature.com/tpj ORIGINAL ARTICLE

A polymorphism of the GTP-cyclohydrolase I feedback regulator alters transcriptional activity and may affect response to SSRI antidepressants

PC McHugh1, PR Joyce2, (BH4) is an essential for synthesis of many 1 1 including . In serotonergic neurons, BH4 is tightly X Deng and MA Kennedy regulated by GTP-cyclohydrolase I feedback regulator (GFRP). Given the pivotal 1Department of Pathology, University of Otago, role of the serotonergic system in mood disorders and selective serotonin Christchurch, New Zealand and 2Department of reuptake inhibitors (SSRIs) antidepressant function, we tested the hypothesis Psychological Medicine, University of Otago, that GFRP gene (GCHFR) variants would modify response to antidepressants in Christchurch, New Zealand subjects with major depression. Two single polymorphisms (rs7164342 and rs7163862) in the GCHFR promoter were identified and Correspondence: Dr PC McHugh, Department of Biology and occurred as two haplotypes (GA or TT). A multiple regression analysis revealed Biochemistry, University of Bath, Claverton that homozygous individuals for the TT haplotype were less likely to respond to Down, Bath BA2 7AY, UK. the SSRI fluoxetine than to the tricyclic antidepressant nortriptyline (P ¼ 0.037). E-mail: [email protected] Moreover, the TT haplotype showed a reduced rate in luciferase reporter gene assays, which may impact on BH4-mediated production, thus suggesting a biological process through which GCHFR promoter variants might influence antidepressant response. The Pharmacogenomics Journal (2011) 11, 207–213; doi:10.1038/tpj.2010.23; published online 30 March 2010

Keywords: tetrahydrobiopterin; pharmacogenetics; neurotransmitters; SSRIs

Introduction

Tetrahydrobiopterin (BH4) is an essential cofactor for a number of including hydroxylase, the rate-limiting step in the of 1 the neurotransmitter serotonin (Figure 1). Furthermore, BH4 can exert its effect on specific membrane receptors to stimulate directly the release of monoamine neurotransmitters, including serotonin.2,3 Several studies have examined the role

of BH4 in the etiology of mood disorders and its treatment. In a previous genetic investigation of the sepiapterin (SPR) gene, the terminal in the

de novo synthesis of BH4 (Figure 1), we showed that variation in the SPR promoter influenced individual risk to mood disorders, as well as a having a potential role in response to selective serotonin reuptake inhibitors (SSRIs) in individuals with major depressive disorder (MDD).4 Curtius et al.5 showed that depressed patients

treated with BH4 showed a marked improvement in mood. Other studies have shown a relationship between BH activity and antidepressants.6–10 Several Received 21 August 2009; revised 3 January 4 2010; accepted 28 February 2010; pharmacogenomic studies investigating mood-regulating drugs have shown 11–13 published online 30 March 2010 altered expression in several components of the BH4 pathway. Together, this Association of GCHFR with fluoxetine response PC McHugh et al 208

Figure 1 Tetrahydrobiopterin biosynthesis pathways in the body. The biosynthesis of BH4 starts de novo from GTP through reactions catalyzed by the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS) and (SPR). Alternatively, the final two-step reduction of the intermediate PPS to BH4 can be effected by aldose reductase (AKR) and carbonyl reductase (CBR). The of these reactions is sepiapterin, which can be salvaged into BH4 through SPR, CBR and (DHFR). Two additional enzymes, -4a- carbinolamine dehydratase (PCD) and dihydropteridine reductase (QDPR) are involved in the regeneration of BH4 from intermediates formed during the of aromatic amino acids. GTPCH activity is modulated by the interaction of GTP cyclohydrolase feedback regulator (GFRP) and effector molecules, BH4 and . Phe, phenylalanine; Tyr, ; Trp, tryptophan; Arg, arginine; Cit, citrulline; PAH, phenylalanine hydroxylase; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase; NOS, synthase; 5-OH-Trp, 5-hydroxytryptophan; AADC, aromatic amino-acid decarboxylase; DBH, b-hydroxylase; NAT, N-acetyltransferase; NAS, N-acetylserotonin; HIOMT, hydroxyindole-O- methyltransferase.

