A multicentre observational study was aimed to assess the prevalence of late-onset Pompe disease (LOPD) in a large high-risk population, using the dried blood spot (DBS) as a main screening tool.
17 ...Italian neuromuscular centres were involved in the late-onset Pompe early diagnosis (LOPED) study. Inclusion criteria were: (1) age ≥5 years, (2) persistent hyperCKaemia and (3) muscle weakness at upper and/or lower limbs (limb-girdle muscle weakness, LGMW). Acid α-glucosidase (GAA) activity was measured separately on DBS by fluorometric as well as tandem mass spectrometry methods. A DBS retest was performed in patients resulted positive at first assay. For the final diagnosis, GAA deficiency was confirmed by a biochemical assay in skeletal muscle, whereas genotype was assessed by GAA molecular analysis.
In a 14-month period, we studied 1051 cases: 30 positive samples (2.9%) were detected by first DBS screening, whereas, after retesting, 21 samples were still positive. Biochemical and molecular genetic studies finally confirmed LOPD diagnosis in 17 cases (1.6%). The median time from the onset of symptoms/signs to diagnosis was 5 years. Among those patients, 35% showed presymptomatic hyperCKaemia and 59% showed hyperCKaemia+LGMW, whereas 6% manifested with LGMW.
LOPED study suggests that GAA activity should be accurately screened by DBS in all patients referring for isolated hyperCKaemia and/or LGMW. A timely diagnosis was performed in five patients with presymptomatic hyperCKaemia, but two had already manifested with relevant changes on muscle morphology and MRI. Consequently, enzyme replacement therapy was started in 14/17 patients, including the 2 patients still clinically presymptomatic but with a laboratory evidence of disease progression.
Summary
We report on our 6-year experience of expanded newborn screening by tandem mass spectrometry in Tuscany (Italy), the first Italian Region to screen all newborns for more than 40 inborn errors ...of metabolism: organization, diseases observed and updates on methods to reduce false-positive and false-negative tests are described. Blood collection is recommended between 48 and 72 h of life. Blood spots are sent daily by courier to laboratory. When a positive result occurs, two subsequent procedures are followed: for disorders with possible acute metabolic decompensation, the baby is immediately recalled and clinical examinations and confirmatory tests are performed; for the other disorders, the nursery provides for a second blood spot. If the test is positive, clinical examinations and confirmatory tests are performed. In both cases, if confirmatory tests are positive, a treatment and a follow-up programme are started. Up to now, spots from 160 000 infants have been analysed and 80 affected patients have been identified (disorders of amino acids, organic acids and fatty acids metabolism). We describe adjustments to cut-off values, the introduction of a second-tier test for propionic acidaemia and for methylmalonic aciduria, the inclusion of succinylacetone in the panel of metabolites, and protocols for premature infants and for newborns on parenteral nutrition or transfused. These changes resulted in a reduction in recalls from 1.37% to 0.32% and consequently of working time and parental stress. Avoiding false-negatives by using more specific markers and minimizing the false-positive rate with second-tier testing is important for a successful newborn screening programme.
Children and adolescents with overt type 1 diabetes (T1D) have been found to show an altered carnitine profile. This pattern has not previously been analyzed in neonates before onset of the disease.
...Fifty children who developed T1D during the first 6 years of life, born and living in the Tuscany and Umbria Regions of Italy, were identified and 200 controls were recruited into the study. All newborns were subjected to extended neonatal screening by mass spectrometry at 48-72 h of life. Four controls for each of the 50 index cases were taken randomly and blinded in the same analytical batch. The panel used for neonatal screening consists of 13 amino acids, free carnitine, 33 acyl-carnitines and 21 ratios. All Guthrie cards are analyzed within 2 days of collection.
Total and free carnitine were found to be significantly lower in neonates who later developed T1D compared with controls. Moreover, the concentrations of the acyl-carnitines - acetyl-L-carnitine (C2), proprionylcarnitine (C3), 3-hydroxyisovalerylcarnitine (C5OH), miristoylcarnitine (C4), palmitoylcarnitine (C16) and stearoylcarnitine (C18) - were also significantly low in the cases vs controls. Furthermore, total amino-acid concentrations, expressed as the algebraic sum of all amino acids tested, showed a trend toward lower levels in cases vs controls.
