TETRAHYDROPIOPTERIN DEFICIENCIES: DIAGNOSIS, TREATMENT AND FOLLOW-UP Alberto Burlina Division of Metabolic Disorders Department of Pediatrics - University Hospital Padova, Italy PHENYLALANINE HYDROXYLATING SYSTEM • Phenylalanine hydroxylase deficiency Phenylketonuria Phenylketonuria responsiveness to tetrahydrobiopterin • Tetrahydrobiopterin deficiencies GTP-CH PTPS SR DHPR PAH PCD DE NOVO BIOSYNTHETIC PATHWAY FROM GTP GFRP + BH4 RECYCLING PATHWAY SYNTHESIS OF TETRAHYDROBIOPTERIN (salvage pathway) TETRAHYDROBIOPTERIN COFACTOR DEFICIENCIES WITHOUT HYPERPHENYLALANINEMIA Segawa disease Sepiapterin reductase deficiency (SR) WITH HYPERPHENYLALANINEMIA GTP cyclohydrolase I deficiency (GTPCH) 6-Pyruvoyltetrahydropterin synthase deficiency (PTPS) Dihydropteridine reductase deficiency (DHPR) Pterin-4-α-carbinolamine dehydratase (PCD) DIFFERENTIAL DIAGNOSIS OF BH4 DEFECTS + + + +/- TETRAHYDROBIOPTERIN METABOLISM PATHWAY GTP GTP-Cyclohydrolase NH2TP Neopterin PTPS 6PTP SR BH4 Tyr Trp DHPR PAH TH TPH L-Dopa 5-HTP qBH2 COMT / MAO AADC MAO HVA Dopamine Serotonin 5HIAA DH SNA HIOMT MHPG Norepinephrine N-acetylserotonin Melatonin LOGISTICS OF CSF INVESTIGATIONS • Sample preparation • Freeze CSF immediately at – – preservatives 70º C – snap freezing • Contamination with blood disturb results (centrifuge – volume immediately before freezing) • Sample storage & transport • Chose the right technique (s) • Certain amount of CSF is (very low levels of metabolites necessary (3-4 ml), collect in at in CSF) least 5 fractions 0,5 – 1 ml • Establish own control values Special tube for pterins (DTE and DETAPAC ) (control values are technique- and age-dependent) • Use always same fraction for same analysis (rostro-caudal • International quality control gradients) important CSF VALUES IN BH4 DEFICIENCIES 5HIAA 5MTHF Disorder Neo (CSF) Bio (CSF) HVA (CFS) (CSF) (CSF) arGTPCH 0.05-3.0 1.5-7.5 61-183 15-48 n PTPS 47-402 1.0-16.0 5-113 5-223 n Mild PTPS 25-230 13-56 n n n PCD 43-117 16-96 n n n DHPR 11-70 43-117 4-75 19-204 low Mild DHPR 11-70 43-117 21-66 n n- DRD 1.1-6.2 3.1-7.6 48-97 120-239 n SR 14-51 72-102* 3-15 49-111 n Controls 15-35 20-70 310-1100 150-800 64-182 * 7,8 – dihydrobiopterin and sepiapterin BH4 DEFICIENCIES BH4 deficiencies 4% 1% 4% 4% PTPS DHPR GTPCH SR PCD Unclassificated 54% 32% TOTAL PATIENTS: 617 BIODEF, 2010 Paroxysmal Eye Movement Abnormalities Athetosis Post-phenylephrine Convergence Reversal of ptosis Spasm Dystonia Fisting Limb Rigidity Torticollis Main Symptoms of BH4 Defects Physiological Aspects Physiological Aspects Dopamine Serotonin – Nigrostriatal pathway Implicated in a wide variety of physiological functions and » Control of movement behaviors. – Mesolimbic system – Feeding » Controls motivational – Sleep behavior – Circadian rhythm – Mesocortical pathway – Neuroendocrine function » Involved in learning – Emotion and memory – Motor – Hypothalamic – Cognitive tuberoinfundibular system – Autonomic » Regulates prolactin secretion PTPS Deficiency forms PTPS Deficiency forms 1%4% 15% severe mild/peripheral transient unknown 81% Mild form Severe form • Normal neurological and mental • Psychomotor retardation development • Low birth weight • Blood prolactine altered (4/5) • Severe depletion of CSF biogenic • CSF neo/Bio increased amines • Decreased of 5-HIAA (starting • Lack of correlation between in from the second year of life) vitro enzyme activity and clinical • PTS genotype : compound phenotype heterozygotes • Possible regulatory role to the physiological skipping of exon 3 (Leuzzi et al, Clin Genet, 2010) DHPR DEFICIENCY • Abnormal signs and symptoms present since birth (low birth weight, microcephaly, in utero damage) •Mild forms are associated at two mutations (G151S, F212G) • Derangement of folate metabolism, characterized by perivascular calcifications and extensive neuronal loss in basal ganglia, central cortex, white matter and seizures. • Early diagnosis and treatment is characterized by very good outcome. SEPIAPTERIN DEFICIENCY: SIGNS AND SYMPTOMS Clinical signs and symptoms % of patients Psycomotor retardation 100 Axial hypotonia 100 Limb Hypertonia 100 Spasticity 100 