Research Article Sepiapterin Reductase Deficiency: a treatable mimic of cerebral palsy

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1 Sepiapterin reductase deficiency mimics CP 1 Research Article Sepiapterin Reductase Deficiency: a treatable mimic of cerebral palsy Jennifer Friedman MD 1, Emmanuel Roze PhD 2, Jose E. Abdenur MD 3, Richard Chang MD 3, Serena Gasperini MD 4, Veronica Saletti MD 5, Gurusidheshwar M. Wali DM 6, Hernan Eiroa MD 7, Brian Neville FRCP 8, Alex Felice PhD 9, Ray Parascandalo MD 10, Dimitrios I. Zafeiriou PhD 11, Luisa Arrabal- Fernandez MD 12, Patricia Dill MD 13, Florian S Eichler MD 14, Bernard Echenne MD 15, L.G. Gutierrez- Solana MD 16, Georg F. Hoffmann MD 17, Keith Hyland PhD 18, Katarzyna Kusmierska PhD 19, Marina A.J. Tijssen MD 20, Thomas Lutz MD 21, Michel Mazzuca MD 22, Johann Penzien MD 23, Bwee Tien Poll- The MD 24, Jolanta Sykut-Cegielska MD 25, Krystyna Szymanska MD 26, Beat Thöny PhD 27, Nenad Blau PhD 28 UCSD Departments of Neurosciences and Pediatrics; Rady Children s Hospital, San Diego, California, USA Department of Neurology, Salpetriere Hospital, APHP, Paris, France; INSERM, UMRS 975 and CNRS CRICM, Hôpital Pitié-Salpêtrière, Paris, France Children s Hospital of Orange County, Orange County, California, USA Pediatric Neurology and Metabolic and Neuromuscular Disorders Units, Neuroscience Department, Anna Meyer Children's Hospital, University of Florence, Italy Developmental Neurology Unit, IRCCS Foundation "Carlo Besta" Neurological Institute, Milan, Italy Director, Neurospecialities Centre, Belgaum, India Servicio de Errores Congenitos del Metabolismo, Hospitalde Pediatria "J.P.Garrahan", Buenos Aires, Argentina Neurosciences Unit, UCL Institute of Child Health, London, UK Laboratory of Molecular Genetics, Department of Physiology and Biochemistry, Faculty of Medicien and Surgery, University of Malta, Msida, Malta 10 Consultant Paediatrician & Senior Clinical Lecturer Department of Paediatrics Mater Dei Hospital Malta 11 1st Department of Pediatrics, Aristotle University of Thessaloniki, Greece 12 Department of Pediatric, Virgen de las Nieves Hospital, Granada, Spain 13 Division of Pediatric Neurology and Developmental Medicine, University Children's Hospital, Basel, Switzerland

2 Sepiapterin reductase deficiency mimics CP 2 14 Department of Child Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA 15 Department of Pediatric Neurology, Gui de Chauliac Hospital, Montpellier, France 16 Seccion de Neurologia, Hospital Infantil Universitario Nino Jesus, Madrid, Spain 17 University Children s Hospital Heidelberg, Heidelberg, Germany 18 Medical Neurogenetics, Atlanta, Georgia, USA 19 Laboratory of Metabolic Disorders,Department of Biochemistry and Experimental Medicine, The Children's Memorial Health Institute, Warsaw, Poland 20 Department of Neurology, Academic Medical Centre Amsterdam, the Netherlands 21 University Children s Hospital Heidelberg, Heidelberg, Germany 22 INSERM U CNRS UMR5292, Lyon Neuroscience Research Center, Brain Dynamics and Cognition Team, Lyon, F-69000, France; University Lyon 1, Lyon, F-69000, France; Service d'audiologie et d'exporations Orofaciales - Hopital Edouard Herriot - Hospices Civils de Lyon, France 23 Klinik für Kinder und Jugendliche Augsburg, Germany 24 Department of Pediatric Neurology, Academic Medical Center Amsterdam, The Netherlands 25 Department of Metabolic Diseases, Children s Memorial Health Institute, Warsaw, Poland 26 Department of Experimental and Clinical Neuropathology, Polish Academy of Science Mossakowski Medical Research Centre, Warsaw, Poland 27 University Children s Hospital Zürich, Division of Clinical Chemistry and Biochemistry, Zürich, Switzerland; Zürich Center for Integrative Human Physiology (ZIHP), Zürich, Switzerland 28 University Children s Hospital Zürich, Division of Clinical Chemistry and Biochemistry, Zürich, Switzerland; Zürich Center for Integrative Human Physiology (ZIHP), Zürich, Switzerland; University Children s Hospital Heidelberg, Heidelberg, Germany Corresponding author: Jennifer Friedman, MD Departments of Neurosciences and Pediatrics University of California San Diego Corresponding author s address: Rady Children s Hospital, San Diego 8010 Frost St, Suite 400 San Diego, California, 92123, USA

3 Sepiapterin reductase deficiency mimics CP 3 Corresponding author s phone and fax: Phone: Fax: Corresponding author s jrfriedman@rchsd.org Running head: Sepiapterin reductase deficiency mimics CP Characters in title 61 Characters in running head: 42 Number of words in abstract: 253 Number of words in main text: 3146 Number of figures: 5 (3 color) Number of tables: 2 plus 1 Supplementary Table Acknowledgments This study was funded in part by the Swiss National Science Foundation grant no A /2 (to NB and BT). Funding source had no role in study design, collection analysis or interpretation of data, or writing of the report. All authors had access to data, commented on drafts and approved the final report. JF and NB had responsibility for submission decision. The authors acknowledge Dr. Fabienne Clot for molecular and Dr. Teodoro Bottiglieri for biochemical analyses. The authors also acknowledge Dr. Marvin Natowicz for commenting on the manuscript. Conflicts of interest NB received research grants from BioMarin Pharmaceutical Inc, Novato, CA and from Merck Serono, Geneva, Switzerland. JF received consultancy fees from BioMarin Pharmaceutical Inc, Novato, CA. KH is Vice President of Medical Neurogenetics, a company that provides biochemical and molecular testing for sepiapterin reductase deficiency.

