Silvio Ferraris

Differential diagnosis of hyperphenylalaninaemia by a combined phenylalanine-tetrahydrobiopterin loading test

We describe a new fully reliable method for the differential diagnosis of tetrahydrobiopterin-dependent hyperphenylalaninaemia (HPA). The method comprises the combined phenylalanine (Phe) plus tetrahydrobiopterin (BH4) oral loading test and enables the selective screening of BH4 deficiency when pterin analysis is not available or when a clear diagnosis has not been previously made. It should be performed together with the measurement of dihydropteridine reductase (DHPR) activity in blood. The new combined loading test was performed in nine patients with primary HPA, three with classical phenylketonuria (PKU), three with DHPR deficiency, and three with 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency. Three hours after oral Phe loading (100 mg/kg body weight), synthetic BH4 was administered orally at doses of either 7.5 or 20 mg/kg body weight. Amino acid (Phe and tyrosine) and pterin (neopterin and biopterin) metabolism and kinetics were analysed. By exploiting the decrease in serum Phe 4 and 8h after administration, a clear response was obtained with the higher BH4 dose (20 mg/kg body weight), allowing detection of all cases of BH4 deficiency, as well as differentiation of BH4 synthesis from regeneration defects. Since DHPR deficient patients who were previously shown to be non-responsive to the simple BH4 loading test gave a positive response, the combined Phe plus BH4 loading test can be used as a more reliable tool for the differential diagnosis of HPA in these patients. Moreover, it takes advantage of being performed while patients are on a Phe-restricted diet.

HDR Syndrome : A Novel de novo Mutation in GATA3 Gene

Human GATA3 haploinsufficiency leads to HDR (hypoparathyroidism, deafness, and renal dysplasia) syndrome. The development of a specific subset of organs in which this transcription factor is expressed appears exquisitely sensitive to gene dosage. We report on a 14‐year‐old patient with symptomatic hypoparathyroidism, sensorineural bilateral deafness, unilateral renal dysplasia, bilateral palpebral ptosis, and horizontal nystagmus. Fundoscopy displayed symmetrical pseudopapilledema, and brain CT scan revealed basal ganglia calcifications. FISH analysis did not disclose any microdeletion in the 22q11.2 or 10p14 regions. GATA3 mutation analysis identified a heterozygous deletion of GG nucleotides at codon 36 and 37 (c.108_109delGG) in exon 2 causing a frameshift with a premature stop codon after a new 15‐aminoacid sequence. Restriction endonuclease analysis performed in parents was negative. Our patient carries a novel “de novo” GATA3 mutation, providing further evidence that HDR syndrome is caused by haploinsufficiency of GATA3, which may be responsible for a complex neurologic picture besides the known triad.

Tetrahydrobiopterin Loading Test in Hyperphenylalaninemia

ABSTRACT: Some cases of primary hyperphenylalaninemia are not caused by the lack of phenylalanine hydroxylase, but by the lack of its cofactor tetrahydrobiopterin. These patients are not clinically responsive to a phenylalanine-restricted diet, but need specific substitution therapy. Thus, it became necessary to examine all newborns screened as positive with the Guthrie test for tetrahydrobiopterin deficiency. Methods based on urinary pterin or on specific enzyme activity measurements are limited in their availability, and the simplest method, based on the lowering of serum phenylalanine after loading with cofactor, was discouraged by the finding that some dihydropteridine reductase-deficient patients were unresponsive. The preliminary observation that this limitation could be overcome by increasing the dose of the administered cofactor prompted us to reevaluate the potential of the tetrahydrobiopterin loading test in hyperphenylalaninemia. Fifteen patients, eight with ultimate diagnosis of phenylketonuria, three with 6-pyruvoyl tetrahydropterin synthase-, and four with dihydropterine reductase-deficiency, have been examined by administering synthetic tetrahydrobiopterin both orally, at doses of 7.5 and 20 mg/kg, and i.v., at a dose of 2 mg/kg. All the tetrahydrobiopterin-deficient patients, unlike those with phenylketonuria, responded to the oral dose of 20 mg/kg cofactor by lowering their serum phenylalanine concentration markedly below baseline to an extent easily detectable by Guthrie cards. This method allows for a simple screening method when enzyme or pterin studies are not available.

Impact of Neonatal Protein Metabolism and Nutrition on Screening for Phenylketonuria

Objectives: Early blood phenylalanine (Phe) elevation after birth enables screening for and anticipation of the diagnosis of phenylketonuria. The differential impact of factors involved in this phenomenon, however, has not been elucidated. To solve this question, phenotype, genotype, dietary Phe intake, timing of blood collection, and Phe metabolism were retrospectively analyzed in 21 phenylketonuria newborns and prospectively in 1. Patients and Methods: Patients were assigned to 1 of 4 classes of phenylalanine hydroxylase (PAH) deficiency (severe, moderate, mild, and benign) on the basis of their Phe tolerance. Phe ingested, tolerated, and released from endogenous catabolism was assessed. Results: From birth to screening test, the amount of Phe tolerated ranged from 704 to 1620 mg, according to the class of PAH deficiency. The amount of Phe ingested ranged only from 204 to 405 mg, whereas the endogenous Phe breakdown ranged from 812 to 1534 mg, resulting in a rate of Phe catabolism ranging from 262 to 341 mg/day, regardless of the class of PAH deficiency. Conclusions: The high rate of protein catabolism is the main determinant of neonatal hyperphenylalaninemia. It is sufficient to turn to positive the screening test in severe and moderate PAH deficiency. In mild and benign PAH deficiency, the outcome of screening procedures can be substantially altered by the concurrence of genetic and peristatic factors. These results imply that the value of blood Phe at the screening test is not fully predictive of the phenylketonuria phenotype, and strengthen concerns regarding the reliability of early screening procedures.

