The Italian screening program for primary congenital hypothyroidism: actions to improve screening, diagnosis, follow-up, and surveillance.

The Italian screening program for primary congenital hypothyroidism (CH) is an integrated system including neonatal screening, diagnosis, treatment, follow-up, and nationwide surveillance of the disease. The aim of the Italian screening program for CH is to identify not only babies with severe permanent CH (core target), but also babies with mild persistent and transient forms of CH who could have a benefit from an early replacement therapy (secondary target). In the last years, despite the important results obtained in terms of standardization of screening and follow-up procedures, it has become clear the need of optimizing the program in order to harmonize the screening strategy and the screening procedures among Regions, and to improve the diagnostic and therapeutic approach in all affected infants. On the basis of available guidelines, the experience of the Italian screening and clinical reference centers, and the knowledge derived from the nation-wide surveillance activity performed by the Italian National Registry of Infants with CH, the Italian Society for Pediatric Endocrinology and Diabetology together with the Italian Society for the Study of Metabolic Diseases and Neonatal Screening and the Italian National Institute of Health promoted actions aimed at improving diagnosis, treatment, follow-up and surveillance of CH in our country. In this paper the most important actions to improve the Italian screening program for CH are described.

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

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 micromol/l in MF (mean +/- SD: 698 +/- 403) and 342-2120 micromol/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.

The pathogenesis of the white matter abnormalities in phenylketonuria. A multimodal 3.0 tesla MRI and magnetic resonance spectroscopy (1H MRS) study.

OBJECTIVE: To gain insights into the nature and pathogenesis of white matter (WM) abnormalities in PKU. METHODS: Thirty-two patients with phenylalanine hydroxylase deficiency (21 with early and 11 with late diagnosis and treatment) and 30 healthy controls underwent an integrated clinical, neuroimaging (3.0 T MRI, diffusion-weighted imaging (DWI), diffusion tensor imaging (DTI)) and neurochemical (1H MRS) investigation. RESULTS: All patients had white matter abnormalities on T2-weighted (T2W) and fluid-attenuated inversion recovery (FLAIR) scans; parietal white was consistently affected, followed by occipital, frontal and temporal white matter. T1-weighted hypointense alterations were also found in 8 of 32 patients. DWI hyperintense areas overlapped with those detected on T2W/FLAIR. The apparent diffusion coefficient (ADC) was reduced and correlated inversely with severity of white matter involvement. Fractional anisotropy index, eigenvalues lambda(min), lambda(middle), lambda(max) obtained from DTI data, and the principal brain metabolites assessed by 1H MRS (except brain phenylalanine (Phe)) were normal. Brain Phe peak was detected in all but two subjects. Brain and blood Phe were strictly associated. Blood Phe at the diagnosis, patient's age, and concurrent brain Phe independently influence white matter alteration (as expressed by conventional MRI or ADC values). CONCLUSIONS: (a) MRI abnormalities in phenylketonuria are the result of a distinctive alteration of white matter suggesting the intracellular accumulation of a hydrophilic metabolite, which leaves unaffected white matter architecture and structure. (b) White matter abnormalities do not seem to reflect the mechanisms involved in the derangement of mental development in PKU. (c) Our data do not support the usefulness of conventional brain MRI examination in the clinical monitoring of phenylketonuria patients.

Treatment monitoring of brain creatine deficiency syndromes: a 1H- and 31P-MR spectroscopy study.