4 Sepiapterin reductase deficiency mimics CP 4 ABSTRACT Objective Sepiapterin reductase deficiency (SRD) is an under-recognized levodopa-responsive disorder. We describe clinical, biochemical and molecular findings in a cohort of patients with this treatable condition. We aim to improve awareness of the phenotype and available diagnostic and therapeutic strategies to reduce delayed or misdiagnosis, optimize management and improve understanding of pathophysiologic mechanisms. Methods Forty-three individuals with SRD were identified from twenty-three international medical centers. The phenotype and treatment response were assessed by chart review using a detailed standardized instrument and by literature review for cases for which records were unavailable. Results In most cases, motor and language delays, axial hypotonia, dystonia, weakness, oculogyric crises and diurnal fluctuation of symptoms with sleep benefit become evident in infancy or childhood. Average age of onset is 7 months with delay to diagnosis of 9.1 years. Misdiagnoses of cerebral palsy (CP) are common. Most patients benefit dramatically from L-dopa/carbidopa often with further improvement with the addition of 5-hydroxytryptophan. Cerebrospinal fluid findings are distinctive. Diagnosis is confirmed by mutation analysis and/or enzyme activity measurement in cultured fibroblasts. Interpretation Common, clinical findings of SRD, aside from oculogyric crises and diurnal fluctuation, are nonspecific and mimic CP with hypotonia or dystonia. Patients usually improve dramatically with treatment. Consequently, we recommend consideration of SRD not only in patients with L-doparesponsive motor disorders, but also in patients with developmental delays with axial hypotonia, and patients with unexplained or atypical CP. Biochemical investigation of cerebrospinal fluid is the preferred method of initial investigation. Early diagnosis and treatment are essential to prevent ongoing injury to developing brain.

5 Sepiapterin reductase deficiency mimics CP 5 INTRODUCTION Defects in monoamine biosynthesis are associated with various phenotypes including developmental delay, diurnal variation of symptoms and dystonia that may improve with levodopa 1. The best known of these disorders is L-dopa-responsive dystonia (DRD; Segawa disease) due to mutations in the GTP cyclohydrolase I gene (Figure 1) 2. Sepiapterin reductase (SR) deficiency (SRD; OMIM#182125) is a more rare condition that may also cause a DRD phenotype 3. Both disorders lead to altered tetrahydrobiopterin (BH4) biosynthesis, and abnormal biogenic amine metabolism (Figure 1). SRD is inherited in an autosomal recessive manner and is caused by mutations in the SPR gene, located on chromosome 2p14-p12 4. Individuals with SRD display a L-dopa-responsive, diurnally fluctuating 3, 5- motor disorder, in most cases associated with cognitive delays and severe neurologic dysfunction 19. The diagnosis of SRD is based on cerebrospinal fluid (CSF) analysis of neurotransmitter metabolites and pterins and can be confirmed by either measurement of SR activity in fibroblasts or mutational analysis of the SPR gene 20. Therapy aims to correct the neurotransmitter and pterin abnormalities using catecholamine and serotonin precursors and/or pathway agonists and inhibitors. Improvement with treatment varies, but in most cases is dramatic. Thirty patients with SRD have been reported as individual cases or small series 3, Detailed, systematic review of the clinical, biochemical and genetic aspects of SRD is lacking. A comprehensive characterization of SRD will enable the development of improved evaluation and care of individuals with SRD as well as an enhanced understanding of the pathophysiology of impaired BH4 metabolism and its impact on neurotransmitter homeostasis in the CNS. We evaluated 43 SR-deficient patients, identified features common to patients with SRD, defined effective treatment strategies and proposed a diagnostic algorithm for this potentially devastating yet treatable disorder. SUBJECTS AND METHODS Standard protocol approvals Approval was obtained from Institutional Review Board of the University of California San Diego.

6 Sepiapterin reductase deficiency mimics CP 6 Cases Forty-three cases with SRD from medical centers in North and South America, Europe and Asia, were identified from three sources: 1. Published reports identified by literature search using MEDLINE (1990 January 2011) using key words: sepiapterin, sepiapterin reductase, SPR. 2. Patient data existing in the BIODEF database ( 3. Cases cared for by the author (JF) or known to the authors (JF, NB) from personal communication. Procedures Clinicians caring for identified cases were asked to provide anonymized clinical, biochemical and molecular data using a standardized survey instrument. For previously published cases, survey responses were compared with literature reports and discrepancies resolved by direct query to the managing physician. Diagnosis was based on characteristic CSF markers in most and confirmed in all by demonstration of reduced fibroblast SR activity and/or pathologic SPR gene mutation. For cases for which survey data was not available (5/43), biochemical, and/or molecular data were extracted from published reports 10, 13 and/or BIODEF database ( Clinical data for these 5 cases were not included in order to maintain uniformity in data collection methods. In this report the following conventions are applied to age of symptom onset: infancy <18 months; childhood 18 months 10 years. Survey Instrument A survey instrument was developed to capture the clinical findings and natural history of SRD, treatment response, and the results of diagnostic studies. The instrument also included open-ended questions to allow ascertainment of previously unreported symptoms and signs and other information relating to therapeutic interventions. RESULTS Forty-three patients with SRD were identified. Surveys were obtained for 38/43 including 11 not previously reported. These include cases from 27 independent families. Both genders were equally affected. Ethnic backgrounds were wide ranging with a probable founder mutation in the Maltese

