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This paper presents findings from studies of EF in individuals with early-treated PKU within the context of recent advances in neuropsychological theory and research. It focuses on means of assessment, contexts of assessment, and the best way to define and investigate EF. Several conclusions can be drawn based on the findings presented here. The first conclusion is that there is clear evidence for phenylalanine-related EF-deficits in early-treated PKU, particularly with respect to prepotent response inhibition and the manipulation or monitoring component of working memory. An important note, however, is that measurement of EF in PKU has become too fragmented, as different researchers and clinicians use different definitions of EF, and subsequently, different instruments to measure EF. This appears to be one of the most important causes of mixed results. A second conclusion is that there appears to be a need to incorporate at least one specific, relatively new taxonomy of EF in PKU-research, i.e. the taxonomy that distinguishes hot and cool EFs, where hot EF is associated with regulation of affect/emotions and motivation, or regulatory functions when the context contains such elements, while cool EF concerns decontextualized regulatory abilities. PKU in adults is increasingly associated with different mental health problems, despite supposedly good treatment standards and adherence throughout childhood and adolescence. Since hot EF is strongly associated with such mental health problems, it is recommended that the hot–cool taxonomy will feature more prominently in future PKU-studies.

Mitochondrial DNA depletion syndrome (MDS) is a clinically heterogeneous group of mitochondrial disorders characterised by a quantitative reduction of the mitochondrial DNA copy number. Three main clinical forms of MDS: myopathic, encephalomyopathic and hepatocerebral have been defined, although patients may present with other MDS associated clinical symptoms and signs that cover a wide spectrum of onset age and disease. We studied 52 paediatric individuals suspected to have MDS. These patients have been divided into three different groups, and the appropriate MDS genes have been screened according to their clinical and biochemical phenotypes. Mutational study of DGUOK , MPV17 , SUCLA2 , SUCLG1 and POLG allowed us to identify 3 novel mutations (c.1048G>A and c.1049G>T in SUCLA2 and c.531 + 4A>T in SUCLG1 ) and 7 already known mutations in 10 patients (8 families). Seventeen patients presented with mtDNA depletion in liver or muscle, but the cause of mtDNA depletion still remains unknown in 8 of them. When possible, we quantified mtDNA/nDNA and CS activity in the same tissue sample, providing an additional tool for the study of MDS. The ratio (mtDNA/nDNA)/CS has shed some light in the discrepant results between the mtDNA copy number and the enzymatic respiratory chain activities of some cases.

To investigate the relationship between neurological complications, neuroradiological findings, and behavioral problems, age at diagnosis and dietary control along the follow-up of the PKU patients in our metabolic unit.Retrospective study of the PKU patients diagnosed and controlled in our unit from 1985 to 2010.Registry of patients in a database with 50 items filled in by review of the clinical histories. Statistical study of the data (SPSS, 19.0 version).121 patients were included (median age: 16.0, range 1 month-46 years). 76% of them were diagnosed through neonatal screening. 12.4% had mild-PKU, 19% moderate-PKU and 68.6% classic-PKU. 88.4% of patients were treated with a protein-restricted diet, and 11.6% with BH4. 97.7% of the early diagnosed patients had normal IQ, while 46.3% of late diagnosed patients had mental retardation, 28.5% were borderline and 25% had normal IQ. In early diagnosed patients, there was a significantly negative correlation between IQ [mean (SD) 100 (11.1)] and the index of dietary control during the first six years of life [median (range) 310 (105-992)] and that of the immediately past year [348 (106-1127)] (p0.0001). The proportion of patients with late diagnosis and neurological and behavioral problems was significantly higher than that of the early diagnosed ones (p0.001). The proportion of early diagnosed patients with neurological and behavioral problems who had good, intermediate or poor dietary control during the first 6 years of life and the immediately past year was significantly different (p0.001).The results show the impact of early diagnosis and good dietary treatment on the IQ and on the percentage of neurological complications and behavioral problems in PKU patients.

Biotinidase deficiency (BD) is an autosomal recessive disorder of biotin metabolism that causes incomplete recycling of free biotin. The resulting depletion of intracellular biotin leads to impaired activities of biotin-dependent carboxylases. The ensuing clinical phenotype includes progressive neurologic deterioration with epileptic seizures, muscular hypotonia as well as skin eczema. BD may be readily diagnosed by analysing enzyme activity in dried blood spots during newborn screening but typically requires molecular confirmation. More than 100 different mutations in the biotinidase gene have been reported to date. To simplify molecular testing we have developed a rapid and accurate denaturing high pressure liquid chromatography (dHPLC) method of the promoter, 3'UTR, all exons including exon/intron boundaries as a first line screen followed by direct sequencing of the respective PCR products. To validate this method we used DNA from 23 different, newly diagnosed patients with biochemically proven BD from Austria, India, Morocco and Spain. A total of 11 mutations, missense 7, frameshift 3 and 1 nonsense, were screened. Six mutations were novel to this study. All mutations revealed distinct dHPLC pattern thus enabling their accurate detection. This study revealed that dHPLC method is robust, automated, economical and above all highly sensitive for the molecular analysis of biotinidase gene and should be used as a pre-analytical tool followed by sequencing of aberrant heteroduplex forming amplicons.