17 evidence supports a link between the BH4 pathway and isothiocyanate extraction. DNA was resuspended in mood control, as well as in antidepressant function. 10 mmol lÀ1 Tris (pH 8.0)/1 mmol lÀ1 EDTA (0.5 ml) and

Because BH4 is essential for such important biological stored at À20 1C. systems, the regulation of the enzyme which is rate limiting The GCHFR gene is approximately 3.6 kb in length and 18 for BH4 synthesis, GTP cyclohydrolase I (GTPCH; Figure 1), consists of three exons. The putative promoter region, could be biologically, pathologically and therapeutically open reading frame, intron–exon boundaries and untrans- important. GTPCH is tightly regulated by GTP cyclohydrolase lated regions of the GCHFR gene were amplified as four PCR I feedback regulator (GFRP).14–16 GFRP mediates feedback fragments of 956, 830, 247 and 770 bp (Supplementary Table inhibition of GTPCH activity by BH4, and the inhibition by S1). PCR was carried out in a total volume of 10 ml, 14 À1 BH4 is reversed by phenylalanine. The GFRP gene is called containing 200 mmol l dNTPs, 2 ml of Q solution (Qiagen GCHFR, and in the rat brain Gchfr expression is abundant in GmbH, Hilden, Germany) for promoter fragments, 0.4 mM of À1 serotonergic neurons of the dorsal raphe nucleus, which each primer, 1.5 mmol l MgCl2, 1 U of Platinum Taq DNA releases serotonin to the rest of the brain, but is undetectable Polymerase (Invitrogen, Carlsbad, CA, USA), 1 ml Platinum in other areas of the central nervous system including Taq reaction buffer and 20–100 ng of genomic DNA. dopamine neurons of the midbrain or Thermal cycling conditions were: 2 min at 96 1C (initial neurons of the coeruleus.15 In this article, we denaturation); 32 cycles of 15 s at 95 1C, 30 s at 60–65 1C, 40 s investigated whether the GCHFR gene influenced antidepres- at 72 1C and a final extension of 2 min at 72 1C. The validity sant response in those with MDD. To examine this relation- of the PCR fragments was determined by DNA sequence ship, we sought to identify polymorphic variants of GCHFR analysis of selected samples. gene and assess the functional significance of these variants and their association with relevant phenotypes. Reporter gene assays Luciferase reporter gene assays were carried out as previously Materials and methods described.19 Briefly, two cell lines were used, COS-7 (ATCC, no. CRL-1651) and RN46A.20 COS-7 cells were maintained Search for GCHFR gene variants in Dulbecco’s modified Eagle’s medium (Invitrogen, Peripheral blood samples were collected from all the Auckland, New Zealand, catalog no. 11971) with 10% fetal 2 patients and genomic DNA was extracted by guanidine calf serum in 75 cm flasks at 37 1C (5% CO2). RN46A cells

