Phenotypic variability, neurological outcome and genetics background of 6-pyruvoyl-tetrahydropterin synthase deficiency

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1 Clin Genet 2010: 77: Printed in Singapore. All rights reserved Short Report 2009 John Wiley & Sons A/S CLINICAL GENETICS doi: /j x Phenotypic variability, neurological outcome and genetics background of 6-pyruvoyl-tetrahydropterin synthase deficiency Leuzzi V, Carducci Ca, Carducci Cl, Pozzessere S, Burlina A, Cerone R, Concolino D, Donati MA, Fiori L, Meli C, Ponzone A, Porta F, Strisciuglio P, Antonozzi I, Blau N. Phenotypic variability, neurological outcome and genetics background of 6-pyruvoyl-tetrahydropterin synthase deficiency. Clin Genet 2010: 77: John Wiley & Sons A/S, 2009 This study aimed to investigate the clinical variability and factors implied in the outcome of 6-pyruvoyl-tetrahydropterin synthase deficiency (PTPSd). Biochemical and clinical phenotype, treatment variables, and 6-pyruvoyl-tetrahydropterin synthase (PTS ) genotype, were explored retrospectively in 19 Italian patients (12 males and 7 females, aged 4 months to 33 years). According to the level of biogenic amines in cerebrospinal fluid (CSF) at the diagnosis, the patients were classified as mild (6) (normal level) or severe (13) (abnormal low level) form (MF and SF, respectively). Blood Phe ranged from 151 to 1053 μmol/l in MF (mean ± SD: 698 ± 403) and μmol/l in SF (mean ± SD: 1175 ± 517) (p = 0.063). Patients with MF showed a normal neurological development (a transient dystonia was detected in one), while all SF patients except one presented with severe neurological impairment and only four had a normal neurological development. The outcome of the SF was influenced by the precocity of the treatment. Serial CSF examinations revealed a decline of 5-hydroxyindolacetic acid in MFs and an incomplete restoration of neurotransmitters in SFs: neither obviously affected the prognosis. PTS gene analysis detected 17 different mutations (seven so far unreported) (only one affected allele was identified in three subjects). A good correlation was found between genotype and clinical and biochemical phenotype. The occurrence of brain neurotransmitter deficiency and its early correction (by the therapy) are the main prognostic factors in PTPSd. V Leuzzi a,b, Ca Carducci a,b, Cl Carducci a,b, S Pozzessere a,b, A Burlina c,rcerone d, D Concolino e, MA Donati f, LFiori g,cmeli h,aponzone i, FPorta i, P Strisciuglio e, I Antonozzi a,b and N Blau j a Department of Child Neurology and Psychiatry, b Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy, c Division of Metabolic Disorders, Department of Pediatrics, University Hospital, Padova, Italy, d University Department of Pediatrics, G. Gaslini Institute, Genoa, Italy, e Department of Pediatrics, Magna Graecia University of Catanzaro, Catanzaro, Italy, f Metabolic Unit, Meyer Children s Hospital, Firenze, Italy, g Department of Pediatrics, San Paolo Hospital, University of Milan, Milan, Italy, h Department of Pediatrics, University of Catania, Catania, Italy, i Department of Pediatrics, University of Torino, Torino, Italy, and j Division of Clinical Chemistry and Biochemistry, University Children s Hospital, Zürich, Switzerland Key words: early onset Parkinsonism movement disorders in children neurotransmitters PKU PTS PTPS deficiency Corresponding author: Vincenzo Leuzzi, Department of Child Neurology and Psychiatry and Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy. Tel: ; fax: ; vincenzo.leuzzi@uniroma1.it Received 16 June 2009, revised and accepted for publication 25 August

