Phenylalanine and tyrosine metabolism in DNAJC12 deficiency: A comparison between inherited hyperphenylalaninemias and healthy subjects.

DNAJC12 deficiency is a new cause of inherited hyperphenylalaninemia (HPA), besides phenylalanine hydroxylase (PAH) deficiency and tetrahydrobiopterin (BH4) deficiencies. Differently from other inherited HPAs, no quantitative data on peripheral phenylalanine (Phe) and tyrosine (Tyr) metabolism are currently available in DNAJC12 deficiency. Phe and Tyr metabolism in a patient with DNAJC12 after a simple Phe oral loading test (100 mg/kg) and a combined Phe (100 mg/kg) + BH4 (20 mg/kg) loading test is presented and compared to patients with disorders of BH4 metabolism, PAH deficiency, and healthy controls. Phe and Tyr metabolism in DNAJC12 deficiency is similar to non-PKU HPA. Differently from BH4 deficiency, BH4 administration in DNAJC12 deficiency does not firmly enhance the rate of Phe hydroxylation. A central effect of BH4 treatment in DNAJC12 deficiency cannot be excluded.

Neonatal phenylalanine wash-out in phenylketonuria.

Phenylketonuria (PKU) is the most common inborn error of amino acids metabolism. PKU management aims to keep as soon as possible blood phenylalanine (Phe), a non-acutely neurotoxic metabolite, within safe ranges through a dietary Phe restriction tailored to individual dietary Phe tolerance. Information on initial neonatal management of PKU, when Phe tolerance is still unknown, is scanty. We reviewed the metabolic data from 304 patients with PAH deficiency detected at newborn screening within the last 37 years. In keeping with the general neonatal management of intoxication-type inborn errors of metabolism, initial management consisted in a Phe wash-out through the exclusive administration of normocaloric Phe-free formulas until normalization of blood Phe. Based on genotype and Phe tolerance assessed at follow-up, 55 patients had classic PKU (18%), 50 mild PKU (17%), and 199 non-PKU hyperphenylalaninemia (HPA) (65%). The duration of Phe wash-out amounted to 7 ± 2 days in classic PKU, 4 ± 2 days in mild PKU, and < 24 h in non-PKU HPA (p < 0.001). After the wash-out, dietary Phe re-introduction and its upwardly titration allowed the assessment of individual metabolic phenotype. During the first 6 years of life, Phe tolerance was stable in classic PKU (~ 200 mg/day) but increased in milder forms, allowing unrestricted diet in non-PKU HPA. Neonatal Phe wash-out in PKU ensures the earliest correction of HPA. This metabolic reset also facilitates the prompt definition of individual Phe tolerance, allowing anticipation of dietary personalization and optimization of longitudinal metabolic control.

Tyrosine metabolism in health and disease: slow-release amino acids therapy improves tyrosine homeostasis in phenylketonuria.

OBJECTIVES: Phenylalanine (Phe) hydroxylase (PAH) deficiency leads to hyperphenylalaninemia (HPA) and tyrosine (Tyr) depletion. We investigated Tyr homeostasis in patients with PAH deficiency and the effect of a slow-release amino acids therapy in phenylketonuria (PKU). METHODS: We performed four complementary investigations: (1) Tyr concentrations were monitored in 114 patients (10.6 ± 11.9 years) with PKU on dietary treatment supplemented with traditional amino acid formulations (n=52, 1175 samples) or non-PKU HPA on a free diet (n=62, 430 samples); (2) Tyr metabolism in PKU was quantitatively evaluated in three patients by a simple Tyr oral loading test (100 mg/kg); (3) diurnal and (4) long-term Tyr concentrations were evaluated in 5 and 13 patients with PKU, respectively, who switched from traditional to slow-release amino acids therapy. RESULTS: 1) Tyr concentrations in the PKU population were subnormal and significantly lower than in non-PKU HPA (p<0.01); (2) the response to a Tyr loading test in PKU was normal, with basal Tyr concentrations reached within 12 h; (3) the diurnal metabolic profile in patients on slow-release amino acids therapy revealed higher morning fasting and nocturnal Tyr concentrations with respect to traditional therapy (p<0.01); (4) this picture was confirmed at follow-up, with normalization of morning fasting Tyr concentrations in patients on slow-release amino acids therapy (p<0.01) and unchanged Phe control (p=0.19). CONCLUSIONS: Slow-release amino acids therapy can improve Tyr homeostasis in PKU. If associated to optimized Phe control, such a metabolic goal may allow long-term clinical benefits in patients with PKU.