BACKGROUND AND PURPOSE: Brain creatine (Cr) deficiencies (BCr-d) are rare disorders of creatine biosynthesis and transport. We performed consecutive measures of total Cr (tCr) and of its phosphorylated fraction, phosphocreatine (PCr), in the brains of children affected by Cr synthesis defects during a long period of therapy. The aim was to identify the optimal treatment strategy for these disorders. MATERIALS AND METHODS: Two patients with guanidinoacetate methyltransferase defect (GAMT-d) were treated with different amounts of Cr and with diet restrictions aimed at reducing endogenous guanidinoacetate (GAA) synthesis. Three patients with arginine:glycine amidinotransferase defect (AGAT-d) were treated with different Cr intakes. The patients' treatments were monitored by means of (1)H- and (31)P-MR spectroscopy. RESULTS: Cr and PCr replenishment was lower in GAMT-d than in AGAT-d even when GAMT-d therapy was carried out with a very high Cr intake. Cr and especially PCr replenishment became more efficient only when GAA blood values were reduced. Adenosine triphosphate (ATP) was increased in the baseline phosphorous spectrum of GAMT-d, and it returned to a normal value with treatment. Brain pH and brain P(i) showed no significant change in the AGAT-d syndrome and at any Cr intake. However, 1 of the 2 GAMT-d patients manifested a lower brain pH level while consuming the GAA-lowering diet. CONCLUSIONS: AGAT-d treatment needs lower Cr intake than GAMT-d. Cr supplementation in GAMT-d treatment should include diet restrictions aimed at reducing GAA concentration in body fluids. (1)H- and especially (31)P-MR spectroscopy are the ideal tools for monitoring the therapy response to these disorders.

Autosomal dominant GTP-CH deficiency presenting as a dopa-responsive myoclonus-dystonia syndrome.

The authors report a kindred in which GTP-CH deficiency resulted in a myoclonus-dystonia syndrome. The proband, a 17-year-old boy, presented with early-onset myoclonus and later, dystonia and bradykinesia. Blood prolactin was increased and CSF homovanillic acid, 5-hydroxyindoleacetic acid, and biopterin were all reduced. L-Dopa/carbidopa administration resulted in clinical improvement. In the paternal branch, the grandfather and three relatives had myoclonus-dystonia and resting or postural tremor of limbs. The authors found a missense mutation in the exon 6 of GCH-1 gene (K224R).

Brain creatine depletion: guanidinoacetate methyltransferase deficiency (improving with creatine supplementation).

The authors describe an Italian child with guanidinoacetate methyltransferase deficiency, neurologic regression, movement disorders, and epilepsy during the first year of life. Brain MRI showed pallidal and periaqueductal alterations. In vivo 1H-MRS showed brain creatine depletion. The assessment of guanidinoacetic acid concentration in biologic fluids confirmed the diagnosis. Clinical, biochemical, and neuroradiologic improvement followed creatine supplementation.

DNA elution and amplification by polymerase chain reaction from dried blood spots.

A quick, sensitive and easily automatizable method for PCR amplification of genomic DNA eluted from dried blood spots is described. DNA is eluted from a 3-mm spot routinely used for neonatal screening of inherited diseases either by boiling or by sonication. A preliminary and brief spot-autoclaving step is mandatory to ensure optimal and reproducible PCR amplifications. Only 1% of the eluted DNA is required for PCR analysis allowing the execution of multiple genetic tests on the same blood spot. The method has been successfully applied to the detection of a known phenylketonuria-causing mutation and will facilitate the analysis of the genetic repository provided by Guthrie's cards stored in neonatal screening laboratories.

Reduced natural killer cell activity in major depression: neuroendocrine implications.

Natural killer cell activity (NKCA) was significantly reduced in a group of depressed patients, melancholic subtype, compared to sex- and age-matched controls. Corticotropin and cortisol values were significantly higher in the depressed subjects than in the controls, but no correlation between high hormone levels and low immunological activity was found in the patients.

The pathobiochemistry of uremia and hyperargininemia further demonstrates a metabolic relationship between urea and guanidinosuccinic acid.