We found that carnitine and amino-acid deficit may be evident before the clinical appearance of T1D, possibly from birth. The evaluation of these metabolites in the neonatal period of children human leukocyte antigen genetically at 'risk' to develop T1D, could represent an additional tool for the prediction of T1D and could also offer the possibility to design new strategies for the primary prevention of the disease from birth.
Aminoacylase 1 (ACY1) deficiency is a rare inborn error of metabolism of which less than 20 observations have been described. Patients exhibit urinary excretion of specific N‐acetyl amino acids and ...manifest a heterogeneous clinical spectrum including intellectual disability, motor delay, seizures, moderate to severe mental retardation, absent speech, growth delay, muscular hypotonia and autistic features. Here, we report the case of ACY1 enzyme deficiency in a 6‐year‐old girl presenting severe intellectual disability, motor retardation, absence of spontaneous locomotor activity and severe speech delay. Urinary excretion of N‐acetylated amino acids was present. Mutational analysis of ACY1 gene identified the new homozygous c.1001_1001+5del6 mutation, which alters the mRNA transcription leading to exon 13 skipping and inclusion of a premature stop codon (p.Lys308Glufs*7). A quantitative fluorescent multiplex‐polymerase chain reaction (QFM‐PCR) assay has been set up and confirmed homozygosity of the mutation in the patient's DNA. Biochemical analysis showed absence of ACY1 enzyme activity in the patient's fibroblasts. The structure of the mutated protein has been defined by homology modeling (HM). Our data endorse the hypothesis of a link between this inborn error of metabolism and the neurological manifestations observed in patients with ACY1 deficiency.
Abstract
Objective: While propranolol pharmacokinetics has been extensively studied in adults, this study reports the first evaluation of propranolol pharmacokinetics in term and preterm neonates.
...Methods: Propranolol concentrations were measured in four term and 32 preterm newborns treated with oral propranolol at the dose of 0.5 or 0.25 mg/kg every 6 h by serial dried blood spots.
Results: The levels of propranolol, although with high inter-individual variability, were proportional with the administered dose. Pharmacokinetic parameters evaluated at the steady state in newborns treated with 0.5 mg/kg/6 h showed values of maximal (71.7 ± 29.8 ng/mL), minimal (42.2 ± 20.8 ng/mL) and average concentration (60.8 ± 25.0 ng/mL), time of maximal concentration (2.6 ± 0.9 h) and area under the time-concentration curve (364.7 ± 150.2 ng/mL/h) similar to those observed in adults. In both dosing groups, elimination half-life was significantly longer (14.9 ± 4.3 and 15.9 ± 6.1 h), and apparent total body clearance (27.2 ± 13.9 and 31.3 ± 13.3 mL/kg/min) lower than those reported in adults, suggesting a slower metabolism in newborns. No differences were observed between newborns with different gestational age or different sex.
Conclusions: Neonates treated with propranolol-exhibited drug concentrations proportional with the dose, with significant long half-life.
In an expanded newborn screening program for inborn errors of metabolism by LC–MS/MS in Tuscany, six newborns out of 169,000 showed decreased blood citrulline levels. In one of them, molecular ...analysis of the OTC gene identified the known p.Trp265Leu mutation, which is correlated with late-onset ornithine transcarbamylase deficiency (OTCD). Hypocitrullinemia is not a reliable marker for OTCD newborn screening, especially for late-onset forms that may exhibit normal citrulline levels. However, when hypocitrullinemia is detected in a newborn in whom intestinal dysfunction and prematurity have been excluded, OTCD should be investigated first because of the OTCD incidence (1:14,000) and the small size of the OTC gene coding sequence.
Summary
Background:
Insulin-resistant hyperglycaemia may occasionally complicate the clinical course of organic acidaemias.
Study Design:
Clinical observation.