7 Sepiapterin reductase deficiency mimics CP 7 population accounting for 7/38 patients 5. Symptoms began at 7 months (range: birth 6 years), with delay to diagnosis averaging 9.1 years. Misdiagnoses of cerebral palsy (CP) were common (19/38). Signs and symptoms All patients (n=38) showed symptoms characteristic of catecholamine and serotonin deficiency. Severity ranged widely with some severely impacted by disabling motor and cognitive deficits and others mildly to minimally symptomatic, identified only because of diagnosis in an affected family member. Features of SRD, present in >65% include motor and speech delay, axial hypotonia, dystonia, weakness and oculogyric crises with diurnal fluctuation and sleep benefit. Frequent clinical features, present in 45-65% include: dysarthria, parkinsonian features (bradykinesia, rigidity, tremor, or masked facies), hyper-reflexia, psychiatric and/or behavioral abnormalities, sleep disturbance, mental retardation, autonomic signs and limb hypertonia. Numerous other symptoms were reported (Figure 2). Cardinal features of DRD, dystonia and diurnal fluctuation (79% - 30/38), were not universal in SRD and were often absent early. Only 40% (12/30) of those developing dystonia and 60% (18/30) of those with diurnal fluctuations had these symptoms in infancy, the latter recognized sometimes only in retrospect. Even among patients up to 4 years of age at diagnosis (n=12), dystonia was not a reliable sign present in only 50% (6/12). In these patients, diurnal fluctuation and oculogyric crises (75% - 9/12) were more consistent findings, the latter sometimes mistaken for seizures, especially when associated with more generalized paroxysmal dystonic episodes. The early clinical phenotype is dominated by non-specific signs - axial hypotonia and developmental delays present in 92% (11/12) of patients diagnosed prior to 4 years of age. Sleep disturbances were common 53% (20/38) often presenting in infancy - 55% (11/20) or in childhood - 35% (7/20). Psychiatric, and/or behavioral abnormalities were also frequent, occurring in 55% (21/38). Though symptom onset could not always be dated, childhood onset was most common - 48% (10/21) (Figure 2). Various types of cognitive disability were also common. Mental retardation occurred in 53% (20/38). Eight percent (3/38) had normal cognition and the remainder had mild to moderate learning disabilities. There was no correlation between degree of cognitive impairment and length of delay

8 Sepiapterin reductase deficiency mimics CP 8 from symptom onset to therapy (data not shown). Comparison of cognitive findings was limited by lack of uniformity in assessment methods. Biochemical and Molecular Investigations (43 patients) CSF analysis was completed in 29/43. All showed low 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA), elevated total biopterin, dihydrobiopterin (BH2), and sepiapterin, consistent with diagnoses of SRD (Figure 3, Supplementary Table 1). SR activity in fibroblasts was reduced or absent in 9/9 patients. Other investigations are summarized in Table 1. 42/43 patients were fully genotyped. Of the 16 mutations detected in the SPR gene (Figure 4) (7 missense, 4 nonsense, 3 frame-shift, 1 splice-site, and 1 in promoter region), the intronic homozygous variant c.596-2a>g is the most common genotype (GF=21.4%), found in a cohort of Maltese patients with DRD 5 and two additional patients. Though we cannot rule out ascertainment bias, the p.r150g mutation, present in 14 patients, possibly arose in the Mediterranean region (Heritage: Spanish 8/14, Turkish 2/14, Italian 1/14, Unspecified Caucasian 3/14). The most common mutations are c.596-2a>g and p.r150g (both AF=20%), p.k251x (AF=17%), and p.v138d (AF=12%). Five novel mutations were detected p.v9gfsx35, p.s136f, p.y196g, p.q206afsx55, and p.k230x. The p.g102c mutation was found in three siblings with normal cognition. There was no other correlation between cognitive function and either specific mutations or CSF metabolite levels (data not shown). Biochemical and molecular data are tabulated in Supplementary Table 1. BIOMDB database contains detailed information on DNA variations. Treatment Thirty-six patients were treated with L-dopa in combination with peripheral decarboxylase inhibitor (carbidopa or benserazide). All patients showed dramatic improvement; three experienced complete resolution of symptoms. Improvements were reported most commonly in motor delays, dystonia and other extrapyramidal symptoms, oculogyric crises, and sleep disturbance, if present. Dose ranged from mg/kg/day L-dopa with most effective dose averaging 3.9 mg/kg/day. Although, dose related dyskinesias were common at initiation or adjustment of therapy (45%), these lessened with reduced dose in most patients. One of nine patients treated for years required increased L- dopa dose (mg/kg) over time. None developed late onset dyskinesias.

9 Sepiapterin reductase deficiency mimics CP 9 Twenty patients were treated with 5-hydroxytryptophan (5-HTP)/carbidopa from 0.14 to 6 mg/kg/d with benefit in 16/20 and most effective dose averaging 2.2 mg/kg/day. A single patient treated with only 5-HTP/carbidopa had marked improvements in motor and sleep symptoms 14. In all others, as 5- HTP was added after L-dopa, independent benefit sometimes was unclear. Most had improvements in motor and sleep symptoms beyond gains achieved with L-dopa. One treated with 5-HTP after 22 years of stable L-dopa dosing had resolution of circadian rhythm disturbance 15. On 5-HTP, 50% developed gastrointestinal side effects including abdominal pain, diarrhea and vomiting. These lessened or resolved with dose reduction or increased carbidopa in some. Though rarely formally assessed both before and after therapy, cognitive skills reportedly improved in 34%(13/38). Two patients improved mildly on L-dopa alone. The other eleven were treated with combination L-dopa/5-HTP. 8/11 received L-dopa and 5-HTP within 1.5 year of each other and thus independent effect of either is unclear. Of these, four had dramatic and four had mild improvements in cognitive skills. The three remaining patients were on L-dopa/carbidopa only for at least eight years prior to introduction of 5-HTP. Two, treated for only a few months, had mild improvement in school performance. The other had resolution of mild dysexecutive syndrome demonstrated by psychometric evaluation 15. Four patients were treated with BH4. Though no clear benefit was noted, the dosage was either low or of short duration. Other beneficial medications were employed less frequently (Table 2). DISCUSSION In this study, we report the evolution of clinical characteristics, biochemical and molecular features and response to therapy in 43 patients with SRD including 13 patients not published previously. The SRD phenotype is characterized by core features including: axial hypotonia, motor and language delays, oculogyric crises, weakness, dystonia and diurnal fluctuation of symptoms with sleep benefit. Parkinsonian features, limb hypertonia, hyper-reflexia, mental retardation, psychiatric and/or behavioral abnormalities, autonomic dysfunction, and sleep disturbances are also common. Numerous rare signs overlap with symptoms noted in other disorders of monoamine neurotransmitter biosynthesis (Figure 2) 1.