Neonatal screening for biotinidase deficiency: A 30-year single center experience.

We reviewed the outcome of newborn screening for biotinidase deficiency performed at our department since 1987. Among 1,097,894 newborns screened, 461 were recalled, and 18 were identified as affected by complete or partial biotinidase deficiency (incidence 1:61,000, false positive rate 0.04%). The common missense mutation Q456H was found in 80% of patients with profound biotinidase deficiency. Of them, one patient harbored the novel mutation M399I in compound heterozygosity (M399I/Q456H). The complex allele A171T/D444H in cis was found in two patients with profound biotinidase deficiency (in homozygosity and in compound heterozygosity with the R211H mutation, respectively) and in one patient with partial biotinidase deficiency (in compound heterozygosity with the protective allele D444H in trans). All detected patients were treated and followed up at our Center until present. Biotin therapy (10-20 mg/day) allowed the full prevention of clinical symptoms in all patients with no adverse effects. These excellent outcomes confirm that newborn screening for biotinidase deficiency is a very effective secondary prevention program.

Early Screening for Tetrahydrobiopterin Responsiveness in Phenylketonuria.

Since 2007, synthetic tetrahydrobiopterin (BH4) has been approved as a therapeutic option in BH4-responsive phenylketonuria (PKU) and since 2015 extended to infants younger than 4 years in Europe. The current definition of BH4 responsiveness relies on the observation of a 20% to 30% blood phenylalanine (Phe) decrease after BH4 administration, under nonstandardized conditions. By this definition, however, patients with the same genotype or even the same patients were alternatively reported as responsive or nonresponsive to the cofactor. These inconsistencies are troubling, as frustrating patient expectations and impairing cost-effectiveness of BH4-therapy. Here we tried a quantitative procedure through the comparison of the outcome of a simple Phe and a combined Phe plus BH4 loading in a series of infants with PKU, most of them harboring genotypes already reported as BH4 responsive. Under these ideal conditions, blood Phe clearance did not significantly differ after the 2 types of loading, and a 20% to 30% decrease of blood Phe occurred irrespective of BH4 administration in milder forms of PKU. Such early screening for BH4 responsiveness, based on a quantitative assay, is essential for warranting an evidence-based and cost-effective therapy in those patients with PKU eventually but definitely diagnosed as responsive to the cofactor.

Long-term safety and effectiveness of pramipexole in tetrahydrobiopterin deficiency.

Tetrahydrobiopterin (BH4) deficiencies are inherited neuro-metabolic disorders leading to monoamine neurotransmitters deficiency. An individualized replacement therapy with neurotransmitters precursors is necessary to restore dopaminergic and serotoninergic homeostasis. The correction of dopaminergic tone is complicated, like in Parkinson disease, by l-dopa short half-life and adverse effects. To improve this picture, since 2009 we introduced the non-ergot dopamine agonist pramipexole as an adjunct to l-dopa therapy in the treatment of the most common causes of BH4 deficiency, 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency and dihydropteridine reductase (DHPR) deficiency. In the short-term period, this approach allowed substantial clinical advantages in affected patients, with amelioration and stabilization of the clinical picture on twice daily treatment administration and no adverse effect. Here we describe the long-term clinical follow-up (83 ± 24 months) of seven patients with BH4 deficiency treated with pramipexole. After a period of good clinical compensation (34 ± 1 months), different impulse control disorders (gambling, compulsive buying, and hypersexuality) were observed in three patients treated with high-dose pramipexole (0.030-0.033 mg/kg/day) beyond adolescence. These psychiatric adverse effects promptly disappeared after curtailing pramipexole dose by 50-60%. Low-dose pramipexole therapy has been safe and effective in the long-term period in all treated patients (59 ± 9 months). High-dose pramipexole therapy in BH4 deficiency can be complicated, like in Parkinson disease, by psychiatric adverse effects. Low-dose pramipexole therapy (∼0.010 mg/kg/day) has been safe and clinically effective on long-term follow-up, representing a helpful therapeutic option in patients with BH4 deficiency.