2 Leuzzi et al. 6-Pyruvoyl-tetrahydropterin synthase (PTPS) deficiency (PTPSd) (MIM# ) is an autosomal recessive disorder of tetrahydrobiopterin (BH4) synthesis, which results in BH4 deficiency, hyperphenylalaninemia (HPA), and brain serotonin and dopamine depletion (1). In spite of the large number of patients reported so far (2), the factors conditioning the outcome and the genetic background of this disorder are only partially known. To contribute to these topics we report clinical presentation and course of 19 Italian patients, who were characterized from a biochemical and genetic point of view. Patients and methods Nineteen Italian PTPSd patients (12 M/7 F, aged 4 months 33 years) from 16 families were enrolled for the study. Cases have been partially reported in previous papers (3,4). The diagnosis of PTPSd was based on the following diagnostic work-up: (i) blood amino acids (19/19), (ii) CSF pterins [Biopterin (Bio) and Neopterin (Neo)] and biogenic amines [5- hydroxyindolacetic acid (5-HIAA) and homovanillic acid (HVA)] (19/19), (iii) Bio and Neo in the urine (19/19), (iv) molecular analysis of 6-pyruvoyl-tetrahydropterin synthase (PTS )gene (19/19), (v) Phe/BH4 loading test (17/19), and (vi) 6-pyruvoyl-tetrahydropterin synthase (PTPS) activity on peripheral cells (blood erythrocytes or fibroblasts) (8/19). In 18 out of 19 subjects, HPA was detected by the neonatal screening, while the diagnosis of PTPSd was achieved later: in 11 cases by the second month of life; in 3 by the fifth month (the delay was due to technical and/or logistic problems in the diagnosis confirmation). Five patients were misdiagnosed as phenylalanine hydroxylase deficiency. In order to evaluate the level of biogenic amine restoration in the brain, serial CSF examinations were performed in most of the patients during the entire follow-up. As part of the diagnostic work-up and therapy monitoring, blood prolactin was also assessed in 18 out of 19 patients according to the recommended guidelines (5). The blood samples were collected 1 h after the awakening, and prolactin was considered increased or normal on the base of more than one assessment. PTS genotype Molecular analysis of patients 10, were previously reported (3,4); the other patients were investigated in the present study, once the parents written consent was obtained. Genomic analysis was performed by exon and intron exon boundaries amplification by PCR, sequencing analysis using BDT v1.1., and automated analysis on 3130 genetic analyzer (Applied Biosystems). Primer sequence and PCR conditions are available on request. DHPLC (Varian Helix) was used to screen 128 normal alleles. RNA was extracted from patients fibroblast cell culture using RNeasy Mini Kit (QIA- GEN) and reverse transcription was performed by High Capacity cdna Reverse Transcription Kit (Applied Biosystems). Computational modeling was performed using Scratch protein predictor (6) and Protein Interfaces, Surfaces and Assemblies service PISA at European Bioinformatics Institute, (7) authored by E. Krissinel and K. Henrick (8). Results The patients were diagnosed as affected by a mild or severe form of PTPS deficiency according to their biochemical phenotype. Whereas, in both conditions Bio was low and/or Neo was high in urine, and CSF with a resulting increase of Neo/Bio ratio, the levels of 5-HIAA and HVA in CSF were normal in the mild form and low in the severe form (Table 1: cases 1 6, and cases 7 19, respectively). All the patients out of one with mild form were normal at the first examination and showed normal neurological and mental development during the entire follow-up (Table 2, cases 1 6). They were treated with BH4, whose dosage was adjusted in order to keep blood Phe in the normal range (Table 2). Five patients (cases 1, 2, 4 6) underwent repeated (1 6) CSF examinations during years of follow-up. While the level of HVA remained in the normal range, an asymptomatic mild decline of 5-HIAA was detected in all subjects from the second year of life. Almost all the patients affected by the severe form were neurologically impaired at the diagnosis, and 8 out of 13 had an abnormal neurological and mental development afterwards (Table 2, cases 7 19). Patients 12 and 13 with the lowest level of CSF biogenic amines at the diagnosis were also the most neurologically compromised. In the only patient born prematurely (Table 1, case 19), CSF examination at the gestational age of 27 weeks disclosed an extremely low level of biogenic amines. Nevertheless, the neurological development of this child was still compatible with its gestational age. The birth weight, that was low in 5 subjects and normal in the others, was 250