10 Sepiapterin reductase deficiency mimics CP 10 Most patients present in infancy with non-specific features including developmental delays and axial hypotonia. When present, oculogyric crises may be a distinguishing sign. Over time there is development of limb hypertonia, hyper-reflexia, dystonia and more apparent diurnal fluctuation and sleep disturbance. Psychiatric and/or behavioral signs may appear later (Figure 2). Though mental retardation is common, many have only mild to moderate learning disabilities. Various psychiatric and psychological symptoms were noted with inattention, irritability, and anxiety most frequent. These symptoms are likely to be under-reported given the severity of motor symptoms that dominate the clinical picture. In addition, early treatment may cause under-estimation of the prevalence of these symptoms not typically evident in very young children. Patients with SRD may pose a diagnostic challenge, especially early and in those with milder phenotypes and/or normal intelligence. Dystonia, a characteristic sign in DRD, is not universal in SRD, 6, 10, and when present may not occur in infancy. Thus, the term DRD, as suggested by some authors 13 may not always apply. In infancy, common features of SRD, hypotonia and developmental delays, are non-specific and mimic other neurologic conditions including, as in this series, CP. Even as children age and dystonia and/or hyper-reflexia emerge, diagnosis may remain elusive. As noted previously in DRD, a striatal (dystonic) toe may simulate a Babinski sign 21. This constellation of findings, in the setting of early developmental delays, may be difficult to distinguish from CP with dystonia. Features atypical for CP 22 (Figure 5), but relatively specific for disorders of biogenic amine metabolism 1, may be unrecognized, mis-interpreted, or not apparent during examinations early in the day. Diurnal variation and sleep disturbance may be difficult to identify, especially in infants. Oculogyric crises, present in only 75% may be mis-interpreted as seizure. Autonomic symptoms may not be routinely queried by neurologists who typically care for these patients. In addition, patients receiving an early diagnosis of CP, are not always re-evaluated to assess for emergence of new signs as they age. Current diagnostic strategies pose additional challenges. Diagnosis requires biochemical and/or molecular investigations that are invasive, costly and/or not universally available. Screening may be helpful to narrow the subset of patients in which to consider further evaluation (Table 1). Though CSF neurotransmitter and pterin analysis is the preferred initial investigation, lumbar puncture has fallen

11 Sepiapterin reductase deficiency mimics CP 11 into disfavor with both physicians and patients. L-Dopa trial is an acceptable, though less informative, alternative when the former is not obtainable (Figure 5). Phenylalanine loading test is a sensitive and specific screen for disorders of BH4 metabolism 23 and was abnormal in 8/8 SRD patients. Blood prolactin and serotonin and urine pterins and catecholamines were abnormal in some patients. Though prolactin level is an accepted surrogate for monitoring L-dopa replacement therapy 24, its use as a sensitive or specific screening tool for disorders of dopamine synthesis has not been established. Too few patients have been evaluated with the other tests to draw conclusions (Table 1). Despite these challenges, it is imperative that clinicians remain vigilant to avoid mis- or delayed diagnosis. Though SRD patients improve dramatically with neurotransmitter precursor supplementation, in most, residual deficits remain. Normal neurotransmitter homeostasis is essential for the developing brain, particularly in the neonatal period; 25 late diagnosis or treatment may potentially lead to irreversible neurologic dysfunction. The extent to which incomplete therapeutic response is due to inadequacies of treatment versus underlying, irreversible brain injury is unclear. At present, it is prudent to begin therapy as early as possible. Sixteen mutations (five novel) were reported in the SPR gene with c.596-2a>g and p.r150g the most common alleles (Figure 4). No clear genotype phenotype correlation is apparent (other than a possible cognition-sparing association with p.g102c). In fact, there may be broad phenotypic variability even among siblings with the same mutations. In a single family, alternative splicing has been proposed as a possible mechanism to account for symptomatic variation 7. Other genetic and/or epigenetic factors likely contribute. Correlation of cognitive level with biogenic amine levels in CSF has also been suggested 7. In our cohort, sample size is too small and there is too much variation in the use of CSF evaluation and the age of therapy initiation to make conclusions regarding correlation between genotype, levels of CSF markers and outcome. It has previously been suggested that p.k251x is associated with a more severe phenotype 7, however, this was not evident in our study with p.k251x noted in homozygous form in individuals both with and without mental retardation. Though most reported patients with SRD have two mutant SPR alleles, a single symptomatic patient with heterozygous mutation and typical DRD phenotype has been reported 10. Although no other symptomatic heterozygous carriers were identified in this study, the possibility of manifesting carriers has been suggested, though not confirmed in several cases with non-genotyped family members with dystonia 17.

12 Sepiapterin reductase deficiency mimics CP 12 In most patients, therapeutic approaches include dopamine and serotonin precursor supplementation. In isolated cases other strategies augmenting dopaminergic and/or serotonergic neurotransmission or BH4 levels were utilized. Most patients display dramatic response to low-dose L-dopa/carbidopa with improvement primarily in motor and sleep symptoms. Dose-related dyskinesias are common and generally resolve with dose reduction. Though independent benefit of 5-HTP was difficult to assess, in many there were added benefits to motor and sleep deficits and variable additional cognitive improvements. A subset of patients had dramatic improvements in cognitive abilities on combination L-dopa/5HTP. In all cases, the effects of therapy on cognition, independent of improvements in disordered sleep, which may have negatively impacted performance, could not be separated. In many, despite dramatic improvements with L-dopa/carbidopa, persistent problems with mild motor and significant cognitive problems remain. Even with these unresolved deficits, 5-HTP was tried in only 53%. Given potential benefit, we recommend 5-HTP, beginning with low dosage, in all cases without complete symptom resolution on L-dopa/carbidopa. Similarly, very few patients with residual deficits on L-dopa/carbidopa/5-HTP undergo additional treatment trials though these medications were successful in a subset (Table 2). In a related disorder of BH4 synthesis, 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, monoamine-a oxidase (MAO-A) inhibitors and dopamine agonists offer additional therapeutic benefits In SR-deficient patients, these agents should be considered when deficits remain on dopamine and serotonin precursor therapy alone. No conclusion can be reached regarding the use of BH4 (sapropterin dihydrochloride) as experience is limited. Although, BH4 can cross the blood-brain barrier 28, high dosage (>10 mg/kg/d) is need to stimulate neurotransmitter production 29,. Trimethoprim, sulfamethoxazole and methotrexate, potential inhibitors of dihydropteridine reductase (DHPR), an enzyme involved in BH4 regeneration, may result in hyperphenylalaninemia in addition to neurotransmitter deficiency 30. These agents should be avoided in SRD. In addition, similar to patients with aromoatic L-amino acid decarboxylase deficiency, some SRD patients supplemented with highdosage L-dopa may potentially develop cerebral folate deficiency and need supplementation with folinic acid (NB personal observation) 31.