Unresponsiveness to tetrahydrobiopterin of phenylalanine hydroxylase deficiency

Conflicting results have been reported concerning the efficacy of tetrahydrobiopterin (BH4), the cofactor of phenylalanine hydroxylase, for reducing phenylalanine (Phe) concentration in phenylketonuria (PKU). We aimed to test quantitatively the effects of BH4 in PKU patients. Seven fully characterized patients were selected among a population of 130 PKU subjects as harboring PKU mutations predicted as BH4 responsive and previously considered responsive to a cofactor challenge. They received a simple Phe (100 mg/kg) and 2 combined Phe (100 mg/kg) and BH4 (20 mg/kg) oral loading tests. Cofactor was administered either before or after the amino acid. The concentrations of Phe, tyrosine (Tyr), and biopterin were measured over 24 hours after loading. The comparative analysis of the loading tests showed that in all patients plasma Phe concentrations peaked within 3 hours, and fell within 24 hours by about 50% in benign, 20% in mild, and 15% in severe phenylalanine hydroxylase deficiency regardless of BH4 administration. A consistent or moderate increase of plasma Tyr, again independent of the cofactor challenge, was observed only in the less severe forms of PAH deficiency. Mean blood biopterin concentration increased 6 times after simple Phe and 34 to 39 times after combined loading tests. The administration of BH4 does not alter Phe and Tyr metabolism in PKU patients. The clearance of plasma Phe after oral loading and, as well as Tyr production, is not related to cofactor challenge but to patients phenotype. The assessment of BH4 responsiveness by the methods so far used is not reliable, and the occurrence of BH4-responsive forms of PKU still has to be definitely proven.

Hair anomalies as a sign of mitochondrial disease

In 8 out of 25 children with a mitochondrial disorder, slow growing, sparse and fragile hair was observed as an early sign of their disease. Microscopic examination of the hair showed the presence of trichorrhexis nodosa and pili torti. Hair abnormalities can be added to the wide clinical spectrum of mitochondrial disorders. Conclusion:microscopic hair examination is an easy, first level diagnostic tool that can lead to a suspected mitochondrial defect in the early stages of the disease, before symptoms of progressive multi-organ involvement develop.

Dipylidium caninum in an infant

Sir: The article dealing with Dipylidium caninum infection in an infant by Reid et al. [4] offers us the opportunity to report an additional case and to discuss some epidemiological and therapeutic aspects of this tapeworm infection. The mother of a 3-month-old girl noted some motile, ricelike objects passed in her childs nappy during the previous 3 days. The infant was well, except for a slight reduction in appetite and in weight gain during the previous week. Neither the parents nor the grandmother living together showed similar symptoms. The family pets, an outdoor dog and two indoor cats, had been regularly checked by a veterinary surgeon. A few weeks before the dog was found to be infected with D. caninum and treated with niclosamide, while the cats were

Different strategies in the treatment of dihydropteridine reductase deficiency

Inherited deficiency of dihydropteridine reductase (DHPR, EC 1.66.99.7) impairs the regeneration of tetrahydrobiopterin (B H 4), the essential cofactor of phenylalanine (Phe) , tyrosine (Tyr), and trypthophan (Trp) hydroxylases, which is oxidized to qBH2 during a coupled reaction with these enzymes ( 1). Hyperphenylalaninemia and scarce production of monoamine neurotransmitters derived from Tyr and Trp-dopamine, norepinephrine, and serotonin-are the main metabolic derangements caused by DHPR deficiency. Untreated patients early develp a severe an progressive neurological picture , which is shared by other inborn errors of BH4 metabolism (1 ). The control of hyperphenylalaninemia and biogenic amine deficiency is necessary to improve their prognosis, together with folinic acid supplementation to avoid folate depiction (2 ) . DHPR deficiency, however, is a heterogeneous disease both at clinical and molecular level. The dietary tolerance to Phe is highe r than in classical phenyketonuria, but shows interindividual differences, as well as age-dependent increase ( 1 ). The control of hype rphenylalaninemia can be achieved either by a Phe-restricted diet or by administration of synthetic cofactor. As in these patients some recycling of BH4 do occur (3), a daily dosage of 8-20 mg/ kg is sufficient to attain nor-