3 PTPS defect: phenotype and genotype Table 1. Patients with mild (1 6) and severe (7 19) form: genotype and biochemical phenotype a ID Genotype Blood PHE (μmol/l) Ur Bio r.v (mmol/mol Creatinine) Ur Neo r.v (mmol/mol Creatinine) CSF Bio (r.v.) (nmol/l) CSF Neo (r.v.) (nmol/l) CSF HVA (r.v.) (nmol/l) CSF 5-HIAA (r.v.) (nmol/l) Prl e 1 b K91E/? 780 < (20 70) 103 (15 35) 646 ( ) 420 ( ) 2 b K91E/? 151 < (20 70) 85 (15 35) 660 ( ) 603 ( ) 3 K129E/K129E 1051 < (20 70) 74 (15 35) 618 ( ) 355 ( ) 4 c P87L/V103A 1053 < (20 70) 59 (15 35) 568 ( ) 301 ( ) 5 c P87L/V103A 917 < (20 70) 81 (15 35) 475 ( ) 383 ( ) 6 I18T/N47D (20 70) 50 (15 35) 544 ( ) 184 ( ) N 7 T67M/K129E (15 40) 99 (12 30) 108 ( ) 41 ( ) N 8 T67M/K129E (15 40) 138 (12 30) 253 ( ) 64 ( ) 9 c.82 83Ins A/IVS1-3 c>g (15 40) 289 (12 30) 78 ( ) 5 ( ) 10 P87L/P87L 1270 < <0.01 (15 40) 168 (12 30) 20 ( ) 140 ( ) 11 R9C/P87L f 3.5 g 24 (10 30) 67 (9 20) 30 (98 450) 69 (45 135) N 12 T67M/? 1320 < <0.01 (10 30) 449 (9 20) <0.01 ( ) 57 ( ) 13 c.393dela/t106m (15 40) 210 (12 30) 25 ( ) 9 ( ) 14 N52S/N52S d (20 70) 106 (15 35) 217 ( ) 143 ( ) 15 N52S/N52S d 707 < (20 70) 153 (15 35) 231 ( ) 153 ( ) 16 V57del/IVS1-3C>G 1330 < (15 40) 206 (12 30) 158 ( ) 21 ( ) 17 T67M/D136V 1341 < (15 40) 141 (12 30) 134 ( ) 38 ( ) 18 IVS3-37insG/IVS3-37insG (20 70) 69 (15 35) 22 ( ) 13 ( ) 19 N47D/H49R (20 70) 38.0 (15 35) 6 ( ) 4 ( ) a Reference values from N Blau, M Duran, ME Blaskovics, KM Gibson (2003) Physician s Guide to the Laboratory Diagnosis of Metabolic Disorders, 2nd edn, Springer Verlag. Berlin, Heidelberg. In case 18, reference values for HVA and 5-HIAA were provided by the laboratory of A.B. b,c,d siblings. e Prolactin: normal (N), mild increase ( ); marked increase ( ). f r.v mmol/mol creatinine. g r.v mmol/mol creatinine. 251

4 Leuzzi et al. not obviously associated with the outcome of the disease. Among the five subjects who started the treatment by the age of 2 months, three showed an almost normal neurological development and two a moderate mental retardation associated with motor impairment. In late (>2 months) treated subjects, two out of eight had a normal mental development with minor motor abnormalities, while all the others were neurologically impaired. Phenylketonuria (PKU) was diagnosed in patient 11 at the age of 13 months when he was examined because of a progressive psychomotor retardation and paroxysmal dystonic movements of the trunk. At the age of 13, as a consequence of the diet discontinuation, he experienced neurological deterioration and recurrence of a paroxysmal trunk dystonia. The diet was resumed and continued until the age of 32, when he developed profound asthenia, restlessness, sleep disorder, and social withdrawnness. On examination he showed: moderate mental retardation, hypokinetic-asthenic syndrome, trunk rigidity, mild dystonia and tremor of upper limbs, mild balance disorders, and dystimia. The diagnostic work-up leaded to the definitive diagnosis of PTPSd. The treatment of the patients with the severe form is summarized in Table 2 (cases 7 19). An obsessive-compulsive syndrome, associated with low CSF level of 5-HIAA and improving with the increase of 5-hydroxytryptophan dosage, was observed in case eight during the adolescence. In patient 11 the attempt to increase Levodopa (L- DOPA)/Carbidopa and 5-hydroxytryptophan over the dosage of 1/0.25 and 1 mg/kg bw day, respectively, or to add a dopamine agonist (Pramipexole), resulted in a marked restlessness, panic