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were maintained in Dulbecco’s modified Eagle’s medium/ At the baseline assessment, patients were evaluated using F12 (Invitrogen, catalog no.10565) with 5% fetal calf serum the Structured Clinical Interview for Depression for DSM-IV and 250 mgmlÀ1 G418 (Geneticin; Invitrogen) in 75 cm2 (SCID),22 and rated for depression severity on the Hamilton 23 flasks at 33 1C (5% CO2). Depression Rating Scale (HDRS) and Montgomery and For reporter constructs, a 1408 bp PCR product was Asberg Depression Rating Scale (MADRS)24. Patients were amplified from both alleles of the GCHFR promoter region also assessed for axis II disorders, completed a range of self- using primers engineered with BglII and HindIII sites report questionnaires and had a detailed neurobiological (Supplementary Table S1). Amplified products were cloned assessment that included neuroendocrine, and into the firefly luciferase reporter vector pGL3-basic (Pro- plasma measures. mega Corporation, Madison, WI, USA), using the BglII and After the baseline assessment, patients were initially HindIII sites. Constructs were confirmed by DNA sequencing randomized to treatment with either fluoxetine or nortrip- and large-scale DNA preparations were carried out with tyline for a period of 6 weeks. If patients were randomized to AxyPrep Miniprep Kit (Axygen Biosciences, Union fluoxetine the starting dosage was 20 mg per day. Clinicians City, CA, USA). of constructs into COS-7 and had flexibility to adjust dosage down to 10 mg or up to RN46A cells were performed in 24-well plates seeded at 60 mg based on their clinical judgment. The usual dosing 5 Â 105 cells per well before . Transfections were regime was 20 mg for 3 weeks, with an increase to 40 or performed using FuGene (Roche Diagnostics GmbH, Man- 60 mg after 3 weeks if no or minimal response. At 6 weeks nheim, Germany) for COS-7 and Effectene (Qiagen) for the fluoxetine doses ranged from 10 to 60 mg with a mean RN46A, as per manufacturer’s instructions. To control for daily dosage of 28.1 mg. For patients randomized to variation in transfection efficiency among replicates, we co- nortriptyline, the protocol was to increase from 25 to transfected promoter constructs with the Renilla luciferase 75 mg over the first three heights. After 1 week a blood vector, pRL-TK (Promega Corporation). At 48 h after trans- level for nortriptyline was obtained, and clinicians then fection, COS-7 and RN46A cells were harvested and firefly adjusted nortriptyline dosages based on clinical response, and Renilla luciferase chemiluminescence were measured side effects and blood levels to try and ensure an optimal using the Dual-Luciferase Reporter Assay System (Promega) clinical trial over 6 weeks. At 6 week the daily nortriptyline on a PerkinElmer (Norwalk, CT, USA) VICTOR3 Wallac 1420 doses ranged from 50 to 175 mg with a mean of 93.5 mg. microplate reader. Promoter activity was calculated as the During this time clinicians saw patients at least weekly and ratio of firefly to Renilla luciferase chemiluminescence. Each had flexibility to adjust dosages to try and achieve optimal construct was tested in triplicate with at least three clinical outcomes. For patients on nortriptyline, this independent transfection experiments. To evaluate statisti- included blood levels after 1 week and as clinically cal significance, we used two-tailed t-test with 95% con- indicated. The HDRS and MADRS were repeated after 3 fidence interval on GraphPad Prism software (GraphPad, and 6 weeks on the initially prescribed antidepressant San Diego, CA, USA). medication. The primary outcome was percentage improve- ment on the MADRS over the initial 6 weeks. The secondary outcome was a dichotomous response/nonresponse out- Study cohorts come based on achieving a X60% improvement on the Clinical trial sample. A total of 195 depressed outpatients MADRS. were recruited for a long-term clinical study examining patterns and predictors of remission, response, recovery, Family sample. Antidepressant response in this study was relapse and recurrence. The clinical study began with a rated retrospectively by participants with a history of MDD, randomization to fluoxetine or nortriptyline. Full details of who had been recruited for a family study of depression and the patient characteristics, treatment protocol and 6-week personality disorder. Antidepressant response, in those who outcome have been fully elucidated in an earlier study.21 Of had received at least 20 mg daily for 6 weeks, was coded as these 195 patients, 57% were women and the mean age was 1 ¼ very good response, 2 ¼ good response, 3 ¼ slight 31.6 year. Of the 100 patients randomized to fluoxetine, 86 response or 4 ¼ minimal, no or negative response. For this completed an adequate 6-week antidepressant trial, whereas study, responders were those who rated their response as of the 95 randomized to nortriptyline, 68 completed an very good (1) or good (2). adequate 6-week antidepressant trial. Exclusion criteria were Participants for this family study were either probands, or minimized but included a diagnosis of schizophrenia or siblings or parents of the probands. The recruitment strategy bipolar I disorder, a principal current diagnosis of severe involved advertising for participants who had been treated alcohol or drug dependence, severe antisocial personality for a depressive episode. Probands then identified living disorder that was judged likely to interfere with cooperation parents (preferably) and/or all siblings, with the objective of with the research protocol or major physical illness. At the recruiting family trios. All consenting subjects were then baseline assessment, patients were also required to have seen by a trained interviewer, who was supervised by one been free of all psychotropic medication for at least 2 weeks research psychiatrist. The interview as based on the Mini- or five drug half-lives, except for an occasional hypnotic for International Neuropsychiatric Interview,25 except that the sleep. This study was approved by the Canterbury Ethics SCID,26 was used for the assessment of alcohol, and drug use Committee (Christchurch, New Zealand). and dependence. In addition to the Mini-International