Newborn feeding and screening for phenylketonuria

Sir, Identification of phenylketonuria (PKU) is anticipated by the rapid phenylalanine (Phe) elevation in affected newborns. Traditionally, the measurement of blood Phe by bacterial or chemical assay before the neonate was discharged from the hospital, i.e. on the third or fourth day of life, allowed a reliable screening test. By coupling high sensitivity and specificity, it enabled the detection of all types of PKU, irrespective of the severity of phenylalanine hydroxylase (PAH) deficiency. The growing practice of early neonatal discharge, however, has raised concern about the reliability of identifying affected newborns by early screening, i.e. on the first or second day of life, before or shortly after feeding has been started (1). Essentially, 35 y after the introduction of screening, the mechanisms leading to blood Phe elevation in PKU newborns are still poorly understood. The relationships between the type and amount of feeding received prior to the screening test and the extent and time course of hyperphenylalaninaemia after birth have been thoroughly examined, with questionable results (2). No study, however, was devised with this view by taking into account the impact on blood Phe elevation of the patients’ reduced Phe tolerance, the main factor responsible for hyperphenylalaninaemia in PKU. Eleven patients, originally found positive at the neonatal screening for PKU (Guthrie bacterial inhibition assay, cut-off value of 30 mg/l), were selected on the basis of the accuracy of nursery records with reference to the type and exact amount of feeding prior to the blood specimen collection. Ultimate diagnosis of either classical PKU, with s vere PAH deficiency (6 patients), or mild PKU, with partial PAH deficiency (5 patients), was obtained after exclusion of tetrahydrobiopterin deficiency (3). Patient phenotype was assessed according to the criteria previously employed (4), including pre-treatment blood Phe value, velocity of blood Phe normalization after diet, and dietary Phe tolerance. Because PAH activity reaches adult levels as early as the second trimester of pregnancy (5), it was possible to evaluate the individual Phe tolerance in the neonatal period retrospectively in each patient. This tolerance level was taken as the minimal daily Phe intake required to increase blood Phe a level above the cut-off value of the screening test during the first 2 mo of life. The assessment of this kind of Phe tolerance allowed approximation of the cumulative amount of Phe potentially tolerated by each newborn from birth to the time of blood specimen collection for the initial screening test. The total Phe dietary intake in the same period was calculated for each newborn on the basis of the amount and Phe content of colostrum (6), and/or the administered milk formula, as provided by the manufacturer. Data listed in the table distinctly show that all PKU newborns shared a cumulative Phe tolerance largely exceeding the dietary Phe intake received during the days preceding the Guthrie test. As a consequence, none

Mental retardation, peculiar facial dysmorphism and HbH inclusions in a 4-year-old boy.

A 4-year-old boy was referred because of severe mental retardation.Hewas the product of a 35-week pregnancy to a 35-year-old woman.Hewas delivered by caesarean section because of breech presentation. Amniocentesis for advancedmaternal age revealed a normal 46,XY karyotype. Birth weight was 2050 g, length 46 cm, head circumference 32.5 cm: all the parameters were appropriate for gestational age. His Apgar scores were 6 and 8 at 1 and 5 min respectively. He did require oxygen in the delivery room for approximately 1 min. He went to the regular nursery and did not breast feed well, but reportedly fed well from the bottle. He had transient hypoglycaemia and was discharged at 6 days of age. He was hospitalised at 6 weeks of age because of vomiting and dehydration. During this episode, metabolic screening was done and was normal. He has had severe developmental retardation: he walked alone at 25 months of age and he hasneverdevelopedanyintelligible speech.Heneverhadseizures. Family history revealed that the patient had a 2-year-old sibling in good health. Both sides of the pedigree were negative for birth defects, mental retardation, recurrent miscarriages, and still births. On physical examination, microcephaly and a facial phenotype characterised by epicanthus, a low nasal bridge, small triangular, upturned nose, everted lower lip, giving the mouth a ‘‘carp-like’’ appearance and widely spaced frontal incisors were observed (Fig. 1 and Fig. 2). He had a mild dorsal kyphosis. Genitalia were normal. An EEG and eye examination were normal. Review of his medical records indicated that he had negative fragile X testing and his urine mucopolysaccharide screen was negative. Fluorescent in-situ hydridisation (FISH) for chromosome 22 microdeletion was negative. FISH and methylation testing for Prader-Willi/Angelman syndromes were negative. Sterol analysis for Smith-Lemli-Opitz syndrome was also negative. A CT scan of the brain showed a mild dilatation of the lateral ventricles and subarachnoidal spaces. The spine X-ray film showed no abnormalities. The hand X-ray film revealed tufting of the distal phalanges. We first considered the Coffin-Lowry syndrome but no mutations in the RSK2 gene were found by SSCP analysis [1]. As a possible differential diagnosis of Coffin-Lowry syndrome we then considered another X-linked disorder with similar features. A haemoglobin electrophoresis was performed and it showed a small amount of HbH. A search for HbH inclusions (Heinz bodies) on a blood smear following incubation with cresyl blue was positive. The follow-up at 5 years of age revealed frequent episodes of vomiting and rumination during acute illnesses. The vomiting was not self-induced and subsided after appropriate rehydration. Eur J Pediatr (2002) 161: 627–628 DOI 10.1007/s00431-002-1049-y