Molecular analysis of PKU-associated PAH mutations: a fast and simple genotyping test

Abstract: Neonatal screening for phenylketonuria (PKU, OMIM: 261600) was introduced at the end of the 1960s. We developed a rapid and simple molecular test for the most frequent phenylalanine hydroxylase (PAH, Gene ID: 5053) mutations. Using this method to detect the 18 most frequent mutations, it is possible to achieve a 75% detection rate in Italian population. The variants selected also reach a high detection rate in other populations, for example, 70% in southern Germany, 68% in western Germany, 76% in Denmark, 68% in Sweden, 63% in Poland, and 60% in Bulgaria. We successfully applied this confirmation test in neonatal screening for hyperphenylalaninemias using dried blood spots and obtained the genotype in approximately 48 h. The method was found to be suitable as second tier test in neonatal screening for hyperphenylalaninemias in neonates with a positive screening test. This test can also be useful for carrier screening because it can bypass the entire coding sequence and intron–exon boundaries sequencing, thereby overcoming the questions that this approach implies, such as new variant interpretations

In vitro study of uptake and synthesis of creatine and its precursors by cerebellar granule cells and astrocytes suggests some hypotheses on the physiopathology of the inherited disorders of creatine metabolism

<p>Abstract</p> <p>Background</p> <p>The discovery of the inherited disorders of creatine (Cr) synthesis and transport in the last few years disclosed the importance of blood Cr supply for the normal functioning of the brain. These putatively rare diseases share a common pathogenetic mechanism (the depletion of brain Cr) and similar phenotypes characterized by mental retardation, language disturbances, seizures and movement disorders. In the effort to improve our knowledge on the mechanisms regulating Cr pool inside the nervous tissue, Cr transport and synthesis and related gene transcripts were explored in primary cultures of rat cerebellar granule cells and astrocytes.</p> <p>Methods</p> <p>Cr uptake and synthesis were explored in vitro by incubating monotypic primary cultures of rat type I astrocytes and cerebellar granule cells with: a) D₃-Creatine (D₃Cr) and D3Cr plus β-guanidinopropionate (GPA, an inhibitor of Cr transporter), and b) labelled precursors of Guanidinoacetate (GAA) and Cr (Arginine, Arg; Glycine, Gly). Intracellular D3Cr and labelled GAA and Cr were assessed by ESI-MS/MS. Creatine transporter (<it>CT1</it>), L-arginine:glycine amidinotransferase (<it>AGAT</it>), and S-adenosylmethionine:guanidinoacetate N-methyltransferase (<it>GAMT</it>) gene expression was assessed in the same cells by real time PCR.</p> <p>Results</p> <p>D3Cr signal was extremely high in cells incubated with this isotope (labelled/unlabelled Cr ratio reached about 10 and 122, respectively in cerebellar granule cells and astrocytes) and was reduced by GPA. Labelled Arg and Gly were taken up by the cells and incorporated in GAA, whose concentration paralleled that of these precursors both in the extracellular medium and inside the cells (astrocytes). In contrast, the increase of labelled Cr was relatively much more limited since labelled Cr after precursors' supplementation did not exceed 2,7% (cerebellar granule cells) and 21% (astrocytes) of unlabelled Cr. Finally, <it>AGAT, GAMT </it>and <it>SLC6A8 </it>were expressed in both kind of cells.</p> <p>Conclusions</p> <p>Our results confirm that both neurons and astrocytes have the capability to synthesize and uptake Cr, and suggest that at least in vitro intracellular Cr can increase to a much greater extent through uptake than through <it>de novo </it>synthesis. Our results are compatible with the clinical observations that when the Cr transporter is defective, intracellular Cr is absent despite the brain should be able to synthesize it. Further research is needed to fully understand to what extent our results reflect the in vivo situation.</p

Executive function impairment in early - treated PKU subjects with normal mental development