attacks, and delusion. Serial (1 14) CSF samples were performed in 8 out of 13 subjects along 2 16 years of followup. Only in two cases (8, 16), a normal level of biogenic amines was restored by the treatment; in all the others CSF HVA and/or 5-HIAA remained well under the reference values. Nevertheless, in three of them (cases 7, 15, 17) neurological development was normal. Focusing on the other biochemical markers, CSF HVA/5HIAA ratio at the diagnosis was normal in the patients with the mild form and in 7 out of 13 affected by the severe form, and was not associated with the clinical outcome. CSF Bio was normal in 4 out of 6 cases with the mild form (and only mildly reduced in 2 cases), and in 2 out of 13 with the severe form. CSF Neo was increased in all patients and higher in patients with the severe form ( ± nmol/l) than in those with the mild form (75.3 ± 18.9 nmol/l) (p = 0.068). Urine excretion of Neo was increased in all patients, while Bio was reduced in all out of two. No significant difference was found between patients with mild and severe form. HPA was found in all the subjects at the diagnosis (mild form: 698 ± 403 μmol/l; severe form: 1175 ± 517 μmol/l; p = 0.063). In patient 2, blood Phe was no far from the cut off value (120 μmol/l) adopted by the Italian screening program. Blood prolactin was higher than normal in 15 out of 18 subjects at the diagnosis (4 out of 5 with the mild form and 11 out of 13 with the severe form) (Table 1). PTS genotype A high allelic heterogeneity was found in patients with PTPSd: 17 pathogenetic mutations were found in the 35 alleles carrying a sequence alteration: five patients were homozygous; all the others were compound heterozygotes (Table 1 and Fig. 1). In three cases the second mutation was not found. Seven mutations are so far unreported: p.r9c (c.25c>t), p.i18t (c.52a>c), c.82 83insA, p.h49r (c.146a>g), p.k91e (c.271a>g), p.v103a(c.308t>a) and IVS3-37insG (c insg). Four were associated with SF phenotype and three with MF. For eight patients (cases 1, 3, R9C I18T c.82_83insa V57del IVS3-37insG P87L K91E V103A ex1 ex2 ex3 ex4 ex5 ex6 Fig. 1. PTS mutations detected in the patients. The seven novel mutations are expressed in bold. IVS1-3C>G N47D H49R N52S T67M T106M K129E c.393dela D136V 252

5 PTPS defect: phenotype and genotype Table 2. Patients with mild (1 6) and severe (7 19) form: clinical phenotype ID Age years/ months Age at diagnosis Sex Birth weight Treatment age at onset/therapy Neurological status at (mg/kg bw/day) a diagnosis Present neurological status month M 2400 g 1 month/bh4: 5 6 Normal Normal neurological status and IQ month M 2450 g 1 month/bh4; 5 6 Normal Normal neurological status and IQ 3 4/5 20 days M n.d. 1 month/bh4: 5 6 Normal Normal neurological status and development 4 1/7 13 days F 3070 g 13 days/bh4: 5 6 n.d. Normal neurological status and IQ 5 3/11 1 month F 2800 g 1 month/bh4: 5 6 Normal Normal neurological status and IQ 6 4/6 1 month M 3450 g 3 months/bh4: 10 Mild dystonic posture of upper limbs 7 17/1 <1 month:hpa 17 months: PTPSd 8 14/5 <1 month:hpa 17 months: PTPSd 9 3/8 <1 month:hpa 4 months: PTPSd M 2300 g 1 month/diet 20 months/bh4: 3 5 L-DOPA: OH-Tript: F 2950 g 18 months/bh4: 6 7 L-DOPA: OH-Tript: M 2800 g 4 months/bh4: 11 L-DOPA: 3 5 5OH-Tript: 3 5 selegiline: /4 2 months M 2480 g 2 months/bh4: 2 L-DOPA: months: HPA 32 years: PTPSd 5OH-Tript: 3 5 M 3200 g 13 months/diet 32 years/bh4: 5 L-DOPA: 1 5OH-Tript: 1 Severe trunk hypotonia developmental delay (DQ 44), athetosis Clumsiness, very mild dystonia of upper limbs, normal IQ Borderline mental development (IQ 76) Hypotonia, developmental delay Moderate mental and language retardation, disturb of coordination, OCD Left esotropia, hypokinesia, hypotonia, developmental delay Developmental delay, trunk rigidity, dystonia, tremor, oculo-gyric crisis, epileptic seizures Trunk rigidity ad dystonia, limb tremor, balance disorders, dysthymia, hypokinetic-asthenic syndrome, sleep disorders Left esotropia, moderate mental and language retardation Moderate mental retardation (IQ 42), bradylalia, dysarthria, clumsiness, limb dystonia, diurnal fluctuation Moderate mental retardation (IQ 54), dyskinesias (myoclonus, chorea), panic attacks 253

