Parkinson’s disease in GTP cyclohydrolase 1 mutation carriers

Mutations in the gene encoding the dopamine-synthetic enzyme GTP cyclohydrolase-1 (GCH1) cause DOPA-responsive dystonia (DRD). Mencacci et al. demonstrate that GCH1 variants are associated with an increased risk of Parkinson's disease in both DRD pedigrees and in patients with Parkinson's disease but without a family history of DRD.


Introduction
Parkinson's disease is a common neurodegenerative disease mainly characterized by severe loss of dopaminergic neurons in the substantia nigra pars compacta and by the formation of -synuclein positive aggregates (Lees et al., 2009). Nigral neuron degeneration and consequent decrease in dopaminergic striatal innervation result in classic Parkinson's disease motor symptoms. Symptomatic treatment with levodopa or dopamine agonists is effective in alleviating these symptoms, although, along with disease progression, levodopa-induced motor complications (e.g. dyskinesias, wearingoff, on-off fluctuations) may appear.
In recent years several Mendelian loci have been unequivocally linked to hereditary forms of Parkinson's disease (Houlden and Singleton, 2012) and genome-wide association studies have succeeded in identifying many common, low risk variants (Plagnol et al., 2011).
The GCH1 gene (14q22.1-q22.2; OMIM 600225) encodes GTP cyclohydrolase 1, the enzyme controlling the first and rate-limiting step of the biosynthesis of tetrahydrobiopterin (BH 4 ), the essential cofactor for the activity of tyrosine hydroxylase, and for dopamine production in nigrostriatal cells (Kurian et al., 2011). Mutations in GCH1 are the most common cause of DOPA-responsive dystonia (DYT5; OMIM#128230) (Clot et al., 2009), a rare movement disorder that presents typically in childhood with lower limb dystonia and subsequent generalization (Nygaard, 1993b). The hallmark of the disease is an excellent and sustained response to small doses of levodopa, generally without the occurrence of motor fluctuations (Trender-Gerhard et al., 2009). Reduction of CSF levels of pterins, dopamine and serotonin metabolites (Assmann et al., 2003), or an abnormal phenylalanine-loading test (Bandmann et al., 2003) are supportive findings in the diagnosis of DOPA-responsive dystonia. Inheritance is usually autosomal dominant with incomplete penetrance (Furukawa et al., 1998), though recessive cases have been described (Opladen et al., 2011). Dominant GCH1 mutations result in a significant reduction of GCH1 activity through a dominant negative effect of the mutant protein on the normal enzyme (Hwu et al., 2000).
Neuropathological examination in a limited number of cases with DOPA-responsive dystonia, revealed marked reduction of melanin pigment and dopamine content in nigrostriatal neurons, but no evidence of nigral cell loss or degeneration (Furukawa et al., 1999).
Parkinsonian features are frequently detected in patients with DOPA-responsive dystonia (Tassin et al., 2000) and family studies have shown that carriers of GCH1 mutations may develop adultonset parkinsonism in the absence of dystonia (Nygaard et al., 1990). Based on previous studies, the prevailing hypothesis was that parkinsonism represented an atypical, age-specific, presentation of DOPA-responsive dystonia without nigral degeneration (Nygaard and Wooten, 1998).
The aim of this study was to further explore the relationship between GCH1 mutations and parkinsonism and consider whether adult GCH1 mutation carriers are at increased risk of developing neurodegenerative Parkinson's disease.
We first describe the clinical, genetic and nigrostriatal dopaminergic imaging findings of DOPA-responsive dystonia pedigrees in which pathogenic GCH1 variants were identified in family members with adult-onset parkinsonism. We subsequently explore the hypothesis that GCH1 variants might be associated with an increased risk for Parkinson's disease, even without a family history for DOPA-responsive dystonia, through examination of wholeexome sequencing data from a large cohort of cases and controls.