Executive functions were studied in 14 early and continuously treated PKU subjects (age 10.8 years, range 8-13) in comparison with controls matched for IQ, sex, age and socioeconomic status. Brain MRI examination was normal in all PKU patients. Neuropsychological evaluation included Wisconsin Card Sorting Test, Rey-Osterreith Complex Figure Test, Elithorn's Perceptual Maze Test, Weigl's Sorting Test, Tower of London, Visual Search and Motor Motor Learning Test. Whatever the IQ, PKU subjects performed worse than controls in tests exploring executive functions. Subgrouping the PKU subjects according to the quality of dietary control for the entire follow-up period (using 400 micromol/L as cut-off value for blood phenylalanine (Phe) concentration) showed that patients with worse dietary control performed more poorly than both the PKU group with the best dietary control and the control group. However, a mild impairment of executive functions was still found in PKU patients with a good dietary control (Phe 400 micromol/L) compared to controls. Concerning the PKU group as a whole, no linear correlation was found between neuropsychological performance and historical and concurrent biochemical parameters. We conclude that (a) PKU patients, even when treated early, rigorously and continuously, show an impairment of frontal lobe functions; (b) a protracted exposure to moderately high levels of Phe can affect frontal lobe functions independently of the possible effect of the same exposure on IQ; (c) in order to reduce the risk of frontal lobe dysfunction, the target of dietary therapy should be to maintain blood Phe concentration below 400 micromol/L

Case report: Childhood epilepsy and borderline intellectual functioning hiding an AADC deficiency disorder associated with compound heterozygous DDC gene pathogenic variants

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive neurometabolic disorder leading to severe combined serotonin, dopamine, norepinephrine, and epinephrine deficiency. We report on a female patient with borderline functioning and sporadic clear-cut focal to bilateral seizures from age 10 years. A neuropsychological assessment highlighted a mild impairment in executive functions, affecting attention span and visual-spatial abilities. Following the diagnosis of epilepsy with a presumed genetic etiology, we applied a diagnostic approach inclusive of a next-generation sequencing (NGS) gene panel, which uncovered two variants in trans in the DOPA decarboxylase (DDC) gene underlying an AADC deficiency. This compound heterozygous genotype was associated with a mild reduction of homovanillic acid, a low level of the norepinephrine catabolite, and a significant reduction of 5-hydroxyindoleacetic acid in cerebrospinal fluid. Remarkably, 3-O-methyldopa (3-OMD) and 5-hydroxytryptophan were instead increased. During the genetically guided re-evaluation process, some mild signs of dysautonomic dysfunction (nasal congestion, abnormal sweating, hypotension and fainting, excessive sleepiness, small hands and feet, and increased levels of prolactin, tiredness, and fatigue), more typical of AADC deficiency, were evaluated with new insight. Of the two AADC variants, the R347Q has already been characterized as a loss-of-function with severe catalytic impairments, while the novel L391P variant has been predicted to have a less severe impact. Bioinformatic analyses suggest that the amino acid substitution may affect affinity for the PLP coenzyme. Thus, the genotype corresponds to a phenotype with mild and late-onset symptoms, of which seizures were the clinical sign, leading to medical attention. This case report expands the spectrum of AADC deficiency phenotypes to encompass a less-disabling clinical condition including borderline cognitive functioning, drug-responsive epilepsy, and mild autonomic dysfunction

Intellectual Disability and Brain Creatine Deficit: Phenotyping of the Genetic Mouse Model for GAMT Deficiency

none9noGuanidinoacetate methyltransferase deficiency (GAMT-D) is one of three cerebral creatine (Cr) deficiency syndromes due to pathogenic variants in the GAMT gene (19p13.3). GAMT-D is characterized by the accumulation of guanidinoacetic acid (GAA) and the depletion of Cr, which result in severe global developmental delay (and intellectual disability), movement disorder, and epilepsy. The GAMT knockout (KO) mouse model presents biochemical alterations in bodily fluids, the brain, and muscles, including increased GAA and decreased Cr and creatinine (Crn) levels, which are similar to those observed in humans. At the behavioral level, only limited and mild alterations have been reported, with a large part of analyzed behaviors being unaffected in GAMT KO as compared with wild-type mice. At the cerebral level, decreased Cr and Crn and increased GAA and other guanidine compound levels have been observed. Nevertheless, the effects of Cr deficiency and GAA accumulation on many neurochemical, morphological, and molecular processes have not yet been explored. In this review, we summarize data regarding behavioral and cerebral GAMT KO phenotypes, and focus on uncharted behavioral alterations that are comparable with the clinical symptoms reported in GAMT-D patients, including intellectual disability, poor speech, and autistic-like behaviors, as well as unexplored Cr-induced cerebral alterations.openRossi, Luigia; Nardecchia, Francesca; Pierigè, Francesca; Ventura, Rossella; Carducci, Claudia; Leuzzi, Vincenzo; Magnani, Mauro; Cabib, Simona; Pascucci, TizianaRossi, Luigia; Nardecchia, Francesca; Pierigè, Francesca; Ventura, Rossella; Carducci, Claudia; Leuzzi, Vincenzo; Magnani, Mauro; Cabib, Simona; Pascucci, Tizian

safety and efficacy of galactogogues substances that induce maintain and increase breast milk production