Genealogy of breastfeeding.

Decline and resurgence of breastfeeding (BF) characterized last century. Several factors influencing BF outcome were identified. Despite the huge literature on BF, no data on its matrilineal transmission are available. BF practice was prospectively followed in 2546 Italian mothers. Lactation and BF outcome were related, besides to known factors interfering with BF, to the occurrence of previous maternal and paternal BF. Recalls of grandmaternal and grand-grandmaternal BF behaviours allowed the construction of familiar pedigrees of BF across three generations. Having been breastfed was the strongest factor addressing successful BF establishment (odds ratio (OR) 9.33; 95% confidence interval (CI) 7.40-11.84; p < 0.0001) and BF duration (at 6 months: OR 3.79; 95% CI 3.11-4.64; p < 0.0001). The hazard ratio for breastfed vs non-breastfed mothers was 0.46 (95% CI 0.41-0.50; log-rank p < 0.0001). The rate of BF failures was fivefold higher in non-breastfed mothers, mostly occurring during lactogenesis when the let-down reflex becomes essential. CONCLUSION: At any generation, mothers are likely to have daughters repeating their BF experience. Differently from the intergenerational effects of environmental factors responsible for the BF secular trend, this trait is transgenerationally transmitted and reversible, with temporal and clinical features of lactation failure. Accordingly, we speculate that epigenetic mechanisms might alter offspring's oxytocinergic receptor signalling. WHAT IS KNOWN: Several cultural and socio-demographic factors are known to influence breastfeeding outcome. The generational effects of breastfeeding itself have not been investigated so far. WHAT IS NEW: Maternal breastfeeding is the most important factor addressing daughters' breastfeeding outcome. This behavior is transmitted transgenerationally, with features suggesting epigenetic mechanisms.

Target Prolactin Range in Treatment of Tetrahydrobiopterin Deficiency.

The introduction of dopamine agonists for treating tetrahydrobiopterin deficiency imposes the evaluation of peripheral prolactin as the sole reliable biochemical marker of dopaminergic homeostasis. Here we provide the clinical interpretation of the previously described short prolactin profile, based on the longitudinal monitoring of 8 patients with tetrahydrobiopterin deficiency.

Newborn screening for galactosemia: a 30-year single center experience.

BACKGROUND: Galactosemia due to complete or near-complete galactose-1-phosphate uridyltransferase (GALT) deficiency was the first disorder added to the pioneering newborn screening panel besides phenylketonuria. In the last 50 years, many criticisms have been focused on the opportunity of its inclusion. Consequently, long-term single center experiences with this issue are generally lacking. METHODS: We reviewed the outcome of newborn screening for hypergalactosemia performed at our department since 1982 and the correspondent long-term clinical outcome. RESULTS: Among 1 123 909 newborns screened for hypergalactosemia, 33 showed abnormal results confirmed at second tier test. Thirteen patients were affected with classic galactosemia, 8 partial GALT deficiency, 3 severe galactokinase deficiency, 7 transient galactosemia, one congenital porto-systemic shunt, and one glucose transporter 2 deficiency. Acute neonatal liver failure in the late first week of life (5.8±1.1 days) unavoidably complicated the clinical course of classic galactosemia, unless in three second-born siblings treated on the basis of presumptive diagnosis immediately after newborn screening sample collection on day 3. Despite early treatment and long-term steadily normal peripheral blood galactose, 77% of patients with severe GALT deficiency present mild to severe intellectual disabilities. All patients with partial GALT deficiency showed normal intellectual development on a regular diet, as well as patients with galactokinase deficiency under treatment. CONCLUSIONS: Availability of screening results within the fifth day after birth would allow the prevention of acute decompensation in classic galactosemia. A systematic diagnostic work-up in all positive newborns is essential to unravel the etiology of hypergalactosemia.