Family study Pedigrees
The clinical and demographic features of the pedigrees with GCH1 mutations involved in this study are described in the 'Results' section. DOPA-responsive dystonia pedigrees were included in the study, where family members affected with adult-onset parkinsonism were available for clinical and genetic examination and in whom dopaminergic studies had been performed. Local ethics committees approved the study and informed consent for genetic testing was obtained in all cases.

Genetic analysis
Genomic DNA was extracted from peripheral blood leucocytes using standard procedures. Probands were screened for GCH1 mutations (NCBI transcript NM_000161.2) by standard bi-directional Sanger sequencing of all six coding exons and exon-intron boundaries (primer sequences available on request). Dosage analysis for GCH1 exonic deletions and duplications was performed by multiplex ligation-dependent probe amplification (MLPA, MRC).

Dopamine transporter imaging studies
Dopaminergic striatal innervation was evaluated as dopamine reuptake transporter (DAT) density by means of single photon computed tomography (SPECT) and [ 123 I]N-!-fluoropropyl-2b-carbomethoxy-3b-(4-iodophenyl) tropane ( 123 I-FP-CIT). SPECT data acquisition and reconstruction has been described in detail elsewhere (Isaias et al., 2010). To obtain comparable measurements among different centres, 123 I-FP-CIT binding values for the caudate nucleus and putamen were calculated by means of the basal ganglia matching tool (Nobili et al., 2013).

Whole-exome sequencing study Participants and study design
The study initially involved 1337 unrelated subjects with Parkinson's disease and 1764 control subjects of European origin or North American of European descent that underwent whole-exome sequencing. Cases, originating mainly from the USA, UK, Holland and France, were recruited by the International Parkinson Disease Genomics Consortium (IPDGC), an international collaboration to understand the genetics of Parkinson's disease.
A further 190 cases with Parkinson's disease were recruited through a community-based epidemiological study of Parkinson's disease in Estonia (University of Tartu, Estonia). Cases with Parkinson's disease were clinically diagnosed according to the UK Parkinson's Disease Society Brain Bank (UKPDSBB) criteria (Hughes et al., 1992).
Control samples were collected by the UCL-exomes, a consortium of researchers within University College London (London, UK) designed to share raw read level data from multiple exome sequencing projects. Control subjects had no diagnosis of Parkinson's disease, DOPAresponsive dystonia or any other movement disorder. Whole-exome sequencing data from an additional 4300 North American individuals of European descent were analysed from the publicly available NHLBI Exome Sequencing Project Exome Variant Server (EVS) database (http://evs.gs.washington.edu/EVS/).

Procedures
Paired-end sequence reads (TruSeq chemistry sequenced on the Illumina HiSeq 2000) were aligned with Burrows-Wheeler Aligner (for IPDGC) and novoalign (for UCL-exomes) against the reference human genome (UCSC hg19). Duplicate read removal, format conversion, and indexing were performed with Picard (http://picard.source forge.net/). The Genome Analysis Toolkit was used to recalibrate base quality scores, perform local realignments around possible indels, and to call and filter the variants. ANNOVAR software was used to annotate the variants (Wang et al., 2010).

Statistical analysis
Frequencies of coding and splice-site GCH1 variants in cases and controls were compared by means of Fisher's exact (statistical significance set at P-value 5 0.05 using a two-tailed test) and odds ratios (OR) and 95% confidence intervals (CI) were calculated. Analyses were performed using the statistical analysis program R (http://www.r-pro ject.org/).

