Concetta Meli

A European multicenter study of phenylalanine hydroxylase deficiency: Classification of 105 mutations and a general system for genotype-based prediction of metabolic phenotype

Phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are allelic disorders caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). Previous studies have suggested that the highly variable metabolic phenotypes of PAH deficiency correlate with PAH genotypes. We identified both causative mutations in 686 patients from seven European centers. On the basis of the phenotypic characteristics of 297 functionally hemizygous patients, 105 of the mutations were assigned to one of four arbitrary phenotype categories. We proposed and tested a simple model for correlation between genotype and phenotypic outcome. The observed phenotype matched the predicted phenotype in 79% of the cases, and in only 5 of 184 patients was the observed phenotype more than one category away from that expected. Among the seven contributing centers, the proportion of patients for whom the observed phenotype did not match the predicted phenotype was 4%-23% (P<.0001), suggesting that differences in methods used for mutation detection or phenotype classification may account for a considerable proportion of genotype-phenotype inconsistencies. Our data indicate that the PAH-mutation genotype is the main determinant of metabolic phenotype in most patients with PAH deficiency. In the present study, the classification of 105 PAH mutations may allow the prediction of the biochemical phenotype in >10,000 genotypes, which may be useful for the management of hyperphenylalaninemia in newborns.

Autism and Phenylketonuria

Phenylketonuria (PKU) has been also reported in children with infantile autism (IA); however, the frequency of this association is variably reported. Patients with various forms of hyperphenylalaninemia (HPA) were evaluated applying two methods: the Autism Diagnostic Interview-Revised (ADI-R) and the Childhood Autism Rating Scale (CARS). A total of 243 patients were investigated, 97 with classical PKU, 62 identified by neonatal screening, and 35 late diagnosed. None out of 62 patients with classic PKU diagnosed early met criteria for autism. In the group of 35 patients diagnosed late, two boys (5.71%) ages 16 and 13 years fulfilled the diagnostic criteria for autism. The present study confirms that classical PKU is one of the causes of autism, but the prevalence seems to be very low.

Long-term Follow-up and Outcome of Phenylketonuria Patients on Sapropterin: A Retrospective Study

OBJECTIVE: Sapropterin dihydrochloride, the synthetic form of 6R-tetrahydrobiopterin (BH4), is an approved drug for the treatment of patients with BH4-responsive phenylketonuria (PKU). The purpose of this study was to assess genotypes and data on the long-term effects of BH4/sapropterin on metabolic control and patient-related outcomes in 6 large European countries. METHODS: A questionnaire was developed to assess phenotype, genotype, blood phenylalanine (Phe) levels, Phe tolerance, quality of life, mood changes, and adherence to diet in PKU patients from 16 medical centers. RESULTS: One hundred forty-seven patients, of whom 41.9% had mild hyperphenylalaninemia, 50.7% mild PKU, and 7.4% classic PKU, were followed up over ≤12 years. A total of 85 different genotypes were reported. With the exception of two splice variants, all of the most common mutations were reported to be associated with substantial residual Phe hydroxylase activity. Median Phe tolerance increased 3.9 times with BH4/sapropterin therapy, compared with dietary treatment, and median Phe blood concentrations were within the therapeutic range in all patients. Compared with diet alone, improvement in quality of life was reported in 49.6% of patients, improvement in adherence to diet was reported in 47% of patients, and improvement in adherence to treatment was reported in 63.3% of patients. No severe adverse events were reported. CONCLUSIONS: Our data document a long-term beneficial effect of orally administered BH4/sapropterin in responsive PKU patients by improving the metabolic control, increasing daily tolerance for dietary Phe intake, and for some, by improving dietary adherence and quality of life. Patient genotypes help in predicting BH4 responsiveness.

Evidence for genetic heterogeneity in D-2-hydroxyglutaric aciduria†

We performed molecular, enzyme, and metabolic studies in 50 patients with D‐2‐hydroxyglutaric aciduria (D‐2‐HGA) who accumulated D‐2‐hydroxyglutarate (D‐2‐HG) in physiological fluids. Presumed pathogenic mutations were detected in 24 of 50 patients in the D‐2‐hydroxyglutarate dehydrogenase (D2HGDH) gene, which encodes D‐2‐hydroxyglutarate dehydrogenase (D‐2‐HGDH). Enzyme assay of D‐2‐HGDH confirmed that all patients with mutations had impaired enzyme activity, whereas patients with D‐2‐HGA whose enzyme activity was normal did not have mutations. Significantly lower D‐2‐HG concentrations in body fluids were observed in mutation‐positive D‐2‐HGA patients than in mutation‐negative patients. These results imply that multiple genetic loci may be associated with hyperexcretion of D‐2‐HG. Accordingly, we suggest a new classification: D‐2‐HGA Type I associates with D‐2‐HGDH deficiency, whereas idiopathic D‐2‐HGA manifests with normal D‐2‐HGDH activity and higher D‐2‐HG levels in body fluids compared with Type I patients. It remains possible that several classifications for idiopathic D‐2‐HGA patients with diverse genetic loci will be revealed in future studies. Hum Mutat 31:1–5, 2010.