Results:
Two term infants, one ...suffering from acute early-onset methylmalonic acidaemia, the other suffering from acute early-onset propionic acidaemia, presented acutely with dehydration, ketoacidosis, and hyperammonaemia. Urinary organic acid, plasma amino acids, and blood and plasma acylcarnitine analysis allowed the diagnosis of methylmalonic and propionic acidaemias. The detection of the novel c.481G>A (p.Gly161Arg) and the known c.655A>T (p.Asn219Tyr)
MUT
gene mutations identified the first patient as affected by methylmalonic acidaemia
mut
type. The high increase of propionylcarnitine after carnitine administration in both patients suggested a greatly elevated metabolic intoxication. Both newborns showed insulin-resistant hyperglycaemia. Patient 1 died, but patient 2, after a strong reduction of glucose administration, survived. To our knowledge, this is the only patient with this complication who survived.
Conclusion:
Insulin-resistant hyperglycaemia complicating neonatal onset of methylmalonic and propionic acidaemias is probably a marker of a serious disease. One patient with this complication survived after a strong reduction of glucose administration. Even if this is probably only a partial intervention, we hypothesize that in this situation a reduction of glucose administration can reduce almost the risk of persistent hyperglycaemia. Further studies are required to confirm our hypothesis.
Ferri L, Guido C, la Marca G, Malvagia S, Cavicchi C, Fiumara A, Barone R, Parini R, Antuzzi D, Feliciani C, Zampetti A, Manna R, Giglio S, Della Valle CM, Wu X, Valenzano KJ, Benjamin ER, Donati MA, ...Guerrini R, Genuardi M, Morrone A. Fabry disease: polymorphic haplotypes and a novel missense mutation in the GLA gene.
Fabry disease (FD) is an X‐linked lysosomal storage disorder with a heterogeneous spectrum of clinical manifestations that are caused by the deficiency of α‐galactosidase A (α‐Gal‐A) activity. Although useful for diagnosis in males, enzyme activity is not a reliable biochemical marker in heterozygous females due to random X‐chromosome inactivation, thus rendering DNA sequencing of the α‐Gal‐A gene, alpha‐galactosidase gene (GLA), the most reliable test for the confirmation of diagnosis in females. The spectrum of GLA mutations is highly heterogeneous. Many polymorphic GLA variants have been described, but it is unclear if haplotypes formed by combinations of such variants correlate with FD, thus complicating molecular diagnosis in females with normal α‐Gal‐A activity. We tested 67 female probands with clinical manifestations that may be associated with FD and 110 control males with normal α‐Gal‐A activity. Five different combinations of GLA polymorphic variants were identified in 14 of the 67 females, whereas clearcut pathogenetic alterations, p.Met51Ile and p.Met290Leu, were identified in two cases. The latter has not been reported so far, and both mutant forms were found to be responsive to the pharmacological chaperone deoxygalactonojirimycin (DGJ; migalastat hydrochloride). Analysis of the male control population, as well as male relatives of a suspected FD female proband, permitted the identification of seven different GLA gene haplotypes in strong linkage disequilibrium. The identification of haplotypes in control males provides evidence against their involvement in the development of FD phenotypic manifestations.
Abstract
Background
Atrial fibrillation (AF) is the most common sustained arrhythmia diagnosed at an advanced age. The most important complications of AF are stroke, heart failure and dementia. The ...pathophysiology of the arrhythmia is complex and multifaceted and several aspects are still not completely understood.
Purpose
Our project aimed at exploring the molecular characteristics of AF in older patients using an untargeted metabolomics approach, an analytical technique that provides an insight of the global metabolic profile at an individual level. As secondary endpoints, we evaluated the association between metabolomics and acylcarnitines, which are mediators of myocyte electrical instability, and difference in metabolomics profile between healthy controls (C) and AF patients (AF-P).