Family study Family A
The proband (Case III-1, Fig. 1A) is a British 18-year-old male who had a difficult caesarean birth, with perinatal distress and subsequent developmental delay. At 18 months he developed inward turning of his feet with walking difficulties and frequent falls. He was diagnosed clinically with DOPA-responsive dystonia at the age of 3 years and administration of levodopa (300 mg/day) markedly improved his symptoms. [ 123 I]FP-CIT SPECT, performed at age 17, was normal (data not shown).
The proband's father (Case II-1), who was initially thought to have cerebral palsy due to a birth injury, was subsequently diagnosed, at the age of 42, with DOPA-responsive dystonia. The proband's grandfather (Case I-1) is a 65 year-old male with a 6-year history of progressive asymmetric rest tremor in the right upper limb. Examination showed signs of typical Parkinson's disease with hypomimia, unilateral rest tremor and asymmetric bradykinesia. He did not present signs of dystonia. 123 I-FP-CIT SPECT showed bilateral reduced tracer uptake more marked on the left (Fig. 1A), consistent with nigrostriatal dopaminergic denervation. He responded well to levodopa therapy (300 mg/day).
GCH1 analysis revealed a heterozygous splice site mutation (c.343 + 5G4C) in the three affected individuals. We previously detected c.343 + 5G4C in a recessive pedigree, carried by the unaffected mother of two very severely affected children who also inherited the K224R mutation from their unaffected father (Bandmann et al., 1996b;Trender-Gerhard et al., 2009). However the c.343 + 5G4C mutation has not been previously described in DOPA-responsive dystonia dominant pedigrees, making its pathogenicity uncertain. Complimentary DNA analysis showed aberrant splicing resulting in a premature stop codon and retention of intron 1 in a proportion of mutant transcripts, confirming the loss-of-function effect of the variant. See Supplementary material for details of the RNA analysis.

Family B
The proband (Case III-1; see Fig. 1B) is a 12-year-old right-handed female of German origin with DOPA-responsive dystonia, with an onset at age 11, with writing and foot dystonia. Her mother (Case II-1) presented at age 39 with progressive loss of dexterity and slowness in her right arm and dystonic posturing of the right foot. Examination showed an asymmetric rigid-akinetic parkinsonian syndrome without tremor and severe right foot fixed dystonia. Levodopa therapy resulted in marked improvement of both dystonic and parkinsonian symptoms. 123 I-FP-CIT SPECT revealed an asymmetric bilateral reduced tracer uptake, more marked in the left striatum. There was sustained response to levodopa therapy although there was an increase in dose requirement (up to 800 mg/day). Levodopa-induced dyskinesias developed 6 years after initiation of levodopa. Examination of the proband's 66-year-old grandmother (Case I-1) revealed oromandibular GCH1 screening in this family revealed two variants: c.68C4T;p.P23L (carried by Cases III-1 and II-1) and c.312C4A;p.F104L (carried by Cases II-1 and I-1). There were no GCH1 exonic rearrangements. F104L is absent in public control data sets and has been previously reported in association with DOPAresponsive dystonia (Clot et al., 2009). P23L (rs41298432) is a benign polymorphism present in population controls at a frequency of 1-2% (Jarman et al., 1997;Hauf et al., 2000).
To confirm GCH1 deficiency, phenylalanine-loading test (100 mg/kg) was performed in Cases I-I and II-I and showed pathologically elevated phenylalanine/tyrosine ratios in both ( Supplementary Fig. 2). CSF analysis, performed in Case III-I, displayed low levels of BH 4 (13 nmol/l; 18-53 nmol/l) and neopterin (6 nmol/l; 10-31 nmol/l), consistent with GCH1 deficiency. Given the benign nature of P23L, we hypothesize that the GCH1 deficiency confirmed in this patient may be the result of an-as yet-unidentified non-coding causative mutation.

Family C
The proband (Case II-1, Fig. 1C) is a German 41-year-old female, affected by DOPA-responsive dystonia, who presented at age 4 years with bilateral foot inversion on walking. Her father (Case I-1) is a 67-year old male with a 1-year history of typical Parkinson's disease with left hand rest tremor, bilateral rigidity and bradykinesia and mild gait difficulties. There was no dystonia. 123 I-FP-CIT SPECT examination revealed asymmetrically reduced DAT-density in the striatum. Rasagiline and pramipexole were started with good response. The mother (Case I-2), aged 62 years, had a normal neurological examination.
The proband was compound heterozygous for two GCH1 missense variants, c.610G4A;p.V204I, inherited from the asymptomatic mother, and the novel variant c.722G4A;p.R241Q, which was paternally inherited. R241Q is absent in public control data sets, is predicted deleterious by all in silico prediction tools and involves an amino acid residue conserved down to invertebrate species. Furthermore a pathogenic mutation at the same residue has already been reported (Bandmann et al., 1998).