Pharmacokinetics of Sapropterin in Patients with Phenylketonuria

AbstractBackground and objective: Untreated phenylketonuria is characterized by neurocognitive and neuromotor impairment, which result from elevated blood phenylalanine concentrations. To date, the recommended management of phenylketonuria has been the use of a protein-restricted diet and the inclusion of phenylalanine-free protein supplements; however, this approach is often associated with poor compliance and a suboptimal clinical outcome. Sapropterin dihydrochloride, herein referred to as sapropterin, a synthetic formulation of 6R-tetrahydrobiopterin (6R-BH4), has been shown to be effective in reducing blood phenylalanine concentrations in patients with phenylketonuria. The objective of the current study was to characterize the pharmacokinetics and pharmacokinetic variability of sapropterin and to identify the characteristics that influence this variability. Patients and methods: This was a 12-week, fixed-dose phase of an open-label extension study. The study was conducted at 26 centres in North America and Europe.Patients with phenylketonuria were eligible to participate if they were ≥8 years of age and had received ≥80% of the scheduled doses in a previous 6-week, randomized, placebo-controlled study or had been withdrawn from that study after exceeding a plasma phenylalanine concentration of ≥1500 μmol/L to ≥1800 μmol/L, depending on the subject’s age and baseline plasma phenylalanine concentration. A total of 78 patients participated. Patients received oral once-daily doses of sapropterin (Kuvan®) 5, 10 or 20 mg/kg/day.Blood samples for the pharmacokinetic analysis were obtained during weeks 6, 10 and 12. A D-optimal sparse sampling strategy was used, and data were analysed by population-based, nonlinear, mixed-effects modelling methods. Main outcome measure: In a prospectively planned analysis, the apparent clearance, apparent volume of distribution, absorption rate constant and associated interindividual variabilities of each parameter were estimated by modelling observed BH4 plasma concentration-time data. Results: The best structural model to describe the pharmacokinetics of sapropterin was a two-compartment model with first-order input, first-order elimination and a baseline endogenous BH4 concentration term. Total bodyweight was the only significant covariate identified, the inclusion of which on both the apparent clearance (mean = 2100 L/h/70 kg) and central volume of distribution (mean = 8350 L/70 kg) substantially improved the model’s ability to describe the data. The mean (SD) terminal half-life of sapropterin was 6.69 (2.29) hours and there was little evidence of accumulation, even at the highest dose. Conclusion: These findings, taken together with the observed therapeutic effect, support bodyweight-based, once-daily dosing of sapropterin 5–20 mg/kg/day.

Levels of L-asparagine in CSF after intramuscular administration of asparaginase from Erwinia in children with acute lymphoblastic leukemia.

PURPOSE As part of a study on the pharmacokinetics associated with the administration of asparaginase (ASNase) from Erwinia to the CNS, we determined the levels of asparagine in the CSF of children with acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS Twenty children received eight standard doses of intramuscular ASNase (10,000 IU/m2) every 3 days as part of induction therapy. In the postremission phase of therapy, the children were randomized to receive either 20 courses of high-dose intramuscular ASNase (25,000 IU/m2) weekly (n = 8) or four courses of standard-dose intramuscular ASNase (10,000 IU/m2) every 3 days (n = 12). RESULTS All patients had detectable levels of L-asparagine in the CSF at the time of diagnosis. The levels of L-asparagine in CSF were undetectable in 15 of 20 (75%) and in seven of 19 (36.8%) children 3 and 5 days, respectively, after administration of standard-dose ASNase. After administration of high-dose ASNase, the levels of L-asparagine in the CSF were undetectable in five (62.5%) and two (25%) of eight children after 3 and 5 days, respectively. CONCLUSION In this study 60% to 70% and 25% to 35% of children had complete depletion of L-asparagine from the CSF after 3 and 5 days, respectively, after administration of both schedules of ASNase from Erwinia. In the remaining patients, administration of ASNase may have resulted in a suboptimal antileukemic effect at the CNS level.