Short prolactin profile for monitoring treatment in BH4 deficiency.

Tetrahydrobiopterin (BH4) deficiency causes hyperphenylalaninemia and impaired synthesis of serotonin and dopamine, leading to brain degeneration and early death if left untreated. Replacement therapy with neurotransmitters precursors is the cornerstone of treatment, relying on 5-hydroxytryptophan and L-dopa administration. Effective restoration of dopaminergic activity is thickened, like in Parkinson's disease, by the pulsatile pharmacokinetic profile of L-dopa. Monitoring of L-dopa therapy in BH4 deficiency is generally based upon clinical observation and periodical measurement of homovanillic acid (HVA) concentration in the cerebrospinal fluid (CSF). According to the finding that dopamine is the natural inhibitor of prolactin (PRL) incretion, we introduced the use of peripheral PRL measurement as an index of dopaminergic homeostasis, so avoiding the need of repeated lumbar punctures in patients with BH4 deficiency. As a single PRL evaluation can be misleading, due to the dependency of PRL fluctuations on L-dopa administration schedule, here we show that a short PRL profile is suitable for monitoring these patients. Together with the assessment of patients' clinical symptoms, this standardized tool will ensure an objective non-invasive reference to the management of dopaminergic replacement therapy in BH4 deficiency, even in patients treated with dopamine agonists.

Dopamine agonists in dihydropteridine reductase deficiency.

The traditional treatment of severe disorders of tetrahydrobiopterin (BH4) metabolism is based on the replacement therapy with BH4, 5-hydroxytryptophan, and L-dopa. Major problems are encountered with L-dopa therapy, especially with increasing age when higher doses are necessary, because of its short half-life and adverse effects. Consequently, different L-dopa-sparing strategies have been successively introduced, with partial reduction of L-dopa dosage and amelioration of the clinical outcome. Recently, we demonstrated that the dopamine agonist pramipexole improves the therapeutic effect of L-dopa in 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency, the most common disorder of BH4 metabolism. Here we report its effectiveness in two patients (males, 7 and 22 years) with dihydropteridine reductase (DHPR) deficiency, the second most frequent cause of BH4 deficiency. Both patients experienced residual symptoms of dopamine deficiency, movement and behavioral disability, and complications of L-dopa therapy, associated with fluctuating hyperprolactinemia. They had full clinical and biochemical assessment, by an adapted Unified Parkinson's Disease Rating Scale (UPDRS) and measurement of diurnal plasma prolactin (PRL) profile before and after a trial with pramipexole. Besides allowing the reduction of L-dopa daily dosage (-58%) and administrations (from three to two) in one patient and to stop L-dopa therapy in the other, the introduction of pramipexole markedly improved and stabilized clinical and biochemical picture in both patients, as revealed by reduction of UPDRS scores and normalization of diurnal plasma prolactin profiles. Dopamine agonists can improve or even replace L-dopa therapy in disorders of synthesis and regeneration of BH4.

Analgesic techniques in minor painful procedures in neonatal units: a survey in northern Italy.