Poor production of breast milk is the most frequent cause of breast lactation failure. Often, physician prescribe medications or other substances to solve this problem. The use of galactogogues should be limited to those situations in which reduced milk production from treatable causes has been excluded. One of the most frequent indication for the use of galactogogues is the diminution of milk production in mothers using indirect lactation, particularly in the case of preterm birth. The objective of this review is to analyze to the literature relating to the principal drugs used as galactogogues (metoclopramide, domperidone, chlorpromazine, sulpiride, oxytocin, growth hormone, thyrotrophin releasing hormone, medroxyprogesterone). Have been also analyzed galactogogues based on herbs and other natural substances (fenugreek, galega and milk thistle). We have evaluated their mechanism of action, transfer to maternal milk, effectiveness and potential side effects for mother and infant, suggested doses for galactogogic effect, and recommendation for breastfeeding

Early alterations of cortical thickness and gyrification in migraine without aura:a retrospective MRI study in pediatric patients

BACKGROUND: Migraine is the most common neurological disease, with high social-economical burden. Although there is growing evidence of brain structural and functional abnormalities in patients with migraine, few studies have been conducted on children and no studies investigating cortical gyrification have been conducted on pediatric patients affected by migraine without aura. METHODS: Seventy-two pediatric patients affected by migraine without aura and eighty-two controls aged between 6 and 18 were retrospectively recruited with the following inclusion criteria: MRI exam showing no morphological or signal abnormalities, no systemic comorbidities, no abnormal neurological examination. Cortical thickness (CT) and local gyrification index (LGI) were obtained through a dedicated algorithm, consisting of a combination of voxel-based and surface-based morphometric techniques. The statistical analysis was performed separately on CT and LGI between: patients and controls; subgroups of controls and subgroups of patients. RESULTS: Patients showed a decreased LGI in the left superior parietal lobule and in the supramarginal gyrus, compared to controls. Female patients presented a decreased LGI in the right superior, middle and transverse temporal gyri, right postcentral gyrus and supramarginal gyrus compared to male patients. Compared to migraine patients younger than 12 years, the ≥ 12-year-old subjects showed a decreased CT in the superior and middle frontal gyri, pre- and post-central cortex, paracentral lobule, superior and transverse temporal gyri, supramarginal gyrus and posterior insula. Migraine patients experiencing nausea and/or vomiting during headache attacks presented an increased CT in the pars opercularis of the left inferior frontal gyrus. CONCLUSIONS: Differences in CT and LGI in patients affected by migraine without aura may suggest the presence of congenital and acquired abnormalities in migraine and that migraine might represent a vast spectrum of different entities. In particular, ≥ 12-year-old pediatric patients showed a decreased CT in areas related to the executive function and nociceptive networks compared to younger patients, while female patients compared to males showed a decreased CT of the auditory cortex compared to males. Therefore, early and tailored therapies are paramount to obtain migraine control, prevent cerebral reduction of cortical thickness and preserve executive function and nociception networks to ensure a high quality of life

Parietal resting-state EEG alpha source connectivity is associated with subcortical white matter lesions in HIV-positive people

Objective Parietal resting-state electroencephalographic (rsEEG) alpha (8–10 Hz) source connectivity is abnormal in HIV-positive persons. Here we tested whether this abnormality may be associated with subcortical white matter vascular lesions in the cerebral hemispheres. Methods Clinical, rsEEG, and magnetic resonance imaging (MRI) datasets in 38 HIV-positive persons and clinical and rsEEG datasets in 13 healthy controls were analyzed. Radiologists visually evaluated the subcortical white matter hyperintensities from T2-weighted FLAIR MRIs (i.e., Fazekas scale). In parallel, neurophysiologists estimated the eLORETA rsEEG source lagged linear connectivity from parietal cortical regions of interest. Results Compared to the HIV participants with no/negligible subcortical white matter hyperintensities, the HIV participants with mild/moderate subcortical white matter hyperintensities showed lower parietal interhemispheric rsEEG alpha lagged linear connectivity. This effect was also observed in HIV-positive persons with unimpaired cognition. This rsEEG marker allowed good discrimination (area under the receiver operating characteristic curve &gt; 0.80) between the HIV-positive individuals with different amounts of subcortical white matter hyperintensities. Conclusions The parietal rsEEG alpha source connectivity is associated with subcortical white matter vascular lesions in HIV-positive persons, even without neurocognitive disorders. Significance Those MRI-rsEEG markers may be used to screen HIV-positive persons at risk of neurocognitive disorders