Our aim was to establish reference values for cerebrospinal fluid (CSF) pyridoxal 5′-phosphate (PLP) in a paediatric population for the diagnosis of pyridox(am)ine 5′-phosphate oxidase (PNPO) deficiency. For reference values, CSF samples from 113 paediatric controls (age range: 1 day–18 years) from Barcelona and London were analysed. Cerebrospinal fluid PLP and biogenic amine concentrations were analysed by HPLC with fluorescence and electrochemical detection. Pyridoxal 5′-phosphate concentrations in 4 patients with PNPO deficiency were determined. A negative correlation between CSF PLP values and age of controls was observed in both populations ( r =−0.503; p r =−0.542; p =0.002). Reference values were stratified into 4 (Barcelona) and 3 age groups (London). For the newborn period, CSF PLP reference intervals were 32–78 and 44–89nmol/L for the Barcelona and London centers, respectively). No correlation was observed in the different age groups between PLP values and biogenic amines metabolites. PLP values in neonates with PNPO deficiency were clearly decreased (PLP=3.6, 12.0, 14.0 and 18.0nmol/L) compared with our reference ranges. In conclusion, reference values for CSF PLP should be stratified according to age. No association was observed between PLP values and biogenic amines metabolites. In our 4 cases with PNPO deficiency, CSF PLP values were clearly below the reference values.

We report a recessive mutation in the tyrosine hydroxylase gene (TH) promoter (c.1-71C>T), present at homozygosity in a patient with dopa-responsive encephalopathy. The change lies in a cAMP response element (CRE) and alters a binding site for the CREM transcription factor. Previous studies support that the CRE in the TH gene is essential for its transcription, suggesting that mutations within this consensus motif may cause an impairment of catecholamine biosynthesis and lead to a pathogenic phenotype.

The clinical, nutritional, and neuropsychological data of 11 mild/moderate PKU patients after one year of treatment with BH4 are evaluated. BH4 monotherapy was introduced at 5 mg/kg/day in 14 PKU patients. In 11/14 patients, Phe tolerance increased significantly from 356 ± 172 to 1546 ± 192 mg/day (p = 0.004), and special PKU formula was gradually reduced until complete removal. In them, mean plasma Phe concentrations remained below 360 μmol/L at 5 mg BH4/kg/day (7 mg/kg/day in one patient). BH4 therapy was stopped in three patients (V388M/P362T and R243Q/IVS10-11G > A genotypes) because it was not possible to improve Phe tolerance and to remove formula intake. Serum micronutrients were not significantly different at the start of treatment and at one year follow-up, except for selenium, which increased significantly after one year of therapy (p = 0.017). Anthropometric, and nutritional measurements were within the age- and sex-specific percentiles for a healthy population after one year therapy. Neuropsychological follow-up indicated that intelligence scores persisted within normal limits. In terms of patients’ genotype, we confirmed that the P275S mutation combined with R408W was associated with long-term BH4 responsiveness, while the combination of P362T/V388M, and R243Q/IVS10-11G > A resulted in poor metabolic control in long-term BH4 therapy. In summary, our data confirm that BH4 is a safe, and effective therapy in a selected group of mild, and moderate PKU patients who respond to the BH4 loading test. Low doses of BH4 in monotherapy permit withdrawal of the special formula and guarantee a good clinical and nutritional outcome with no adverse side effects in PKU patients.

We summarize the diagnosis, outcome, and molecular studies of five Mediterranean patients with citrullinemia: four neonatal classical forms and one subacute form, who also suffers from Down syndrome and presented with severe hepatic encephalopathy at age 7. Mutational analysis revealed three alleles with a common mutation and five new mutations: two Moroccan siblings are homozygous for G390R, the subacute form is compound heterozygous for G390R/G117D (new mutation), and the two other neonatal forms are compound heterozygous for four new mutations: V69A/E270Q and T119I(R108L)/?.

The molecular detection of heterozygotes for hyperphenylalaninemia is difficult due to the large number of mutations in the PAH gene. For this reason, various indexes that measure plasma concentrations of phenylalanine (Phe) and tyrosine (Tyr), as an expression of Phe metabolizing capacity, have been used for the detection of carriers for mutations in the PAH gene. In this study, we contrast the biochemical and the molecular data in order to know if this is an accurate method. Familial genetic analysis of the PAH gene in 93 parents of hyperphenylalaninemia patients allows the study of the biochemical expression of the different mutant alleles. Molecular study was performed by SSCP and DGGE analyses of PAH genes, and plasma amino acid analysis by ion-exchange chromatography. Then the biochemical and molecular data were compared by the Student t test. The results found show a relationship between the severity of PKU/HPA mutations in the PAH gene and their biochemical phenotype (Phe/Tyr, Phe2/Tyr) as an expression of the residual enzymatic activity. The study adds further information about the prevalent Mediterranean allele mutations.