Method
We evaluated 50 consecutive older AF-P with persistent AF, waiting to be treated with elective electrical cardioversion of the arrhythmia, and 22 healthy controls (C). In all patients, the cardiological evaluation was integrated with tools of the Geriatric Multidimensional Assessment (GMA) to describe neurocognitive function (Mini-Mental State Examination, MMSE), depressive symptoms (15-item Geriatric Depression Scale, GDS), and physical performance (Short Physical Performance Battery, SPPB). Interleukin-6 (IL-6), a marker of low-grade inflammation, was measured with ELISA assays. Metabolomics analysis and acylcarnitine measurements were carried out using gas chromatography coupled with mass spectrometry (GC-MS).
Results
AF-P and C did not differ by age (76 ± 6 vs. 70 ± 14 years, p = 0.055); patients had a lower proportion of women (AF-P: 32% vs. C: 64%, p = 0.012). Cluster analysis applied to metabolomic data in all population identified two subset of subjects, characterized by a different body mass index (BMI; 23.57 vs. 32.53 Kg/m², p = 0.046). When studying only the AF-P group, the simultaneous presence of a higher CHA2DS2-VASc score (4.17 ± 1.32 vs. 2.88 ± 1.83, p = 0.007), a lower physical function (SPPB; 8.97± 2.22 vs. 10.75 ± 1.13, p = 0.009), and higher IL-6 levels (4.42 vs. 2.39 pg/mL, p = 0.002) described the cluster with a worse clinical phenotype and a specific metabolomics pattern. Furthermore, an association between IL-6 and medium- long-chain acylcarnitines emerged (acylcarnitine 12:1, β = 31.7 ± 9.4, R = 0.446, p = 0.001).
Conclusions
Present analysis demonstrated that a specific metabolomics profile was characteristic of older AF patients with a frail phenotype, identified by higher CHA2DS2-VASc score and IL-6 concentration, and lower physical performance. Future studies are needed to reveal the single molecular pathways responsible of these behaviour, potentially useful to guide a targeted therapy. Last, inflammation per se could contribute to electrical instability in AF, while body mass index, a surrogate measure of undernutrition, could identify different metabolic pathways in controls and in patients with the arrhythmia.Metabolomics by clinical phenotypes
A prerequisite for the efficacy of chemotherapy is that it reaches the tumor mass at a therapeutic concentration. In brain tumors this phenomenon is hampered by the presence of the blood brain ...barrier (BBB) which limits the spread of chemotherapeutic agents within the brain. It is lately emerged as this Multi Drug Resistance (MDR) phenomenon is explained through the cooperation of P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2), two "gatekeeper" transporters that work in tandem on the BBB and are also present on the plasma membrane of certain brain tumors. Recently, we have attempted to improve the therapeutic efficacy of pharmacological treatment in malignant brain tumors by safe and temporary BBB permeabilization. We have demonstrated that morphine and, to a lower level, ondansetron and dexamethasone allow an accumulation of doxorubicin within the rat brain by LC-MS/MS mass spectrometry. All these drugs are substrates of P-gp and BCRP efflux pumps. The aim of the current proposal is to expand our preliminary observation, to understand the mechanism of action of BBB "permeabilization" induced by morphine or other drugs, and to exploit this method for the "treatment" of brain tumour in an animal model. i) Quantifying the level drugs that do not usually cross the BBB (mitoxantrone or melphalan) after morphine pre-treatment in a preclinical model. Verifying the cytotoxic effect of morphine plus doxorubicin (and other chemotherapeutic agents) treatment by using MTT and TUNEL analysis in glioblastoma cell lines ii) Quantifying the level of drugs that do not usually cross the BBB after morphine pre-treatment in an artificial BBB through a monolayer of MDCKII cells over-expressing the human P-gp or BCRP. iii) Investigating the regulatory role of certain microRNA in MDR mechanism by RT-PCR and western blot analysis of P-gp, BCRP, miR-21, miR-27a and miR-451. Our data suggest that blocking efflux transporters by pretreatment with morphine, ondansetron or desamethasone is able to allow doxorubicin penetration inside the brain. This is not associated with acute cardiac or renal toxicity. These preliminary results will enable us to novel therapeutic approaches to refractory or recurrent brain tumors in which molecules usually stopped by the BBB may have a therapeutic impact.
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