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Neuropsychiatric Interview questions the Mood Disorder tion rate between the GA and TT haplotypes in both COS-7 Questionnaire27 was also used to assist with the diagnosis of and RN46A cells, with the TT haplotype showing lower bipolar disorders. Further details about the study are expression levels than the GA haplotype (Figures 2a and b). available in other studies.19,28 For this study, we have limited the eligible participants to Genetic analysis those with a lifetime diagnosis of MDD (that is, excluding To genotype this promoter polymorphism in clinical those with bipolar disorders, or those who used an SSRI for cohorts, we developed a restriction fragment length poly- reasons other than depression), who received adequate trials morphism assay. This assay was applied to all individuals in of SSRI antidepressant drugs. the trial study and the family study (n ¼ 156 and 578, respectively). The polymorphism was in Hardy–Weinberg GCHFR genetic analysis equilibrium (P ¼ 0.08), with frequencies of 0.52 for the GA For GCHFR genotyping we developed a restriction fragment haplotype and 0.48 for the TT haplotype. length polymorphism assay. Briefly, a 638 bp PCR product We explored associations of promoter haplotypes with was generated as described above (Supplementary Table S1). antidepressant response in individuals with MDD who were Amplicon (4 ml) was digested in a total volume of 10 ml, with studied in the context of the randomized clinical trial of the 1U of Tsp509I restriction enzyme (New England Biolabs antidepressants fluoxetine and nortriptyline. Table 1 shows Inc., Ipswich, MA, USA), 1 ml of NEB Buffer 1 and incubated the number of subjects by drug and haplotype, and their at 65 1C for 1 h. The digested fragments were separated on response to treatment. Multiple regression analysis revealed 3% agarose gels. The restriction pattern was GA/GA: 415, that there was no direct effect of drug (F ¼ 0.24, P ¼ NS) or of 223; GA/TT: 415, 299, 223, 116; TT/TT: 299, 223, 116. genotype (F ¼ 0.13, P ¼ NS), but that there was a significant drug  genotype interaction (P ¼ 0.037). Thus individuals Statistical analyses The associations between the rs7164342 and rs7163862 polymorphisms and antidepressant response in the clinical trial were initially examined by multiple linear regression analysis with percent improvement on the MADRS as the dependent variable. Logistic regression was then used to examine categorical outcome response variables across both the clinical trial and the family study. It should be noted that of participants with MDD in the family study, some might have received an adequate trial of more than one of the SSRIs. Hardy–Weinberg equilibrium was calculated using SHEsis.29

Results

Search for GCHFR gene variants The GCHFR gene was analyzed for DNA sequence variation in 24 unrelated individuals of Caucasian descent. The genetic screen included 1.3 kb of putative promoter, exonic regions including intronic boundaries and the 50 and 30 untranslated regions. The only variants observed in the regions screened were the adjacent À1012G4T and À1011A4T transversions of the putative promoter region. Although two existing independent dbSNP entries (rs7164342 and rs7163862) correspond to these variants, our analysis indicated these adjacent single nucleotide polymorphisms were in total linkage disequilibrium and behaved as one polymorphism with two haplotypes (GA and TT). This observation was based on the initial DNA sequence analysis of 24 individuals as well as genotyping of the two cohorts, totaling 734 individuals.

Functional analysis Figure 2 Reporter gene analysis of GTP-cyclohydrolase I feedback To assess whether this polymorphism affected basal GCHFR regulator (GCHFR) promoter haplotypes. The expression levels of the promoter activity, we cloned the variant promoters into a two GCHFR promoter constructs containing haplotypes GA or TT were luciferase reporter vector. Reporter gene assays of the two compared in (a) COS-7 and (b) RN46A cells. The expression levels of constructs showed a significant difference in basal transcrip- each haplotype with standard errors are shown. *Po0.05.

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Table 1 Treatment outcome of individuals with major depression randomized to fluoxetine or nortriptyline in a clinical trial

AD medication Fluoxetine Nortriptyline Genotype GA/GA GA/TT TT/TT GA/GA GA/TT TT/TT

No. randomized 22 38 19 24 33 20 No. completed 21 31 16 15 26 14 Female/Male 12/10 18/20 10/9 17/7 21/12 9/11 Age 31 (12) 33 (11) 34 (12) 32 (11) 32 (12) 28 (11) MADRS Week 0 31.6 (7.5) 31.9 (5.8) 30.9 (6.0) 26.8 (7.7) 30.2 (6.3) 31.3 (6.6) Week 6 9.2 (8.8) 12.7 (9.8) 16.3 (12.9) 12.5 (10.1) 13.7 (12.5) 10.4 (8.3) Mean % improvement 73 (24) 61 (29) 49 (35) 53 (34) 56 (38) 67 (25)

Abbreviations: AD, antidepressant; MADRS, Montgomery and Asberg Depression Rating Scale. Results are shown as mean (+s.d.). In this table the percentage of responders is based upon an intent–to-treat analysis.