Family D
The proband is an Italian 58-year-old female (Case II-1, Fig. 1D), who developed progressive tremor and clumsiness in the right arm at age 44 years. Clinical examination showed typical Parkinson's disease with hypomimia, hypophonia and asymmetrical bradykinesia and rigidity. Action dystonic tremor (right 4 left), poor postural reflexes and slow gait were also evident and there was a sustained response to levodopa. The dose was gradually increased up to 400 mg/day, after which rotigotine 4 mg/day was added.
Dyskinesias and wearing-off symptoms developed 6 years after levodopa initiation. 123 I-FP-CIT SPECT revealed asymmetrically reduced DAT binding values in the striatum.
Her sister (Case II-2; Fig. 1D), aged 60, had a childhood onset of mild walking difficulties. At age 55, she developed exerciseinduced left foot dystonia and dystonic tremor in both arms. She had no bradykinesia or other parkinsonian signs. Low-dose levodopa (100 mg alternate days) was started with excellent symptom control. 123 I-FP-CIT SPECT was normal. Their father was reported to have a tremulous condition, but was not available for clinical or genetic examination. GCH1 sequencing revealed that both sisters were heterozygous for the previously reported pathogenic mutation c.626 + 1G4C (Garavaglia et al., 2004).
The main clinical features of the GCH1 mutation carriers with adult-onset parkinsonism and abnormal 123 I-FP-CIT SPECT imaging are summarized in Table 1. Their clinical features fully met the UKPDSBB criteria for definite Parkinson's disease diagnosis. None of these cases presented significant diurnal fluctuations, worsening of symptoms in the evening or substantial sleep benefit, features often recognized in cases with DOPA-responsive dystonia (Kurian et al., 2011). DAT binding values are reported in Supplementary Table 1.

