The diagnostic challenge of mild citrulline elevation at newborn screening.

Citrulline is a target analyte measured at expanded newborn screening (NBS) and its elevation represents a biomarker for distal urea cycle disorders and citrin deficiency. Altered ratios of citrulline with other urea cycle-related amino acids are helpful for the differential diagnosis. However, the use of cut-off values in screening programmes has raised the issue about the interpretation of mild elevation of citrulline levels detected at NBS, below the usual range observed in the "classical/severe" forms of distal urea cycle disorders and in citrin deficiency. Herein, we report ten subjects with positive NBS for a mild elevation of citrulline (<100 µmol/L), in whom molecular investigations revealed carriers status for argininosuccinate synthase deficiency, a milder form of argininosuccinate lyase deficiency and two other diseases, lysinuric protein intolerance and dihydrolipoamide dehydrogenase deficiency, not primarily affecting the urea cycle. To guide the diagnostic process, we have designed an algorithm for mild citrulline elevation (<100 µmol/L) at NBS, which expands the list of disorders to be included in the differential diagnosis.

Chronic liver involvement in urea cycle disorders.

The increased survival of urea cycle disorders (UCDs) patients has led the attention to clinical manifestations that characterize the long-term disease course. Acute and chronic liver disease have been anecdotally reported since the very first description of UCDs. However, a detailed analysis of long-term liver involvement in large patient cohorts is still needed. Chronic liver damage in UCDs has probably a multifactorial origin, but the specific underlying mechanisms of liver disease have not yet been well elucidated. In this study, we report on chronic liver involvement and on associated metabolic abnormalities in a large cohort of 102 UCD patients, followed by two reference centers in Italy. Chronic liver involvement was observed in over 60% of UCDs patients, and comparison between individual diseases showed a significant higher frequency in argininosuccinate lyase deficiency (ASLD) and in hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome with elevation of transaminases and of gamma-GT in ASLD, and of alpha-fetoprotein in HHH syndrome. Also, consistent with a chronic hepatic dysfunction, ultrasound examination revealed more pronounced abnormalities in ASLD and in HHH syndrome, when compared to other UCDs. Our study highlights in a large UCDs patients' cohort that chronic liver disease is a common finding in UCDs, often with a distinct phenotype between different diseases. Furthers studies are needed to elucidate the specific involvement of different metabolic pathways in the pathogenesis of liver dysfunction in UCDs.

A Case of Suspected Hyperphenylalaninemia at Newborn Screening by Tandem Mass Spectrometry during Total Parenteral Nutrition.

Phenylketonuria (PKU) is a rare autosomal recessive condition affecting about 1 in 10,000 people in the Europe, with a higher rate in some countries, like Ireland and Italy. In Italy, newborn screening (NBS) by MS/MS allows the diagnostic suspicion of PKU and its variants (Hyperphenylalaninemia (HPA), Tetrahydrobiopterin (BH4) synthesis deficiency, and Tetrahydrobiopterin (BH4) recycling deficiency) through the quantification of Phenylalanine (Phe) and the Phenylalanine/Tyrosine (Phe/Tyr) ratio in dried blood Spot (DBS) samples. Here, we report a case of an HPA whose suspicion was possible with expanded NBS, even if the normal-weight newborn was in total parenteral nutrition (TPN). It is known that TPN may present metabolic alterations, mainly for amino acids at NBS in MS/MS, frequently causing false positives. Actually, TPN is considered a special protocol in NBS, requiring several sample collections. In particular, a DBS sample is required before TPN, at basal time point (48 h after birth) and 72 h after the end of the procedure. In the case we report, even if the first DBS sample (before TPN) resulted negative, the repeated NBS tests revealed increased levels of Phe and dramatically high Phe/Tyr ratio. Thus, the newborn was recalled, and the NBS test was repeated several times before that HPA suspicion was confirmed by other specific biochemical tests. This case highlights the importance of Phe/Tyr ratio, only detectable by MS/MS analysis, in supporting the diagnostic suspicion during amino acids administration in the neonatal period.

Identification of nine new IDS alleles in mucopolysaccharidosis II. Quantitative evaluation by real-time RT-PCR of mRNAs sensitive to nonsense-mediated and nonstop decay mechanisms.