To better understand the biosynthesis of guanidinosuccinic acid, we determined urea, arginine, and guanidinosuccinic acid levels in nondialyzed uremic and hyperargininemic patients. These substances were also determined during several years of therapy in one hyperarginiemic patient. Interrelationships of guanidinosuccinic acid levels with their corresponding urea and arginine levels were assessed by linear correlation studies. In uremic patients, a significant positive linear correlation (r = .821, p less than .001) was found between serum urea and guanidinosuccinic acid levels A significant positive linear correlation was also found between serum urea levels and urinary guanidinosuccinic acid levels (r = .828, P less than .001), but not between serum arginine levels and urinary guanidinosuccinic acid levels in hyperargininemic patients. In the intrahyperargininemic patient study, a similar significant positive correlation was found between serum urea levels and the corresponding urinary guanidinosuccinic acid levels (r = .866, P less than .001); the correlation between serum arginine levels and the corresponding urinary guanidinosuccinic acid levels was smaller. The presented analytical findings in uremic and hyperargininemic patients clearly demonstrate a metabolic relationship between urea and guanidinosuccinic acid.

Visual, auditory, and somatosensorial evoked potentials in early and late treated adolescents with phenylketonuria.

Pattern reversal visual, auditory, and somatosensorial evoked potentials were recorded in two groups of phenylketonuric (PKU) adolescents after protracted exposition to high concentrations of phenylalanine following diet discontinuation. The first group consisted of 11 early treated (before age 3 months) PKU patients (ET-PKU); the second group consisted of 11 late detected (after age 8 months), symptomatic, PKU subjects (LT-PKU). Despite the relevant lag between the two groups in mental development and neurological status, no clear-cut difference in evoked potentials could be detected. Only the wave I latency of the brainstem auditory evoked potentials (BAEPs) was significantly shorter in ET- versus LT-PKU children. The P100 latency, I-V interpeak latency (IPL), and I-III IPL seem to discriminate the less severe form of PKU (ET-PKU type 3) from the most severe forms, ET-PKU type 1 plus 2 and LT-PKU. No correlations were found between clinical, biochemical, and neurophysiological parameters. The present data suggest that evoked potentials technique is of limited sensitivity in detecting central nervous system (CNS) alterations in PKU adolescents after diet discontinuation.

Automated high-performance liquid chromatographic method for the determination of homocysteine in plasma samples.

Plasma homocysteine determination is essential for the diagnosis of inborn errors of metabolism of sulfur amino acids and is achieving considerable importance as a possible risk marker in vascular occlusive pathology. The aim of this study was therefore to develop a fast and sensitive method to assay total and free homocysteine and total and free cysteine in plasma samples, using an automated precolumn sample pretreatment including reduction with 2-mercaptoethanol, carboxymethylation of free thiols and derivation with o-phthalaldehyde. The chromatographic separation was accomplished in 7 min, the within-run and between-run R.S.D.s were all less than 4.3%, the response was linear in the range 0.4-150 microM for homocysteine and 4-1000 microM for cysteine and the mean recoveries were higher than 96%. Moreover, with minimal modification, the method allowed the evaluation of methionine, another important marker of transsulfuration and remethylation defects. The method was applied to the diagnosis of inborn errors involving sulfur amino acids metabolism and to detect mild hyperhomocysteinemia.

Automated method for the measurement of amino acids in urine by high-performance liquid chromatography.

An automatic and sensitive HPLC method for the determination of primary and secondary amino acids included cystine and homocystine in urine samples is described. After a simple ultrafiltration, urine samples were subjected to reduction of disulfides, carboxymethylation of free thiols and double precolumn derivatization with o-phthalaldehyde-3-mercaptopropionic acid and 9-fluroenylmethyl chloroformate. All reactions were fully automated by means of an injector programme and were accomplished in 10 min. Since urine samples contain a large number of amino compounds, a good resolution was required. By optimization of the conditions, separation of 40 amino acids in 92 min was achieved. The recovery of amino acids ranged from 83% for TRP to 105% for CIT. The within-run and between-run R.S.D.s of urinary amino acid concentrations were below 10% for most amino acids except for HYL, LYS and ORN. The method was applied to the diagnosis of genetic disorders of amino acid metabolism.

Prolactin responses to domperidone in chronic schizophrenia.