13 Sepiapterin reductase deficiency mimics CP 13 The pathophysiology underlying SRD is incompletely understood. Reduced SR enzymatic activity causes impaired BH4 biosynthesis and subsequent reduction in monoamine neurotransmitter production. Reduced neurotransmitter levels likely account for most symptoms as features in SRD overlap those in other conditions affecting monoamine neurotransmitter biosynthesis 1. In SRD, in contrast to these other conditions, there is an accumulation of BH2 and sepiapterin (Figure 1). These changes could potentially affect other metabolic pathways including tyrosine and tryptophan hydroxylase and NOS 32. It has been postulated that altered ratios of BH4:BH2 33 may lead to uncoupling of NOS and increased free radical generation 20. The potential impact is unclear but suggests that further study is necessary to establish whether BH4 supplementation or other strategies to reduce oxidant stress are warranted in SRD. Additional key issues requiring further study include: (1) possible utility of screening evaluations to narrow the subset of patients in which to consider SRD; (2) delineation of the entirety of the phenotypic spectrum, especially the mildest cases; (3) potential correlations of phenotypes with SPR genotypes and/or levels of CSF metabolites; (4). the impact of L-dopa and/or 5-HTP supplementation on cognition and behavior; and (5) the role for serial CSF evaluations to monitor neurotransmitter, pterins, and/or folate levels. There are inherent limitations to retrospective studies. Because of varying amounts of clinical observation and documentation, there is a reduced ability to comprehensively evaluate the natural history of SRD for all patients. This may result in underestimation of some symptoms and signs. Further, there may be a bias toward reporting motor and expected symptoms rather than unexpected or non-neurologic symptoms that may not be considered part of the phenotype. In addition, as most patients received treatment during childhood, the natural history and evolution of symptoms is altered. Despite these limitations, this report offers insights into the genetic and biochemical basis of SRD, the clinical spectrum and responses to therapy in this disorder, and the role of BH4 and monoamine neurotransmitter biosynthesis in human disease. Increased awareness of these issues will improve diagnostic evaluation and management and provide new insights into the care of patients with this uncommon but treatable and under-recognized condition. Given the potential for dramatic clinical improvement, we recommend consideration of SRD or other neurotransmitter disorder in all patients with a dopa-responsive motor disorder, patients with developmental delays and axial hypotonia, and in patients with unexplained or atypical cerebral palsy especially if hypotonia or dystonia are present

14 Sepiapterin reductase deficiency mimics CP 14 (Figure 5). A lumbar puncture and biochemical investigations of CSF are essential for an early and accurate diagnosis. ACKNOWLEDGEMENT This study was funded in part by the Swiss National Science Foundation grant no A /2 (to NB and BT). The authors acknowledge Dr. Fabienne Clot for molecular and Dr. Teodoro Bottiglieri for biochemical analyses. The authors also acknowledge Dr. Marvin Natowicz for commenting on the manuscript.

15 Sepiapterin reductase deficiency mimics CP 15 REFERENCES 1. Kurian MA, Gissen P, Smith M, Heales SJ, Clayton PT. The monoamine neurotransmitter disorders: an expanding range of neurological syndromes. Lancet Neurol Aug;10(8): Ichinose H, Ohye T, Takahashi E, et al. Hereditary progressive dystonia with marked diurnal fluctuation caused by mutation in the GTP cyclohydrolase I gene. Nature Genet. 1994;8: Bonafé L, Thöny B, Penzien JM, Czarnecki B, Blau N. Mutations in the sepiapterin reductase gene cause a novel tetrahydrobiopterin-dependent monoamine neurotransmitter deficiency without hyperphenylalaninemia. Am J Hum Genet. 2001;69: Thöny B, Heizmann CW, Mattei MG. Human GTP cyclohydrolase I gene and sepiapterin reductase gene map to region 14q21-q22 and 2p14-p12, respectively, by in situ hibridization. Genomics. 1995;26: Neville BG, Parascandalo R, Farrugia R, Felice A. Sepiapterin reductase deficiency: a congenital dopa-responsive motor and cognitive disorder. Brain Oct;128(Pt 10): Abeling NGGM, Duran M, Bakker HD, et al. Sepiapterin reductase deficiency an autosomal recessive DOPA-responsive dystonia. Mol Genet Metab. 2006;89: Arrabal L, Teresa L, Sanchez-Alcudia R, et al. Genotype-phenotype correlations in sepiapterin reductase deficiency. A splicing defect accounts for a new phenotypic variant. Neurogenetics Mar Echenne B, Roubertie A, Assmann B, et al. Sepiapterin reductase deficiency: clinical presentation and evaluation of long-term therapy. Pediatr Neurol Nov;35(5): Kusmierska K, Jansen EE, Jakobs C, et al. Sepiapterin reductase deficiency in a 2-year-old girl with incomplete response to treatment during short-term follow-up. J Inherit Metab Dis Jan 7:DOI /s Steinberger D, Blau N, Goriuonov D, et al. Heterozygous mutation in 5'-untranslated region of sepiapterin reductase gene (SPR) in a patient with dopa-responsive dystonia. Neurogenetics Sep;5(3): Verbeek MM, Willemsen MA, Wevers RA, et al. Two Greek siblings with sepiapterin reductase deficiency. Mol Genet Metab Aug;94(4): Wali GM, Thony B, Blau N. Sepiapterin reductase deficiency: Two Indian siblings with unusual clinical features. Mov Disord Mar 10;25 (7): Clot F, Grabli D, Cazeneuve C, et al. Exhaustive analysis of BH4 and dopamine biosynthesis genes in patients with Dopa-responsive dystonia. Brain Jul;132(Pt 7): Friedman J, Hyland K, Blau N, MacCollin M. Dopa-responsive hypersomnia and mixed movement disorder due to sepiapterin reductase deficiency. Neurology Dec 12;67(11): Leu-Semenescu S, Arnulf I, Decaix C, et al. Sleep and rhythm consequences of a genetically induced loss of serotonin. Sleep Mar 1;33(3): Dill P, Wagner M, Somerville A, Thöny B, Blau N, Weber P. Paroxysmal stiffening, upward gaze, and hypotonia in infancy: clinical hallmarks of sepiapterin reductase deficiency, a very rare but treatable disorder of neurotransmitter metabolism. Neurology. 2011:in press. 17. Bainbridge MN, Wiszniewski W, Murdock DR, et al. Whole-genome sequencing for optimized patient management. Sci Transl Med Jun 15;3(87):87re Mazzuca M, Christa L, Damaj l, et al. Dopa-responsive hypersomnia in combined sepiapterin reductase (SR) and methyl malonyl CoA epimerase (MCEE) deficiency. J Inerit Metab Dis. 2010;33:S Thibert R, Hyland K, Chiles J, Steinberg S, Eichler F. Levodopa response reveals sepiapterin reductase deficiency in a femal heterozygote with adrenoleukodysrophy. J Inerit Meta Dis. 2011(in press).