The present study aimed to characterize mutant alleles in Mucopolysaccharidosis II and evaluate possible reduction of mRNA amount consequent to nonsense-mediated or nonstop mRNA decay pathways. A combination of different approaches, including real-time RT-PCR, were used to molecularly characterize seventeen patients. Fifteen alleles were identified and nine of them were new. The novel alleles consisted of three missense mutations (p.S71R, p.P197R, p.C432R), two nonsense (p.Q66X, p.L359X), two frameshifts (p.V136fs75X, p.C432fs8X), one allele carrying two in-cis mutations [p.D252N;p.S369X], and a large deletion (p.G394_X551). Analysing these results it emerged that most of the alterations resulted in mutants leading to mRNAs with premature termination codons, and therefore, potentially sensitive to mRNA surveillance pathway. By using real-time RT-PCR, the mRNAs resulting (i) from substitutions that changed one amino acid to a stop codon (L359X, and S369X), or caused the shifted reading frame with premature introduction of a stop codon (C432fs8X), (ii) from large deletion (p.G394_X551) that included the termination codon, seemed to be subject to degradation by nonsense-mediated (i) or nonstop decay (ii) mechanisms, as mRNA was strongly underexpressed. On the contrary, two mutations (Q66X and V136fs75X) produced transcripts evading mRNA surveillance pathway despite both of them fulfilled the known criteria. These results confirm the wide variability of the mRNA expression levels previously reported and represent a further exception to the rules governing susceptibility to nonsense-mediated decay. A close examination of the molecular basis of the disease is becoming increasingly important for optimising the choices of available or forthcoming therapies such as, enzyme replacement therapy or enzyme enhancement therapy.

Fruit-induced FPIES masquerading as hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) symptoms develop at first introduction of fruit during weaning. We report on an infant with suspected HFI who presented with repeated episodes of vomiting and hypotension after ingestion of fruit-containing meals. The first episode occurred at age 4 months. Despite negative genetic testing for HFI, strict avoidance of fruit ingestion resulted in lack of recurrence of symptoms. Oral-fructose-tolerance testing conducted with an apple mousse did not determine hypoglycemia or fructosuria but caused severe hypotension. Allergy evaluations were negative, and the history was diagnostic for fruit-induced food protein-induced enterocolitis syndrome. Because this non-immunoglobulin E-mediated gastrointestinal food hypersensitivity manifests as profuse, repetitive vomiting, often with diarrhea, leading to acute dehydration and lethargy, it may be misinterpreted as HFI. We advise pediatricians to consider food protein-induced enterocolitis syndrome in the differential diagnosis when there is a suspicion of HFI.

Cognitive findings and behavior in children and adolescents with phenylketonuria.

OBJECTIVE: The primary aim of this study was to assess cognitive development, in particular that of executive functions (EFs), and behavioral findings for patients with early treated phenylketonuria (PKU). Furthermore, we evaluated the relationships of our findings with plasma levels of Phe and adherence to dietary prescriptions. METHODS: A cross-sectional design was adopted. Patients who had early treated PKU, who were older than 4 years, and who were regularly seen by a physician were enrolled in the study. Cognitive development was assessed with Wechsler Scales appropriate to the chronological age of subjects in the study. Executive functions were assessed using the Tower of London test, and behavioral findings were quantified with the Child Behavior Checklist. RESULTS: Thirty-five patients were enrolled (mean age 11.5 years, SD ± 6.2). The mean Full Scale intelligence quotient was in the normal range (93.4 ± 17.4), without significant difference between the verbal intelligence quotient (mean, 94.3 ± 16.1) and performance intelligence quotient (mean, 93.9 ± 18.0). The majority of patients showed a deficit within the EF domain of cognitive abilities (mean lower than 1.8 SD of the normal mean), 8 of them showing a score <2 SD lower than the normal mean. Internalizing problems were also observed in 12 patients (38.7%) and were higher in adherent patients and in patients with a lower intelligence quotient. CONCLUSION: After early diagnosis and treatment of PKU, residual problems can be found in EFs of patients not achieving satisfactory Phe levels, while scores of internalizing behaviors were higher in compliant patients.

Living with phenylketonuria from the point of view of children, adolescents, and young adults: a qualitative study.

OBJECTIVE: To explore the experiences of patients affected by phenylketonuria, a genetic metabolic disease currently screened during the neonatal period, in whom treatment was begun shortly after birth. Dietary treatment of the metabolic abnormality can prevent effects on the central nervous system through infancy, adolescence, and adulthood, but the associated challenges for personal and social life, such as everyday problems of adherence to a strict regimen, have been inadequately studied. METHOD: A qualitative study was performed using semistructured interviews involving 20 patients aged between 8 and 23 years and referred to a pediatric hospital for phenylketonuria. A thematic framework was formulated in advance which identified the main issues to be investigated, and a list of questions was devised. The interviews were audiotaped and were all transcribed and analyzed by 3 investigators with the support of NVivo software. RESULTS: Patients showed a fair knowledge of their condition but did not feel that they were truly ill. However, they adhered to their treatment regimen although they perceived no direct, immediate, adverse effects of their disease. A long-standing habit of following a demanding diet from infancy emerged from the interviews. A major problem for these patients is the difference perceived when comparing themselves with their peers, and the fear of stigmatization can prevent patients from participating in social occasions during which food is shared. One coping strategy patients may use is to disclose their condition to peers to overcome isolation. CONCLUSIONS: The impact of long-standing, demanding health behaviors on the social life of patients affected by a chronic metabolic disease must be recognized, taking into particular consideration both social functioning and adherence to dietary treatment.