6 Leuzzi et al. Table 2. Continued ID Age years/ months Age at diagnosis Sex Birth weight Treatment age at onset/therapy Neurological status at (mg/kg bw/day) a diagnosis Present neurological status month: HPA 3 years: PTPSd 13 14/10 2 months: HPA 4 months: PTPSd M 2050 g 3 years/bh4: L-DOPA: OH-Tript: selegiline: M 1690 g 8 months/bh4: 20 L-DOPA: 1 5 5OH-Tript: /6 8 days F 3540 g 8 days/bh4:5 6.4 L-DOPA: OH-Tript: /5 1 month M 3170 g 1 month/bh4: L-DOPA month: HPA 5 months: PTPSd month: HPA 12 months: PTPSd 5OH-Tript: selegiline: 0.1 M 2900 g 5 months/bh4 L-DOPA OH-Tript: Selegilina: M 2900 g 12 months/bh4: L-DOPA OH-Tript: 1 5 Selegilina: /6 1 month F months/bh4: 21 L-DOPA 1.5 5OH-Tript: /4 20 days F 1020 g 25 days/bh4: 10 L-DOPA: 1 5OH-Tript: 1 Trunk hypotonia and limb rigidity, spastic tetraparesis, severe mental retardation Progressive psychomotor retardation, hypotonus, hypokinesia Trunk hypotonia, limb rigidity, lethargy Trunk hypotonia, limb rigidity, lethargy Severe mental retardation, spastic tetraparesis, seizures, self- and hetero-injurious conducts; deceased at 20 years Severe mental retardation, coreoathetosis, mild limb dystonia Psychomotor retardation Normal mental development (IQ 105), mild language disorder and attention deficit Trunk hypotonia Normal mental development, mild upper limbs coordination disorder Progressive psychomotor retardation, seizures Normal mental development, bradylalia with diurnal fluctuation Mild psychomotor retardation Mild psychomotor retardation, mild distal limb dystonia Normal for gestational age Normal psychomotor development HPA, hyperphenylalaninemia; OCD, obsessive-compulsive disorder; PTPSd, PTPS deficiency. a Range of the dosages administered during the course of the disease; L-DOPA was associated with Carbidopa (ratio 4:1). 254

7 PTPS defect: phenotype and genotype 5, 6, 7, 9, 18, and 19) the allelic segregation was confirmed in the parents. To evaluate the pathogenicity of the novel variations and to rule out the presence of polymorphisms, 128 normal alleles were screened for the presence of the seven new mutations by using DHPLC and none of them was found in the examined alleles. The alignment of the protein among different species showed that the five residues interested by the missense mutations were conserved with the exception of Isoleucine 18. To evaluate the effects of c.82 83insA and c insg insertions, RNA was extracted from patient s fibroblasts and, the cdna obtained by reverse transcription, was divided into two fragments by nested PCRs and analyzed by sequencing. The c.82 83insA causes a frame shift, which creates a stop codon at amino acid position 29. Furthermore, as it occurs at the second-last nucleotide of exon 1, it probably also affects RNA splicing. At the RNA level, we observed three species: a fragment carrying the complete skipping of exons 2 and 3, a fragment carrying the A insertion and a fragment, due to the second allele (IVS1-3C>G), carrying a skipping of 12 nucleotides of exon 2. The intronic insertion (IVS3-37insG), instead, showed only the complete skipping of exon 3. Discussion PTPS deficiency is the most common alteration of BH4 metabolism and one of the more frequent causes of autosomal recessive Parkinsonism in childhood (1). The extension of newborn screening in new emerging countries has revealed some important population based differences in the frequency of this disease (9,10). As HPA is generally, also if not obligatorily [see, for example, case 1 in (11)], one of the metabolic markers of this disorders, PTPSd diagnosis can be precociously addressed according to the diagnostic work-up flow chart