Whole-exome sequencing study
We hypothesized that pathogenic variants in GCH1 could be found in subjects with Parkinson's disease without a family history for DOPA-responsive dystonia. To investigate this we examined whole-exome sequencing data of a large cohort of patients predominantly affected by early-onset or familial Parkinson's disease and controls. After quality control checks (removal of gender mismatches, duplicate, related and non-Caucasian samples, samples with low call rate or excess of heterozygosity), 1318 cases with Parkinson's disease and 1635 controls remained. Additional control data (n = 4300) were obtained from the publically available Exome Variant Server (EVS) data set.
In total 1318 cases and 5935 controls were analysed for the presence of GCH1 coding (including small insertions/deletions, missense and stop-gain changes) or splice-site variants ( AE 5 base pairs from the coding exons). The mean age of subjects with Parkinson's disease was 55.7 AE 13.9 years (range 17-101; data available for 970 cases) and the mean age at onset was 46.7 AE 13.8 years (range 6-98; data available for 1194 cases). Four hundred and twenty-three of 1194 (35.4%) were earlyonset cases (age at onset 4 40 years) and $630 were familial cases (positive family history for Parkinson's disease in a first or second-degree relative).
Coverage of the six GCH1 coding exons (NCBI transcript NM_000161.2) was comparable in the three data sets (IPDGC, UCL-ex and EVS; Supplementary Table 2). No common variants (frequency 41%) were identified. The benign polymorphisms P23L (rs41298432) and P69L (rs56127440), detected at similar frequencies in cases and controls, were not included in the analysis.
One case was heterozygous for the GBA mutation E326K. This is a relatively common variant ($1-2% Caucasians) that was recently shown to be associated with a modest but significant increase in the disease risk (Duran et al., 2013). The main features of the 10 cases with Parkinson's disease with pathogenic or possibly pathogenic GCH1 variants are listed in Table 3.
The age at onset of GCH1-mutated cases was 43.2 AE 13.4 years (range 17-61). Seven had a positive family history of Parkinson's disease. DNA of other family members was available for only one case and we showed segregation of the same GCH1 mutation (Q110X) in the affected sister of the index case. All cases exhibited a variable combination of asymmetrical bradykinesia, rigidity, rest and postural tremor, walking difficulties, postural instability and excellent response to dopaminergic treatment, consistent with a clinical diagnosis of Parkinson's disease.
The two subjects with the youngest age at onset of symptoms (Cases 4 and 9, who developed symptoms at age 32 and 17, respectively) presented with dystonic features in the lower limbs at onset, a well recognized characteristic of young-onset Parkinson's disease cases (Bozi and Bhatia, 2003). Case 5 developed lower limb dystonia in off periods over the course of the disease. The remainder did not present with any symptoms or signs of dystonia.
Detailed information about treatment was available for eight cases: the two cases (Cases 1 and 7) with the shortest disease duration (45 years) were treated only with a dopamine-agonist, whereas the other cases were taking a combination of levodopa and other anti-parkinsonian drugs. Mean disease duration was 17.6 AE 15.4 years (range 4-56). Cases with longer disease duration displayed a more severe clinical picture with some degree of postural instability (Hoehn and Yahr score 5 3), indicating disease progression in spite of the dopaminergic treatment.
In those patients taking levodopa and for whom follow-up information was available (n = 7), all developed clinically relevant motor complications of chronic levodopa treatment, including wearing off, motor fluctuations and dyskinesias. Dyskinesias in Case 4 were so disabling that he required treatment with deep brain stimulation of the subthalamic nuclei at age 60.

Family study
We report here four unrelated DOPA-responsive dystonia pedigrees in which loss-of-function GCH1 mutations (two splice-site mutations and two missense mutations, confirmed to be pathogenic by metabolic or CSF studies) were found in individuals, asymptomatic for DOPA-responsive dystonia during childhood, who developed adult-onset parkinsonism. They all met the UKPDSBB clinical criteria for a definite diagnosis of Parkinson's disease and had imaging evidence of a Parkinson's disease-like nigrostriatal dopaminergic denervation.
A parkinsonian syndrome in the absence of dystonia has been reported in adults who are first-degree relatives of children with DOPA-responsive dystonia. In a series of 21 families, Nygaard showed that 7/50 (14%) individuals older than 40 years had parkinsonism (Nygaard, 1993a) and Hagenah et al. (2005) reported that 8/23 (34.7%) patients of their series had a positive family history for Parkinson's disease. GCH1 mutations have also been shown to segregate in pedigrees with multiple individuals affected by isolated parkinsonism (Irie et al., 2011).
Our study provides evidence that in most of the cases the parkinsonian phenotype in adult GCH1 mutation carriers is likely due to nigrostriatal degeneration, rather than being simply part of the phenotypic spectrum of metabolic GCH1-related striatal dopamine deficiency. This is consistent with other previous isolated reports of adult-onset parkinsonism in GCH1 mutation carriers with abnormal nigrostriatal imaging (features summarized in Table 1) (Kikuchi et al., 2004;Hjermind et al., 2006;Eggers et al., 2012;Ceravolo et al., 2013).
Our imaging findings are, however, in apparent contrast to a previous report by Nygaard et al. (1992). The authors described a large DOPA-responsive dystonia pedigree, in which three subjects had a late-onset benign parkinsonism, two of which had normal nigrostriatal dopaminergic function determined by means of 18 F-fluorodopa PET. Compensatory mechanisms at the presynaptic level (e.g. increased dopamine-intake and dopamine-decarboxylation activity) may result in relatively higher striatal 18 F-fluorodopa uptake in the initial phase of Parkinson's disease, underestimating the degree of nigral cell decrease (Nandhagopal et al., 2011). DAT values are therefore a more precise indicator of dopaminergic innervation loss (Ito et al., 1999). We speculate that GCH1parkinsonian cases with normal 18 F-fluorodopa-PET scan could have upregulated compensatory dopaminergic activity at the presynaptic level, possibly masking the presence of striatal denervation.
In agreement with our findings, Gibb and Lees reported in 1991 a case that presented with juvenile-onset parkinsonism and dystonia with good response to levodopa (commenced at the age of 30) and occurrence of disabling dyskinesias after 1 year of treatment. The patient died at 39 years and pathological examination showed a striking combination of low melanin content in nigral neurons and devastating neuronal loss with reactive gliosis. Furthermore, Lewy bodies were found in surviving nigral cells and in the locus coeruleus (Gibb et al., 1991). This case was subsequently demonstrated to be carrier of a heterozygous mutation in GCH1 (c.276delC) (Segawa et al., 2004).