16 Sepiapterin reductase deficiency mimics CP Blau N, Bonafé L, Thöny B. Tetrahydrobiopterin deficiencies without hyperphenylalaninemia: Diagnosis and genetics of Dopa-responsive dystonia and sepiapterin reductase deficiency. Mol Genet Metab. 2001;74: Nygaard TG, Waren SP, Levine RA, Naini AB, Chutorian AM. Dopa-responsive dystonia simulating cerebral palsy. Pediatr Neurol. 1994;11: Neville B. Congenital DOPA-responsive disorders: a diagnostic and therapeutic challenge to the cerebral palsies? Dev Med Child Neurol Feb;49(2): Opladen T, Okun JG, Burgard P, Blau N, Hoffmann GF. Phenylalanine loading in pediatric patients with dopa-responsive dystonia: revised test protocol and pediatric cut-off values. J Inerit Metab Dis. 2010;33: Spada M, Ferraris S, Ferrero GB, et al. Monitoring treatment in tetrahydrobiopterin deficiency by serum prolactin. J Inherit Metab Dis. 1996;19(2): Frederick AL, Stanwood GD. Drugs, biogenic amine targets and the developing brain. Dev Neurosci. 2009;31(1-2): Schuler A, Kalmanchey R, Barsi P, et al. Deprenyl in the treatment of patients with tetrahydrobiopterin deficiency. J Inherit Metab Dis. 2000;23: Porta F, Mussa A, Concolino D, Spada M, Ponzone A. Dopamine agonists in 6-pyruvoyl tetrahydropterin synthase deficiency. Neurology Aug 25;73(8): Kapatos G, Kaufman S. Peripherally administered reduced pterins do enter the brain. Science. 1981;212: Ponzone A, Ferraris S, Baglieri S, Spada M. Treatment of tetrahydrobiopterin deficiencies. In: Blau N, editor. PKU and BH4: Advances in Phenylketonuria and Tetrahydrobiopterin. Heilbronn: SPS Verlagsgesellschaft; p Blau N, Curtius AC, Kierat L, Leupold D, Kohne E. Hyperphenylalaninemia caused by dihydropteridine reductase deficiency in children receiving chemotherapy for acute lymphoblastic leukemia. J Pediatr. 1989;115: Brun L, Ngu LH, Keng WT, et al. Clinical and biochemical features of aromatic L-amino acid decarboxylase deficiency. Neurology. 2010;75: Zorzi G, Thöny B, Blau N. Reduced nitric oxide metabolites in CSF of patients with tetrahydrobiopterin deficiency. J Neurochem. 2002;80: Takeda M, Yamashita T, Shinohara M, et al. Plasma Tetrahydrobiopterin/Dihydrobiopterin Ratio. Circ J Mar 18;73:

17 Page 21 of 31 Annals of Neurology Table 1. Additional investigations in 38/43 SR-deficient patients. Investigation BLOOD/PLASMA Number Abnormal/ Number Tested Abnormalities Noted Phenylalanine Loading 8/8 Elevated Phenylalanine and Phenylalanine/Tyrosine Ratio Fasting Phenylalanine a 3/25 Elevated mild Prolactin 14/18 Elevated Serotonin 4/4 Reduced URINE Catecholamines 2/3 2- Reduced HVA, 5HIAA and VMA; 1-Reduced Dopamine Pterins b 2/5 Reduced Biopterin IMAGING MRI - brain 5/33 1-Mild Peri-ventricular Leukomalacia; 2-Mild Cortical Atrophy; 2-Delayed Myelination (repeat MRIs normal on therapy) Other Imaging 0/5 No Abnormalities c : 2 CT; 1- MRS; 1-18 F-FDG PET; 1 IBZM-SPECT; 1-(FP)-CIT-SPECT OTHER EEG 8/26 3- Spikes; 3- Non-specific changes; 2-Spike wave discharges Elevations in SRD patients have not been previously reported but likely occur due to impaired hepatic hydroxylation of phenylalanine. Elevations were mild and a phenylalanine-restricted diet was not felt to be necessary. Probably non-specific A single patient had two studies. HVA = homovanillic acid; 5-HIAA = 5-hydroxyindoleacetic acid; VMA = Vanillmandelic acid; CT = computed tomography; MRI = magnetic resonance imaging; 18 F-FDG PET = 18-fluorodeoxyglucose positron emission tomography; IBZM-SPECT = 123I-iodobenzamide single-photon computed emission tomography; (FP)-CIT-SPECT = (123)I-N-ω-fluoropropyl-2βcarbomethoxy-3β-(4-iodophenyl)nortropan single-photon computed emission tomography John Wiley & Sons