designed for subjects with HPA (1). Our data support the importance of assessing the pattern of pterin excretion in any patient with HPA, independently from the level of blood Phe. As a result of the progressive implementation of this procedure, the youngest patients in our series took advantage of a more precocious diagnosis. We also recommend considering this condition in all patients presenting with movement disorders of unknown etiology. According to our data, the first and most relevant prognostic factor in PTPSd is the occurrence of biogenic amines depletion in CSF at the moment of the diagnosis: based on this biochemical feature the patients are classified as affected by generalized (severe) or peripheral (mild) form of PTPSd (12,1). These different biochemical phenotypes reflect the degree of PTPS impairment and the selective sensitivity of phenylalanine, tyrosine, and tryptophan hydroxylases to BH4 deficiency (12,1). Mild form This rare condition, which involves less than 20% of all PTPSd patients (1,2), is generally associated with normal neurological and mental development (1). However, a number of clinical and biochemical data from our patients and the literature suggest the mild form as a condition in which biogenic amine homeostasis in the brain, even if preserved, is more vulnerable and perhaps unstable in time: (i) blood prolactin was altered in four out of five patients at the diagnosis, suggesting a scarcity of Dopamine at level of D 2 receptors located on lactotroph membranes (13) (which was not reflected by HVA dosage in CSF) (14); (ii) CSF Neo/Bio ratio was slightly increased in all patients; (iii) in spite of the positive clinical course, serial assessment of biogenic amines revealed in our cases a decline of 5-HIAA in CSF starting from the second year of life. A similar trend, without any associated symptom, was previously reported either under BH4 treatment (15) or after BH4 discontinuation [(12), patient GT]. On the contrary, the child reported by Scriver et al. [(16); proband L], notwithstanding a normal biogenic amine level in CSF, suffered from irritability, truncal hypotonia and developmental delay, and exhibited reversible neurological deterioration and limb dystonia after BH4 discontinuation. Even if these cases suggest a wider than so far thought phenotypic spectrum, as rule, the prognosis of this condition is good. Moreover, it remains to be established, if the decline of CSF 5-HIAA needs to be corrected by precursor supplementation. In the lack of data concerning long-term outcome of this condition, we suggest monitoring clinically all these patients until the adulthood and treating only those who manifest neurological or behavioral disorders. PTS genotype is reported in a few patients with mild form recorded in the International BH4 Database (2). We characterized four patients, while only a mutant allele (carrying a so far unreported alteration) was identified in two brothers. Homozygous K129E genotype was already found in another Italian patient (3,15), who presented with mild and transient HPA associated with an increase 255

8 Leuzzi et al. of Neo/Bio ratio in urine (while CSF pterins and biogenic amine were normal). In comparison with this case, in our patient the HPA was persistent and Neo/Bio ratio was high both in urine and CSF. Two affected siblings were compound heterozygotes for the severe p.p87l mutation associated with a so far unreported mutation (p.v103a). Computational modeling analysis suggests for this mutation a role only in conformational structure variation that would be compatible with a high residual enzymatic activity. Finally, the association between the new p.i18t mutation, which involves the important phosphorylation motif (17), and p.n47d mutation results in a mild phenotype. A possible dominant negative effect has been supposed for p.n47d in a subject with mild phenotype also carrying p.d116g mutation (18). Severe