Whole-exome sequencing study
We subsequently showed, in a large cohort of patients with Parkinson's disease without family history of DOPA-responsive dystonia, that rare GCH1 coding variants are associated with Parkinson's disease and increase the disease risk by 7-fold on average. Among the GCH1 variants identified by exome sequencing, two (Q110X and K224R) have been shown to cause GCH1 deficiency and DOPA-responsive dystonia in dominant pedigrees (Leuzzi et al., 2002;Saunders-Pullman et al., 2004) and two (V204I and M230I) have been reported in heterozygous sporadic or in recessive cases with DOPA-responsive dystonia (Segawa et al., 2004;Trender-Gerhard et al., 2009;Opladen et al., 2011).
It was not possible to functionally investigate (e.g. phenylalanine-loading test or CSF analysis) the other heterozygous variants identified in this study, therefore their effect on GCH1 activity remains undetermined. However, three of the four novel variants (A120S, D134G and G217V) detected in cases with Parkinson's disease were located at amino acid positions that are fully conserved through species down to invertebrates and were predicted to be pathogenic by all in silico prediction tools, whereas this was not the case for any of the novel mutations present in controls.
Nevertheless, the limitations of prediction tools in reliably distinguishing benign from pathogenic missense changes are well known and therefore we did not exclude any variant from the association test based on predictions scores, possibly underestimating the effect size of GCH1 pathogenic variants.
Previous studies investigating the contribution of rare coding GCH1 variants in small cohorts of cases with Parkinson's disease have reported negative results although these were insufficiently powered to draw conclusions (Bandmann et al., 1996a;Hertz et al., 2006;Cobb et al., 2009). An as-yet unpublished metaanalysis of existing genome-wide association study data has, however, identified GCH1 as a common low-risk locus (Singleton, personal communication), consistent with the hypothesis of a causal role for GCH1 in Parkinson's disease.
The mechanism whereby GCH1 mutations could predispose to nigral cell degeneration is uncertain. Biochemical evidence of GCH1 deficiency and reduced dopamine production has been reported in asymptomatic carriers of GCH1 mutations (Takahashi et al., 1994;Furukawa et al., 2002). We speculate that GCH1 deficiency and the consequent chronic dopamine deficiency could directly predispose to nigral cell death. This would suggest that normal levels of dopamine exert a protective role on the survival of nigral neurons. There is increasing evidence that levodopa is not toxic to nigral neurons as was previously thought (Parkkinen et al., 2011). Furthermore, activation of dopamine receptors may have a strong anti-apoptotic effect and increase survival of dopaminergic neurons (Nair et al., 2003;Vaarmann et al., 2013). In animal models, levodopa has been shown to promote recovery of nigrostriatal denervation (Datla et al., 2001).
Another possibility is that GCH1 mutation carriers who do not develop symptoms of DOPA-responsive dystonia in childhood may have compensatory mechanisms that allow for normal nigrostriatal dopaminergic transmission. The maintenance of these mechanisms may increase nigral cell vulnerability to ageing or other environmental and genetic factors, favouring degeneration.
It is also possible that the reduced striatal basal dopamine levels found in GCH1 mutation carriers may simply lower the threshold of nigral cell loss before parkinsonian symptoms are exhibited. Lastly, we cannot exclude that other yet unrecognized cellular pathways, not related to dopamine synthesis, may be disrupted by GCH1 and BH 4 deficiency. However, the observation that no DOPA-responsive dystonia cases, treated with levodopa since childhood, have been shown to develop nigral cell loss (Snow et al., 1993;Turjanski et al., 1993;Jeon et al., 1998), supports the notion that levodopa may indeed have a role in reducing the risk of degeneration.