18 Page 23 of 31 Annals of Neurology Table 2. Medications used to treat 38 SR-deficient patients. Medication Patients Duration Dose Range Most Effective Side Effects Reported with Average Used Dose (Number of Patients with Benefit (Range) (yrs) (mg/kg/day) (mg/kg/day) Symptom) -dopa 1,2,3 Dyskinesia (16); Vomiting 36/ ( ) (2); Anxiety (1); Impulsivity (1); Increased oculogyric crises (1) -HTP 2,3 Diarrhea (5); Abdominal 16/ ( ) Pain (5); Vomiting (4); Sleepiness (1); Anxiety (1); Transaminase elevation (1) BH 4 4 0/4 2.4 ( ) None Bromocriptine 5 Dyskinesia(1); Increased 2/3 Not Reported Not Reported Not Reported dystonia (1); Pathologic Anticholinergics 1/2 Not Reported Various None gambling (1) Selegeline 7 1/ None ( ) Sertraline 8 1/ Dyskinesia/Myoclonus 9 Melatonin 10 1/ None Two adult patients declined L-dopa trial due to mild symptoms. 2 In combination with peripheral decarboxylase inhibitor. In some patients L-dopa and 5-HTP were initiated simultaneously. Therefore, independent effectiveness and adverse events for these medications could not be determined. Side effects in these patients included: bradykinesia(1); hypertension(1); psychosis(1). The latter was felt to be medication related and resolved with reduction in L-dopa dose. Dosage of up to 4 mg/kg/d in two patients, one for 4 and one for 5 5 years. Dosage of 10 mg/kg/d in one patient for 2 weeks and one patient dosage 10 mg/kg/d for 1 week, 15 mg/kg/d for 1 week and 20 mg/kg/d for 1 week. 5 Dopamine Agonist. 6 Benztropine beneficial in a single patient. Trihexphenidyl not tolerated in a single patient. 7 Monoamine oxidase-a inhibitor. 8 Serotonin Reuptake Inhibitor. 9 Patient on combination sertraline and selegeline. 10 Product of serotonin metabolism. John Wiley & Sons

19 Annals of Neurology Page 24 of 31 Figure 1. Tetrahydrobiopterin (BH4) pathway and its function in the biosynthesis of neurotransmitters dopamine and serotonin. BH4 is synthesized in de novo pathway by three enzymes: GTP cylclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). The final two-steps reduction of 6-pyruvoyl-tetrahdropterin to BH4, catalyzed by SR, proceed via two oxo-intermediates (1 -O- PH4 and 2 -O-PH4) and can be alternatively processed by carbonyl reductase 1 (CBR1), aldose reductase 1B1 (AKR1B1), and 3α-hydroxysteroid dehydrogenase type 3 (AKR1C3). During the hydroxylation of tyrosine to L-Dopa and tryptophan to 5-hydroxytryptophan (catalyzed by tyrosine- and tryptophan hydroxylases, respectively) BH4 is oxidized to a carbinolamine intermediate (HO-BH4), which is subsequently regenerated to BH4 by the enzymes pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR). In patients with SRD the 1 -O-PH4 intermediate is non-enzymatically converted to sepiapterin (Sep), which can be subsequently reduced to 7,8-dihydrobiopterin (7,8-BH2) by CBR1 and accumulate in patients with SRD. Both Sep and 7,8-BH2 accumulate in patients with SRD, however due to the very low activity of dihydrofolate reductase (DHFR) in brain, 7,8-BH2 cannot be reduced back to BH4 (salvage pathway). Enzyme abbreviations: GTPCH, GTP cylclohydrolase I; PTPS, 6-pyruvoyl-tetrahydropterin synthase; SR, sepiapterin reductase; PCD, pterin-4a-carbinolamine dehydratase; DHPR, dihydropteridine reductase; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase; CBR1, carbonyl reductase 1; AKR1B1, aldose reductase 1B1; AKR1C3, 3α-hydroxysteroid dehydrogenase type 3; DHFR, dihydrofolate reductase; AADC, aromatic amino acid decarboxylase. Metabolites abbreviations: NH2TP, dihydroneopterin triphosphate; PTP, 6-pyruvoyl-tetrahdropterin; 1 -O- PH4, 1 -oxo-2 -hydroxypropyl-tetrahydropterin; 2 -O-PH4, 2 -oxo-1 -hydroxypropyl-tetrahydropterin; Sep, sepiapterin; 7,8-BH2, 7,8-dihydrobiopterin; BH4, tetrahydrobiopterin; q-bh2, quinonoide-dihydrobiopterin; HO-BH4, 4a-hydroxy-tetrahydrobiopterin; Tyr, tyrosine; Trp, tryptophan; L-Dopa, dihydroxyphenylalnine; 5- OH-Trp, 5-hydroxytryptophan. 275x190mm (300 x 300 DPI) John Wiley & Sons

20 Page 25 of 31 Annals of Neurology Figure 2. Symptoms in 38 sepiapterin reductase deficiency patients. Symptoms are sorted by frequency. Symptom bars are subdivided to indicate age of onset of each symptom. Where age of onset differed for symptoms within a grouped category, earliest age of onset is represented. Where symptoms are grouped, the number of patients within each subgroup is as follows: Parkinsonian Signs (Tremor 15, Bradykinesia 14, Masked Facies 13, Rigidity 11); Psychiatric/Behavioral Symptoms (Inattention 14, Irritability 11, Anxiety 7, Hyperactivity 5, Aggression 4,Obsessive or Compulsive Features 3, Depression 2, Impulsivity/Disinhibition 2, Panic 1, Psychosis-possibly medication related - 1); Sleep Disturbance (Hypersomnolence 15, Difficulty Maintaining Sleep 10, Drowsiness 6, Difficulty Initiating Sleep 5); Autonomic Symptoms/Signs (Excessive Sweating 12, Ptosis 7, Nasal Congestion 6, Temperature Instability 4); Gastrointestinal Symptoms (Feeding Difficulties 7, Recurrent Vomiting 6, Reflux 3); Eye Movement Abnormalities (Strabismus 6, Oculomotor Apraxia 3, Nystagmus 2); Pulmonary Symptoms (Wheezing 5, Noisy Breathing 1, Laryngospasm 1). The following symptoms, not represented in the figure, were reported in single patients: axial hypertonia, constipation, microcephaly, cataract, premature graying and growth retardation. 193x150mm (300 x 300 DPI) John Wiley & Sons