Table 2 Response rates (numbers of responders/nonrespon- Table 3 Response rates (%) in haplotype homozygotes across ders) to an adequate trial of SSRIs by haplotype in the family both studies in depressed participants who completed ade- study quate antidepressant trials (numbers of participants by haplo- type are in brackets) GA/GA GA/TT TT/TT Drug GA/GA TT/TT OR (95% CI) P-value Fluoxetine 74% (20/7) 61% (37/24) 52% (15/14) Citalopram 77% (10/3) 76% (25/8) 60% (6/4) Drug Nortriptyline 57% (18) 79% (14) 2.75 0.52–14.4 0.23 Paroxetine 70% (14/6) 69% (29/13) 44% (4/5) trial Fluoxetine 76% (31) 44% (16) 0.24 0.06–0.99 0.049

Family Fluoxetine 76% (25) 57% (28) 0.42 0.13–1.38 0.15 with the homozygous TT haplotype had a poorer response study to fluoxetine and a better response to nortriptyline. Citalopram 77% (13) 60% (10) 0.45 0.08–2.74 0.39 We next explored response in the family study. Table 2 Paroxetine 75% (19) 67% (6) 0.71 0.10–5.18 0.74 shows the number of responders and nonresponders to Any SSRI 75% (57) 59% (44) 0.47 0.20–1.10 0.082 fluoxetine, paroxetine and citalopram by haplotype. Logis- tic regression did not show any statistically significant effect Both Any SSRI 76% (78) 55% (60) 0.39 0.19–0.81 0.012 studies of haplotype on SSRI response. However, examination of the response rate in the SSRI Abbreviation: SSRI, selective serotonin reuptake inhibitor. trials across both the clinical trial and family study suggested that there were consistent effects (even if not individually statistically significant) for SSRI response Discussion between the homozygous GA and homozygous TT haplo- types. Table 3 shows the response rates in haplotype In this series of studies we have found in a randomized homozygotes across the drug trial and family study as well clinical trial in major depression that the homozygous TT as the odds ratio (95% confidence intervals) of response. haplotype pair was associated with a lower percentage From this table it can be seen that the odds ratio is above improvement in depressive symptoms in those randomized one for nortriptyline, but consistently ranges from 0.24 to to fluoxetine than in those randomized to nortriptyline. 0.71 for SSRIs. Although this is statistically significant in the Furthermore, across both the clinical trial and the family drug trial for fluoxetine and approaches traditional levels of study that there was a lower response to SSRIs in those with statistical significance for any SSRI trial in the family study, homozygous TT than the homozygous GA haplotype. it is statistically significant for any SSRI trial across both These findings suggest that GCHFR promoter variants may studies, where there is a 76% response rate to any SSRI for influence response to SSRIs. The GFRP protein tightly those with homozygous GA haplotype, but only a 55% regulates BH4 production, and in rat central nervous system response rate to any SSRI in those with homozygous TT this regulation appears to be restricted to serotonergic cells haplotype. of the raphe nuclei.15 Serotonin is implicated in many We also investigated whether there was association neuropsychiatric disorders including major depression. between GCHFR promoter haplotypes and clinical diagnosis Furthermore, SSRIs are believed to exert their effect on in the family study, but none was apparent (data not raphe nuclei serotonin neurons, as well as at their targets.30 shown). Therefore a priori one would predict that GCHFR variants