Limitations of the study
First, dopamine transporter imaging was not available for the cases with Parkinson's disease with GCH1 variants identified in the exome sequencing study. It remains a possibility therefore that some of these cases (in particular Case 9, who presented at age 17, with lower limb dystonia and parkinsonism) may represent DOPA-responsive dystonia cases with a parkinsonian phenotype, which may have been misdiagnosed as Parkinson's disease.
However, removal of the aforementioned case from the statistical analysis did not change substantially the significance of the association (P = 0.0003). Furthermore, most of the patients for whom clinical follow-up data were available showed a progressive disease course with increasing levodopa requirements, emergence of motor complications due to chronic treatment with levodopa and presence of classic non-motor features of Parkinson's disease, strongly supporting nigrostriatal cell loss as the underlying pathology.
Although dyskinesias have been rarely described also in DOPAresponsive dystonia cases, these are significantly different from the ones generally observed in Parkinson's disease. Indeed they tend to appear at the beginning of the treatment and subside after dose reduction without reoccurring with subsequent slow dose increase (Furukawa et al., 2004;Lee et al., 2013). Second, we could not determine at the individual level the effect on pterin and dopamine metabolism of the GCH1 variants detected in the exome sequencing study. Reduced penetrance of GCH1 pathogenic variants for the DOPA-responsive dystonia phenotype is a well-established feature. Nevertheless it has been repeatedly reported, through analysis of brain tissue (Furukawa et al., 2002), CSF (Takahashi et al., 1994) and urine (Leuzzi et al., 2013), that even completely asymptomatic carriers of GCH1 mutations have abnormal metabolism of biopterins and dopamine, although to a lesser extent than DOPA-responsive dystonia cases. This indicates the existence of a metabolic endophenotype, which we speculate could be the pathogenic mechanism underlying the increased risk for Parkinson's disease.
Third, we evaluated a cohort enriched with early-onset and familial Parkinson's disease cases. Thus the frequency of detected GCH1 variants may not reflect the frequency in late-onset sporadic cases. Finally, we did not assess our samples for the presence of GCH1 copy number variants, possibly underestimating the frequency of GCH1 mutations.

Conclusion
We provide evidence that rare GCH1 coding variants should be considered as a risk factor for Parkinson's disease. This is derived both from imaging evidence of striatal dopaminergic denervation in GCH1 pathogenic variant carriers with a clinical diagnosis of definite Parkinson's disease (in DOPA-responsive dystonia pedigrees) and from exome sequencing data that show a significant association between GCH1 coding variants and an increased risk for the disease.
These findings expand the clinical and biological relevance of GCH1 deficiency, suggesting a role not only in biochemical dopamine depletion and DOPA-responsive dystonia, but also in nigrostriatal degeneration. The question as to how the same variants known to cause a Mendelian disease may also exist as risk alleles in Parkinson's disease may be explained by the well-known reduced penetrance of GCH1 pathogenic variants. Whether additional genetic or epigenetic factors play a role in determining the clinical phenotype of GCH1 variant carriers should be addressed by future studies. A better understanding of the relationship between GCH1 deficiency and Parkinson's disease will shed light on the role of dopamine metabolism on nigral neuron survival, with potential therapeutic implications for patients.