Lifestyles and socio-cultural factors among children aged 6-8 years from five Italian towns: The MAPEC-LIFE study cohort

Background: Lifestyles profoundly determine the quality of an individual’s health and life since his childhood. Many diseases in adulthood are avoidable if health-risk behaviors are identified and improved at an early stage of life. The aim of the present research was to characterize a cohort of children aged 6–8 years selected in order to perform an epidemiological molecular study (the MAPEC_LIFE study), investigate lifestyles of the children that could have effect on their health status, and assess possible association between lifestyles and socio-cultural factors. Methods: A questionnaire composed of 148 questions was administered in two different seasons to parents of children attending 18 primary schools in five Italian cities (Torino, Brescia, Pisa, Perugia and Lecce) to obtain information regarding the criteria for exclusion from the study, demographic, anthropometric and health information on the children, as well as some aspects on their lifestyles and parental characteristics. The results were analyzed in order to assess the frequency of specific conditions among the different seasons and cities and the association between lifestyles and socio-economic factors. Results: The final cohort was composed of 1,164 children (50.9 boys, 95.4% born in Italy). Frequency of some factors appeared different in terms of the survey season (physical activity in the open air, the ways of cooking certain foods) and among the various cities (parents’ level of education and rate of employment, sport, traffic near the home, type of heating, exposure to passive smoking, ways of cooking certain foods). Exposure to passive smoking and cooking fumes, obesity, residence in areas with heavy traffic, frequency of outdoor play and consumption of barbecued and fried foods were higher among children living in families with low educational and/or occupational level while children doing sports and consuming toasted bread were more frequent in families with high socio-economic level. Conclusions: The socio-economic level seems to affect the lifestyles of children enrolled in the study including those that could cause health effects. Many factors are linked to the geographical area and may depend on environmental, cultural and social aspects of the city of residence

Adult cognitive outcomes in phenylketonuria:explaining causes of variability beyond average Phe levels

OBJECTIVE: The objective was to deepen the understanding of the causes of individual variability in phenylketonuria (PKU) by investigating which metabolic variables are most important for predicting cognitive outcomes (Phe average vs Phe variation) and by assessing the risk of cognitive impairment associated with adopting a more relaxed approach to the diet than is currently recommended. METHOD: We analysed associations between metabolic and cognitive measures in a mixed sample of English and Italian early-treated adults with PKU (N = 56). Metabolic measures were collected through childhood, adolescence and adulthood; cognitive measures were collected in adulthood. Metabolic measures included average Phe levels (average of median values for each year in a given period) and average Phe variations (average yearly standard deviations). Cognition was measured with IQ and a battery of cognitive tasks. RESULTS: Phe variation was as important, if not more important, than Phe average in predicting adult outcomes and contributed independently. Phe variation was particularly detrimental in childhood. Together, childhood Phe variation and adult Phe average predicted around 40% of the variation in cognitive scores. Poor cognitive scores (> 1 SD from controls) occurred almost exclusively in individuals with poor metabolic control and the risk of poor scores was about 30% higher in individuals with Phe values exceeding recommended thresholds. CONCLUSIONS: Our results provide support for current European guidelines (average Phe value = < 360 μmol/l in childhood; = < 600 μmo/l from 12 years onwards), but they suggest an additional recommendation to maintain stable levels (possibly Phe SD = < 180 μmol/l throughout life). PUBLIC SIGNIFICANCE STATEMENTS: We investigated the relationship between how well people with phenylketonuria control blood Phe throughout their life and their ability to carry out cognitive tasks in adulthood. We found that avoiding blood Phe peaks was as important if not more important that maintaining average low Phe levels. This was particularly essential in childhood. We also found that blood Phe levels above recommended European guidelines was associated with around 30% increase in the risk of poor cognitive outcomes