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could likely influence patient response to SSRIs, and indeed 3 Miwa S, Watanabe Y, Koshimura K, Masaki T. [A novel function of our preliminary observations suggest this may be the case. tetrahydrobiopterin]. Nippon Yakurigaku Zasshi 1992; 100: 367–381. However, a larger study sample will be required to confirm 4 McHugh PC, Joyce PR, Kennedy MR. Polymorphisms of sepiapterin reductase gene alter promoter activity and may influence risk of bipolar these observations. disorder. Pharmacogenet Genomics 2009; 19: 330–337. We also investigated the consequences of GCHFR promo- 5 Curtius HC, Niederwieser A, Levine RA, Lovenberg W, Woggon B, ter haplotypes on transcriptional activity, using luciferase Angst J. Successful treatment of depression with tetrahydrobiopterin. reporter gene assays. In the first instance we assessed Lancet 1983; 1: 657–658. 6 Miura H, Qiao H, Kitagami T, Ohta T. Fluvoxamine, a selective serotonin promoter activity in COS-7, an African green monkey reuptake inhibitor, suppresses tetrahydrobiopterin in the mouse kidney cell line and subsequently in RN46A, an undiffer- hippocampus. Neuropharmacology 2004; 46: 340–348. entiated serotonergic cell line derived from rat raphe nuclei. 7 Miura H, Kitagami T, Ozaki N. Suppressive effect of paroxetine, a We regarded the latter cell line to be of physiological selective serotonin uptake inhibitor, on tetrahydrobiopterin levels and dopamine as well as serotonin turnover in the mesoprefrontal system of relevance to mood disorders, as well as antidepressant mice. Synapse 2007; 61: 698–706. response. The two reporter constructs representing either 8 Hashimoto R, Nagatsu T, Ohta T, Mizutani M, Omura I. Changes in the the GA or the TT haplotype showed similar expression concentrations of tetrahydrobiopterin, the cofactor of tyrosine hydro- profiles in both cell lines (Figures 2a and b), where in each xylase, in blood under physical stress and in depression. Ann NY Acad Sci 2004; 1018: 378–386. line the expression of the TT haplotype was significantly 9 Hashimoto R, Mizutani M, Ohta T, Nakazawa K, Nagatsu T. Changes in lower than the GA haplotype. In the association study, we plasma tetrahydrobiopterin levels of depressives in depressive and observed a preponderance of homozygous TT haplotype remission phases: reconfirmed by measurement with an internal pairs in patients with poor SSRI response. Lower GCHFR standard. Neuropsychobiology 1994; 29: 57–60. 10 Abou-Saleh MT, Anderson DN, Collins J, Hughes K, Cattell RJ, Hamon expression in patients with the TT haplotype may impact on CG et al. The role of in depression and the effects of response to SSRIs, although we do not have direct evidence antidepressive therapy. Biol Psychiatry 1995; 38: 458–463. for this suggestion. It would be of interest to investigate how 11 McQuillin A, Rizig M, Gurling HM. A microarray study patient haplotypes may impact directly on GFRP activity, of the molecular pharmacology of lithium carbonate on mouse brain mRNA to understand the neurobiology of mood stabilization and but at present there is no routine method with which to treatment of bipolar affective disorder. Pharmacogenet Genomics 2007; measure GFRP protein activity in patient samples. 17: 605–617. In conclusion, evidence is presented for a significant 12 McHugh PC, Rogers GR, Loudon B, Glubb DM, Joyce PR, Kennedy MA. association between homozygous TT haplotypes in the Proteomic analysis of embryonic stem cell-derived neural cells exposed to the antidepressant paroxetine. 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GTP cyclohydrolase I feedback regulatory protein is expressed in serotonin neurons on BH4-mediated neurotransmitter production, which in and regulates tetrahydrobiopterin biosynthesis. J Neurochem 1999; turn may influence response to SSRIs. These findings may 72: 669–675. also be of wider relevance to other physiological and 16 Yoneyama T, Wilson LM, Hatakeyama K. GTP cyclohydrolase I feedback pathological pathways that depend on GFRP activity. regulatory protein-dependent and -independent inhibitors of GTP cyclohydrolase I. Arch Biochem Biophys 2001; 388: 67–73. 17 Ciulla TA, Sklar RM, Hauser SL. A simple method for DNA purification from peripheral blood. Anal Biochem 1988; 174: 485–488. Conflict of interest 18 Thony B, Auerbach G, Blau N. Tetrahydrobiopterin biosynthesis, regeneration and functions. Biochem J 2000; 347(Part 1): 1–16. The authors declare no conflict of interest. 19 McHugh PC, Joyce PR, Kennedy MA. 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