PAH deficiency in Italy: correlation of genotype with phenotype in the Sicilian population

SummaryThe results of the neonatal screening for phenylalanine hydroxylase (PAH) deficiency in Sicily show that its incidence is higher than previously reported for mainland Italians and that non-PKU HPA is in excess of classical and mild PKU. The latter finding suggests that a high number of non-PKU HPA mutations would occur in the Sicilian population compared to populations with an inverted PKU/non-PKU HPA ratio. Previous studies have identified 40 mutations accounting for the majority (98%) of mutant alleles underlying PAH deficiency in Sicily. In order to study the molecular basis of the distribution of PAH deficiency phenotypes in the Sicilian population, we have correlated 31 of those mutations with clinical and metabolic phenotypes in 12 mentally retarded patients, 14 treated patients with classic or mild PKU, and 13 subjects presenting the non-PKU HPA phenotype. The present study proposes a tentative classification for a large number (26) of PAH gene mutations which may represent an additional tool for establishing a differential diagnosis for PAH deficiency in the Sicilian population.

Exon deletions of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemics

A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase (PAH) alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods. These methods include denaturing gradient gel electrophoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and direct sequencing. In recent years, it has been shown that a significant proportion of undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes using gene-scanning methods due to a masking effect of the non-deleted allele. To date, no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or mild hyperphenylalaninemia. We used multiplex ligation- dependent probe amplification (MLPA), comparative multiplex dosage analysis (CMDA), and real-time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemia patients. Four deletions removing different phenylalanine hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving exons 4-5-6-7-8 (systematic name c.353-?_912 + ?del) and exon 6 (systematic name c.510-?_706 + ?del) have not been reported previously. In this study, we show that exon deletion of the PAH gene accounts for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics.

Clinical manifestations and management of four children with Pearson syndrome.

Pearson marrow‐pancreas syndrome is a fatal disorder mostly diagnosed during infancy and caused by mutations of mitochondrial DNA. We hereby report on four children affected by Pearson syndrome with hematological disorders at onset. The disease was fatal to three of them and the fourth one, who received hematopoietic stem cell transplantation, died of secondary malignancy. In this latter patient transplantation corrected hematological and non‐hematological issues like metabolic acidosis, and we therefore argue that it could be considered as a useful option in an early stage of the disease.

Nutritional and racial determinants of the increase in plasma homocysteine levels after methionine loading

Abstract Background: Low circulating plasma levels of total homocysteine (tHcy) are associated with a lower prevalence of coronary heart disease among black people than among white people living in Burkina Faso. Objective: The purpose of this study was to provide a rationale for a possible mechanism for the decrease in plasma tHcy levels among black people compared with white people living in Burkina Faso. Methods: Healthy, black, adult, lifelong inhabitants of Burkina Faso and healthy, white adults born in Italy but living in Burkina Faso ≥5 years were eligible for enrollment. Controlled diets were assigned to all subjects for 2 weeks before the study. After an overnight (12-hour) fast, a methionine-loading test was performed in all subjects. Plasma levels of tHcy, cysteine, glutathione, and cysteinylglycine were measured simultaneously using high-performance liquid chromatography after fasting (baseline) and at either 4 and 8 hours (n = 30) or 2, 4, 6, and 8 hours (n = 4) after methionine loading. During the 12 hours after loading, the clinical conditions and adverse events of subjects were monitored. Results were analyzed using the Student t test and Mann-Whitney U test. Results: Seventeen black adults (9 males, 8 females; median age, 21 years) and 17 white adults (8 males, 9 females; median age, 35 years) were enrolled. Mean plasma levels of tHcy, cysteine, and glutathione increased from mean baseline levels more slowly in the black group than in the white group and peaked 8 hours after methionine loading (16.8 ± 3.0 μmol/L, 130.4 ± 25.7 μmol/L, and 68.3 ± 21.2 μmol/L, respectively). In the white group, these levels peaked 4 hours after loading (16.1 ± 4.0 μmol/L, 215.8 ± 18.6 μmol/L, and 38.6 ± 12.4 μmol/L, respectively). Only the mean plasma cysteinylglycine level decreased significantly (from 35.7 ± 11.4 μmol/L to 19.0 ± 6.1 μmol/L; P Conclusions: The findings of this study suggest that, in addition to lower plasma tHcy levels, the metabolism of plasma tHcy is different in black people than in white people after methionine loading. This difference may be due to different alimentary habits associated with a reduced dietary availability of methionine. Moreover, the higher plasma levels of glutathione before and after methionine loading appear to occur exclusively in black people compared with whites and correspond with the variation of cysteinylglycine, suggesting that, in addition to nutritional factors, a racial component may contribute to the difference in plasma levels of tHcy. This difference also might explain, in part, the lower prevalence of coronary heart disease in black people living in Burkina Faso compared with that in other populations.