21 Annals of Neurology Page 26 of 31 Figure 3. CSF concentrations (median and 25th 75th percentile) of key neurotransmitter metabolites in patients with sepiapterin reductase deficiency at the time of diagnosis (age yrs; median 8.3 yrs). Neo: neopterin; Bio: biopterin; BH2: 7,8-dihydrobiopterin; Sep: sepiapterin; 5HIAA: 5-hydroxyindoleacetic acid; HVA: homovanillic acid. Reference ranges are marked as shaded area. Sepiapterin is not detectable in healthy controls. Reference ranges may differ between laboratories. 275x190mm (300 x 300 DPI) John Wiley & Sons

22 Page 27 of 31 Annals of Neurology Figure 4. Physical structure of the SPR gene and spectrum of mutations found in patients with SR deficiency. Accession numbers in Genome Sequence Data Base: AB and AB for human gdna; M for human cdna (for the cdna numbering starts with 1 as A at the ATG-start codon). 254x190mm (300 x 300 DPI) John Wiley & Sons

23 Annals of Neurology Page 28 of 31 Figure 5 Diagnostic algorithm for patients with a possible disorder of neurotransmitter metabolism. SRD and other disorders of neurotransmitter metabolism should be considered in patients with: 1.Developmental delay with hypotonia; 2.Suspect but unexplained CP or CP with atypical features; and 3. Uncharacterized L- dopa-responsive motor disorders. 1. In a patient with developmental delay and hypotonia, if oculogyric crises, diurnal fluctuation, sleep disturbance, extrapyramidal or autonomic signs exist, a disorder of neurotransmitter biosynthesis is likely and CSF analysis* should be done. If no other signs are present, CSF analysis should be considered if standard work up for hypotonia is unrevealing. If CSF analysis is abnormal then mutational screening and/or measurement of enzymatic activity can be targeted to confirm specific disorder suggested by pattern of CSF abnormalities. If CSF evaluation is impractical, alternative evaluation may include L-dopa trial and/or phenylalanine load. If John Wiley & Sons

24 Page 29 of 31 Annals of Neurology negative, CSF analysis must still be done to exclude disorder of neurotransmitter metabolism. If L-dopa trial and/or phenylalanine loading are positive, CSF analysis will allow targeted mutational screening, however, one should keep in mind that Phe-load can be positive in heterozygote carriers for phenylketonuria. Alternatively, CSF analysis may be skipped and broad mutational screening undertaken. Mutational and gene dosage screening may be time consuming, costly and false negatives may still occur. Therefore, this alternative evaluation route should be reserved for cases in which CSF analysis is not available, is declined, or in which other clinical features lead to suspicion of a specific diagnosis. 2. In a patient with unexplained CP or CP with atypical features, a disorder of neurotransmitter metabolism should be considered and diagnostic algorithm, as outlined above, should be followed. Atypical or unexplained features suggesting need for further metabolic investigation in a child with possible CP include lack of adequate antecedent, non-diagnostic MRI, progressive symptoms, familial occurrence, episodic encephalopathy and features not expected in the cerebral palsies eg. diurnal variation, sleep disturbance, autonomic symptoms, or oculogyric crises22 3. All patients with a dopa-repsonsive motor disorder should be evaluated for a disorder of neurotransmitter metabolism. CSF analysis* (after discontinuation of L-dopa therapy for at least 10 days) is the recommended first step. If L-dopa withdrawal is impractical, the results of CSF analyses may still be informative if either pterins or 5HIAA levels are abnormal. Alternatively, molecular investigations can be done, guided either by results of phenylalanine loading test or clinical symptoms with caveats as noted above1. *CSF analysis - homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5HIAA), pterins (neopterin, biopterin, and sepiapterin), and 5-methyltetrahydrofolate. CSF = cerebrospinal fluid; Phe = phenylalanine; SR = sepiapterin reductase. 215x279mm (300 x 300 DPI) John Wiley & Sons

25 Annals of Neurology Page 30 of 31 Supplementary Table 1 Biochemical and molecular data of patients with SR deficiency at the age of diagnosis. BIODEF#* Age** 5HIAA HVA Neo Bio BH2 Sep SR (FB) Allele 1 Allele 2 Ref. yrs nmol/l mu/mg <5 p.k251x p.k251x nd nd nd nd nd nd nd p.g102c p.r150g nd nd nd nd nd nd nd p.g102c p.r150g nd nd nd nd nd nd nd p.r150g p.k251x nd nd nd nd nd nd nd p.r150g p.k251x nd 43 nd nd p.y196g p.y196g <5 p.r150g p.t151gfsx <5 p.q119x p.q119x nd nd 35 nd nd p.r150g p.r150g nd 5.0 <5 p.t151gfsx2 p.t151gfsx nd 7.0 nd p.p163l p.p163l nd c.-13g>a wt nd 13.1 nd K251X p.k251x nd nd nd nd nd nd nd c.596-2a>g c.596-2a>g nd nd nd nd nd nd nd c.596-2a>g c.596-2a>g nd nd nd nd nd nd nd c.596-2a>g c.596-2a>g nd nd nd nd nd nd nd c.596-2a>g c.596-2a>g nd nd nd nd nd nd nd c.596-2a>g c.596-2a>g nd nd nd nd nd nd nd c.596-2a>g c.596-2a>g nd nd nd nd nd nd nd c.596-2a>g c.596-2a>g nd 5.2 nd p.v138d p.v138d nd 12.4 nd p.v138d p.v138d nd nd <5 p.k230x p.k230x nd nd <5 p.k251x p.k251x nd 6.4 nd nd nd nd 20.0 <5 p.k251x p.k251x nd p.n127k p.n127k nd 74 nd 15.1 <5 p.r219x p.r219x nd nd nd nd nd nd nd c.596-2a>g c.596-2a>g nd nd nd nd nd nd nd c.596-2a>g c.596-2a>g nd 6.0 nd p.r150g p.r150g nd 7.8 nd p.g102c p.r150g nd 10.8 nd p.r150g p.q206afsx nd 16.0 nd p.v138d p.v138d , 4, 6 7, , 12 11, 12 11, 12 11, 12 11, 12 11, 12 11, , 18 1 John Wiley & Sons