The prolactin response to 20 mg of domperidone, a peripheral dopamine receptor antagonist, was evaluated in a group of 17 male, drug-free, elderly, chronic schizophrenic patients and 8 age-matched male normal control subjects. Both groups of subjects were receiving a variety of nonpsychotropic medications not known to affect the prolactin response to dopamine receptor antagonists. Basal plasma prolactin levels did not differ between the two groups. However, the prolactin response following domperidone was significantly greater in the schizophrenic patients, although plasma domperidone levels did not differ between the two groups. This effect is opposite to the previously reported effect of domperidone in young schizophrenic patients compared with age-matched control subjects (Nerozzi et al., 1990). The prolactin response to domperidone was markedly smaller in the old compared with the young normal control subjects, whereas the young and old schizophrenic patients had identical responses. Possible explanations for these results are considered, especially the possibility of abnormalities in the release of dopamine and pituitary D2 dopamine receptors in the elderly schizophrenic patients compared with age-matched normal control subjects.

Plasma prolactin response to domperidone in acute schizophrenia and schizophreniform illness.

The prolactin (PRL) response to 20 mg of domperidone, a peripheral dopamine (DA) blocking agent, was evaluated in a group of 16 drug-free, acute, young schizophreniform and schizophrenic males and in a group of age-matched normal males. Although basal plasma PRL levels were normal, the PRL responses following domperidone were blunted in both patient groups. The PRL response was more blunted in the schizophreniform patients than in the schizophrenic patients. Possible explanations for these results include pharmacokinetic factors or abnormalities of the pituitary D2 DA receptors.

[Effects of the length of radioiodine treatment of hyperthyroidism on the distribution of blood levels of calcium and phosphorus as a function of sex and age]. / Effetti a distanza del trattamento con radioiodio dell'ipertiroidismo sulla distribuzione dei livelli ematici del calcio e del fosforo in funzione del sesso e dell'età. Rilievi statistici.

An evaluation was made of the incidence of hypoparathyroidism after 131I management of hyperthyroidism and of the effect of irradiation on the relation between blood calcium, phosphorus and proteins and age in normal subjects. 356 treated patients and 216 controls were examined. Serum calcium was determined from 2 to 6 yr after treatment. It was found that calcium values decrease with age in males, wherease in women this phenomenon is less marked and, indeed, is no longer apparent over the age of 30. In the normal male, phosphrous also decreases with age, while in females there is a fall until the age of 30-40 yr, followed by a rise. Only 1 subject with a value of 8.45 mg calcium/100 ml was noted in the treated group and there was no significant difference between the means for the two groups, suggesting that parathyroid insufficiency is a virtually non-existent complication of the 131I treatment of hyperthyroidism. The relation between blood calcium and phosphorus and age in the treated group was examined with reference to subjects with normal thyroid function only. In the case of calcium, values were no longer related to age after treatment in males, while phosphorus values fell to below those observed in females, coupled with an increase in function of age as in women, though this itself was not statistically significant. Treatment also suppressed the relation between total blood proteins and age noted in the normal male. None of the parameters considered displayed any significant changes in the treated females. It would thus seem that 131I abolishes the differences in blood calcium and phosphorus mean values and age-linked patterns normally found between males and females.

[Screening for aminoacidopathies in newborns by means of an aminoacid analyzer. Reference values and statistical determinations (author's transl)]. / Screening neonatale per le aminoacidopatie con l'uso di un Aminoacid Analyzer. Determinazione dei valori di riferimento e valutazioni statistiche.

Plasmatic concentrations of amino acids valine, methionine, isoleucine, leucine, tyrosine and phenylalanine, in 4100 newborns of age varying between 1 and 18 days have been analyzed. The samples were randomly taken among the newborns of Lazio, Abruzzi and Molise subjected to neonatal screening for aminoacidopathies at the Centro delle Oligofrenie Dismetaboliche, Istituto di Neuropsichiatria Infantile. Average concentrations in mumoles/100 ml of the aminoacids and their correlations with weight and age have been studied. A comparison of the results of this research with the already published data is made, and the cut off points in mass screening for aminoacidopathies are calculated.