Extrapyramidal signs 100 SR Deficiency forms 4% Dystonia, with diurnal fluctuations 100 Tremor, bradikynesia 33 Rigid hypertonia/ cogwheel sign 58 severe mild Chorea athetosis 17 Slurred speech, dysarthria 17 Abnormal eye movements 58 96% Oculogyric crises 42 Autonomic dysfunction 83 Hypersalivation 25 Temperature instability/episodes of sweating and pallor 25 Lethargy/hypersomnolence,with diurnal fluctuations 58 Seizures 33 Cognitive deficit 25 Mood and behavior disturbances 17 Endocrine abnormalities 8 Response to L-dopa 100 GTPCH DEFICIENCY RECESSIVE FORM • Normal Phe neonatal screening (Blau and Burlina,J Ped 1995) • Normal Phe level but abnormal CSF pterines and neurotransmitters (Horvat,MGM 2008) • 7/110 mutant alleles causing the disease Segawa Disease • Normal phe level • Postural dystonia (~ 6 yrs) Ch ild •Action dystonia (~ 7 yrs) re n • Hand tremor (~ 10 yrs) A • Torticollis do le sc en • Postural dystonia (writer´s cramp) ts A du • Tremor without dystonia lts • Writer´s cramp ~ • Parkinsonism DIET AND BH4 THERAPY BH4 dosages – mg/kg/day Diseases Low – phe diet (Doses/day) GTPCH No 5-10 (2) PTPS No 5-10 (2) DHPR Yes 8-20 (2) PCD Yes / • BH4 in DHPR is controversial • BH4 brain level is dose dependent and reduced in presence of high level of dihydrobiopterin CSF/PLASMA PHENYLALANINE LEVELS IN BH4 PATIENTS The diet has to be managed on the basis of the “minimal” Phe tolerance. PHARMACOLOGY OF DOPAMINERGIC TRANSMISSION (Benarroch E, 2006) PTPS DEFICIENCY: THERAPY Age Medication Dosage (mg/kg per day) Dose/day (n) L-dopa/Carbidopa 1-3/10-20% 3-6 Newborn 5-OHtriptophan 1-2 3-6 L-dopa/Carbidopa 4-7/10-20% 3-6 5-OHtriptophan 3-5 3-6 < 1-2 years Selegiline 0,1-0,2 2 Entacapone 7-12 2 Pramipexole 0,03 2 L-dopa/Carbidopa 8-15/10-20% 3-6 5-OHtriptophan 6-9 3-6 > 1-2 years Selegiline 0,1-0,2 2 Entacapone 7-17 2 Pramipexole 0,03 2 (Ponzone, Neurology 2009) GTPCH DEFICIENCY THERAPY PADUA EXPERIENCE 5OH – triptophan Entacapone L-dopa dosages – Carbidopa dosages – dosages – Pts Age mg/kg/day mean % (range) mg/kg/day mean mg/kg/day (range) mean (range) RN 18 yrs 8,4 (6,5-10,5) 10 3,6 (2-5,2) 8,4 (5,5-11) ST 12 mo 8,9 (2-18) 25 / / PM 6 mo 1,9 (1,7-2) 25 / / DHPR THERAPY Age Medication Dosage (mg/kg per day) Dose/day (n) L-dopa/Carbidopa 1-3/10-20% 3-6 Newborn 5-OHtriptophan 1-2 3-6 Folinic acid 15-20 mg/day 1-2 L-dopa/Carbidopa 4-7/10-20% 3-6 < 1-2 years 5-OHtriptophan 3-5 3-6 Folinic acid 15-20 mg/day 1-2 L-dopa/Carbidopa 8-15/10-20% 3-6 > 1-2 years 5-Ohtriptophan 6-9 3-6 Folinic acid 15-20 mg/day 1-2 15 mg/kg/die 0.3 mg/kg/die MONITORING AND FOLLOW UP Blood phenylalanine level not reflect the clinical status not reflect the CSF phe level CSF neurotransmitters levels HVA invasive and painful logistic (difficult to perform) Prolactin screening marker for adjusting therapy (PTPS,DHPR) Clinical improvement Neuro-cognitive (Ogawa, Brain & Development, 2008) (Concolino, JIMD,2007) EXCUTIVE FUNCTION IN BH4 DEFICIENCIES Wisconsin Card Sorting Test (WCST) and Tower of Hanoi Test: effect of therapy. (Tanaka, Dev Med Child Neurology, 2007) PRENATAL DIAGNOSIS OF BH4 DEFICIENCIES Methods Fetal samples GTPCH PTPS DHPR PTERIDINES ANALYSIS ** Amniotic fluid + + + ENZYME ASSAY Erythrocytes - + + Stimulated + - + lymphocytes Cultured amniocytes - + + Chorionic villi - + + DNA ANALYSIS* Cultured amniocytes + + + Chorionic villi + + + * 1st trimester ** 2nd trimester CONCLUSION Selective screening is performed in most parts of the world BH4 loading, urinary pterins, DHPR activity and CSF neurotransmitters in the first week of life Early diagnosis is associated with better outcome Treatment is effective in reducing neurological symptoms Treatment guidelines are still vague Reports of long-term follow-up and outcome are rare and inconsistent ACKNOWLEDGMENTS: Andrea Bordugo Monica Del Rizzo Chiara Cazzorla Chiara Zanco M. Naturale, C. Edini, T Gomiero Alessandro Burlina Centre for Adult Metabolic Diseases, Neurological Unit, San Bassiano Hospital, Bassano del Grappa, Italy Nenad Blau Division of Clinical Chemistry and Biochemistry, University Children’s Hospital, Steinwiesstrasse 75, CH-8032 Zürich, Switzerland BIODEF.