INTRODUCTION: The aim of this survey was to evaluate the current practice regarding pain assessment and pain management strategies adopted in commonly performed minor painful procedures in Northern Italian Neonatal Intensive Care Units (NICUs). METHODS: A multicenter survey was conducted between 2008 and 2009 in 35 NICUs. The first part of the survey form covered pain assessment tools, the timing of analgesics, and the availability of written guidelines. A second section evaluated the analgesic strategies adopted in commonly performed painful procedures. The listed analgesic procedures were as follows: oral sweet solutions alone, non-nutritive sucking (NNS) alone, a combination of sweet solutions and NNS, breast-feeding where available, and topical anesthetics. RESULTS: Completed questionnaires were returned from 30 neonatal units (85.7% response rate). Ten of the 30 NICUs reported using pain assessment tools for minor invasive procedures. Neonatal Infant Pain Scale was the most frequently used pain scale (60%). Twenty neonatal units had written guidelines directing pain management practices. The most frequently used procedures were pacifiers alone (69%), followed by sweet-tasting solutions (58%). A 5% glucose solution was the most frequently utilized sweet-tasting solution (76.7%). A minority of NICUs (16.7%) administered 12% sucrose solutions for analgesia and the application of topical anesthetics was found in 27% of NICUs while breast-feeding was performed in 7% of NICUs. DISCUSSION: This study found a low adherence to national and international guidelines for analgesia in minor procedures: the underuse of neonatal pain scales (33%), sucrose solution administration before heel lance (23.3%), topical anesthetics before venipuncture, or other analgesic techniques. The presence of written pain control guidelines in these regions of Northern Italy increased in recent years (from 25% to 66%).

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 patient's 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.

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.

Breastfeeding or oral sucrose solution in term neonates receiving heel lance: a randomized, controlled trial.

OBJECTIVE: The purpose of this work was to compare the efficacy of breastfeeding versus orally administered sucrose solution in reducing pain response during blood sampling through heel lance. METHODS; We conducted an open-label, randomized, controlled trial at a neonatal unit of a public hospital in northern Italy on 101 term neonates undergoing heel lance with an automated piercing device for routine neonatal screening for congenital disorders. Newborn infants were randomly assigned to breastfeeding during blood sampling or to the oral administration of 1 mL of 25% sucrose solution. We validated the multidimensional acute pain rating scale of the Premature Infant Pain Profile, heart rate increase, oxygen saturation decrease, crying behavior (duration of first cry, cry percentage in 2 minutes, and during blood sampling), duration of sampling, and the number of performed heel lances. RESULTS: Median Premature Infant Pain Profile scores were lower in the breastfeeding group (3.0) than in the sucrose-solution group (8.5), and the median group difference was -5.0. The median heart rate increase, oxygen saturation decrease, and duration of first cry for the breastfeeding group were, respectively, 13.0, -1, and 3 and for sucrose group were 22, -3, and 21. Medians were significantly different between the groups. There were no significant differences in the sampling duration and numbers of heel lances. CONCLUSIONS: This study suggests that breastfeeding provides superior analgesia for heel lance compared with oral sucrose in term neonates.

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 peristaltic 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.

Outcome and long-term follow-up of 36 patients with tetrahydrobiopterin deficiency.

We describe the treatment, the clinical, and biochemical findings and the outcome of 26 patients with 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency and 10 patients with dihydropteridine reductase (DHPR) deficiency. These are the two most common forms of the autosomal-recessively inherited tetrahydrobiopterin (BH4) deficiency. Time of diagnosis, dosage of BH4 and neurotransmitter precursors, folinic acid substitution, and levels of 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) in cerebrospinal fluid (CSF) are essential parameters in the follow-up of patients. Unfortunately, treatment protocols vary greatly among patients and clinical centers, and CSF investigations and outcome assessments are not always available. Seventeen patients with PTPS deficiency and four patients with DHPR deficiency were diagnosed within 2 months after birth. In 14 patients with PTPS deficiency (54%; 9 early and 5 late diagnosed) and 2 patients with DHPR deficiency (20%; all early diagnosed) no developmental delay is observed, while in 10 patients with PTPS deficiency (38%; 6 early and 4 late diagnosed) and 8 patients with DHPR deficiency (80%; 2 early and 6 late diagnosed) development was delayed. Two PTPS-deficient patients died in the newborn period. DHPR deficiency seems to be more severe than PTPS deficiency and it is clearly the onset of treatment that determines the outcome. Our data suggest that diagnosis within the first month of life is essential for a good outcome and that low CSF5 HIAA and HVA values in CSF could be an indicator for the ongoing developmental impairment