form When compared with the mild form, the clinical course of the severe form is characterized by a much more large variability and a poorer prognosis. Similarly to what reported for other autosomal recessive disorders of biogenic amine metabolism (19), a derangement in mental development was generally associated with the disorders of movement. On the contrary, in the autosomal dominant form, due to the defect of GTP-cyclohydrolases (or Segawa disease), mental functions remain usually unaffected (20). This observation confirms the importance of the biogenic amines in the pre-natal and early post-natal brain development and differentiation (21). The occurrence of high percentage of PTPSd patients with a low weight at birth (22,23) has supported the view of a possible pre-natal onset of the disease in the most compromised cases (22). We have detected a severe depletion of CSF biogenic amines as early as 27 weeks of gestational age in a PTPSd child born prematurely. On the practical ground, we suggest to assess repeatedly blood Phe in the pre-term newborns and to look as soon as possible for PTPS deficiency in case of HPA. We confirm the early initiation of treatment as one of the main factors affecting the outcome of severe PTPSd. The beginning of the therapy by the first month of life and high dosages (10 15 mg/kg/ bw/day) of l-dopa have been recently associated with a normal mental development in PTPSd patients (24). Such high dosages could be tolerated only by a few of our patients. The retrospective design of our study as well as the different approach in patient classification (that in our cases included the levels of CSF biogenic amines at the diagnosis) make difficult the comparison of our results with those of this study. The occurrence of psychiatric disorders in PTPSd patients has not been systematically explored so far. In two patients we diagnosed behavior disturbs, such as obsessive-compulsive disorder or depression with panic attacks, revealing a possible serotonergic imbalance, which improved (in a case) by handling the dosage of the biogenic amines precursors. Four new mutations were found in these patients. Two are missense mutations involving the binding site for zinc (p.h49r) and the oligomerization site (p.r9c). The other two are single nucleotide insertions, causing severe molecular modifications. The skipping of 12 nucleotides associated to IVS1-3C>G alteration has been already reported (3, patient 16). Patient 18 carried homozygous c insg mutation, which was confirmed in the parents, who were both heterozygote for the mutation. As the family originated from a small town, the parents consanguinity could not be definitively ruled out. The lack of correlation between in vitro enzyme activity and clinical phenotype (12, 25) coupled with the existence of milder forms suggest that PTS gene expression could be differently regulated in different tissues. In this respect, a possible regulatory role has been ascribed to the physiological skipping of exon 3 (in adjunction of that observed as result of several sequence variations) whose extent is different in different tissues and is probably associated with the adjacent stretch of polypyrimidine (26). This mechanism has been well described in the expression of other genes, such as the CFTR gene, where the presence of polypyrimidine-binding proteins, which regulate exon 9 skipping and therefore the amount of functional transcripts, was demonstrated (27). In conclusion, the main prognostic factor in PTPSd is the occurrence of biogenic amines depletion in the brain as detected in early post-natal period by CSF examination. An early diagnosis and treatment influence the outcome of the disease in subjects with CSF neurotransmitter deficiency. According to the repertoire of the pathogenetic alterations so far reported, genotype generally helps in predicting phenotype. 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