Publications by authors named "Giancarlo la Marca"

123 Publications

Neonatal Screening in Europe Revisited: An ISNS Perspective on the Current State and Developments Since 2010.

Int J Neonatal Screen 2021 Mar 5;7(1). Epub 2021 Mar 5.

International Society for Neonatal Screening (ISNS) Office, 3721CK Bilthoven, The Netherlands.

Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylketonuria. The panel of screened disorders ("conditions") then gradually expanded, with a boost in the late 1990s with the introduction of tandem mass spectrometry (MS/MS), making it possible to screen for 40-50 conditions using a single blood spot. The most recent additions to screening programmes (screening for cystic fibrosis, severe combined immunodeficiency and spinal muscular atrophy) were assisted by or realised through the introduction of molecular technologies. For this survey, we collected data from 51 European countries. We report the developments between 2010 and 2020 and highlight the achievements reached with the progress made in this period. We also identify areas where further progress can be made, mainly by exchanging knowledge and learning from experiences in neighbouring countries. Between 2010 and 2020, most NBS programmes in geographical Europe matured considerably, both in terms of methodology (modernised) and with regard to the panel of conditions screened (expanded). These developments indicate that more collaboration in Europe through European organisations is gaining momentum. We can only accomplish the timely detection of newborn infants potentially suffering from one of the many rare diseases and take appropriate action by working together.
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http://dx.doi.org/10.3390/ijns7010015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006225PMC
March 2021

Upfront Enzyme Replacement via Erythrocyte Transfusions for PNP Deficiency.

J Clin Immunol 2021 Feb 27. Epub 2021 Feb 27.

Department of Pediatrics, Dr. von Hauner Children's Hospital, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany.

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http://dx.doi.org/10.1007/s10875-021-01003-9DOI Listing
February 2021

Morquio B disease: From pathophysiology towards diagnosis.

Mol Genet Metab 2021 03 1;132(3):180-188. Epub 2021 Feb 1.

Molecular and Cell Biology Laboratory, Paediatric Neurology Unit and Laboratories, Neuroscience Department, A. Meyer Children's Hospital, Florence, Italy; Department of Neurosciences, Psychology, Pharmacology and Child Health, University of Florence, Italy. Electronic address:

Morquio B disease is an attenuated phenotype within the spectrum of beta galactosidase (GLB1) deficiencies. It is characterised by dysostosis multiplex, ligament laxity, mildly coarse facies and heart valve defects due to keratan sulphate accumulation, predominantly in the cartilage. Morquio B patients have normal neurological development, setting them apart from those with the more severe GM1 gangliosidosis. Morquio B disease, with an incidence of 1:250.000 to 1:1.000.000 live births, is very rare. Here we report the clinical-biochemical data of nine patients. High amounts of keratan sulfate were detected using LC-MS/MS in the patients' urinary samples, while electrophoresis, the standard procedure of qualitative glycosaminoglycans analysis, failed to identify this metabolite in any of the patients' samples. We performed molecular analyses at gene, gene expression and protein expression levels, for both isoforms of the GLB1 gene, lysosomal GLB1, and the cell-surface expressed Elastin Binding Protein. We characterised three novel GLB1 mutations [c.75 + 2 T > G, c.575A > G (p.Tyr192Cys) and c.2030 T > G (p.Val677Gly)] identified in three heterozygous patients. We also set up a copy number variation assay by quantitative PCR to evaluate the presence of deletions/ insertions in the GLB1 gene. We propose a diagnostic plan, setting out the specific clinical- biochemical and molecular features of Morquio B, in order to avoid misdiagnoses and improve patients' management.
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http://dx.doi.org/10.1016/j.ymgme.2021.01.008DOI Listing
March 2021

[The Newborn Screening Program in Italy: Comparison with Europe and other Countries.]

Rev Esp Salud Publica 2021 Jan 26;95. Epub 2021 Jan 26.

Laboratorio di Screening Neonatale, Biochimica Clinica e Farmacologia. AOU Meyer Dipartimento di Scienze Biomediche, Sperimentali e Cliniche. Università Studi Firenze. Florencia. Italia.

In Italy, since 2016, extended neonatal screening has been mandatory throughout the country for about 40 inherited metabolic diseases. The law contains indications on: the list of pathologies, the information and consent, the methods of collecting and sending samples, the newborn screening system with the elements of its organization, appointed to guarantee the entire path of newborn screening, from the level I test to taking charge of the confirmed positive newborn, the communication and recall procedures for diagnostic confirmation and patient management, training and information initiatives, as well as the criteria for allocating the allocation. Extended neonatal screening has introduced new issues in diagnosis, choice of decision levels, and metabolic disease panels to screen. Of particular relevance in order to a strong reduction of false positives, was the introduction of the second-tier test for some diseases such as leucinosis, isovaleric acidemia, methylmalonic aciduria. As regards the diseases to be screened, the Italian situation differs greatly from what happens in Europe where in the majority of member states there is no legislation / law governing this preventive pediatric service; screening is almost always on a voluntary basis (with the collection of written informed consent from both parents) and applied on the basis of health guidelines or recommendations. In the world, the most complete panel is the US one (RUSP, Recommended Uniform Screening Panel) which currently contains 62 pathologies, 35 of which are defined as core panels and 27 as secondary panels. As the name implies, it is a panel that the US Health Resources and Services Administration -HRSA- recommends that it be applied by every State and that includes new screening in some areas including Pompe disease and MPS I, creatine deficiency. In conclusion, extended neonatal screening represents a real revolution in the metabolic field offering newborns an early diagnosis combined with effective therapeutic treatments capable of radically changing the course of these serious diseases.
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January 2021

High frequency of biotinidase deficiency in Italian population identified by newborn screening.

Mol Genet Metab Rep 2020 Dec 5;25:100689. Epub 2020 Dec 5.

Newborn Screening, Biochemical & Pharmacology Lab, Clinic of Paediatric Neurology, A. Meyer Children's Hospital, Firenze, Italy.

The biotinidase (BTD) enzyme is essential for recycling biotin, a water-soluble B-complex vitamin that is the coenzyme of four carboxylases involved in fatty acid synthesis, amino acid catabolism and gluconeogenesis. If untreated, total or partial BTD deficiencies lead to an autosomal recessive inherited organic aciduria whose clinical features, mainly presenting in the first years of life, include, seizures, skin rash, and alopecia. Based on residual BTD enzyme activity it is possible to identify partial or total biotinidase deficiency. The incidence of profound and partial biotinidase deficiency worldwide is estimated to be about 1 in 60.000. We report twelve years of experience in the newborn screening of biotinidase deficiency on 466.182 neonates. When a positive screening result occurred, a clinical evaluation was made of the patient and genetic counselling was offered to the family. Molecular analysis the BTD gene was carried out in all recalled neonates. Newborn screening lead to the identification of 75 BTD deficiencies with an incidence of about 1:6.300 births, ten times higher than the reported worldwide incidence. BTD deficiency was confirmed at a genomic level in all patients, demonstrating a high frequency of the p.(Asp444His) amino acid substitution and the complex allele p.(Ala171Thr)/p.(Asp444His) in the analyzed Italian newborns. Four new mutations (two small deletions, one stop mutation and one missense mutation) and a new combined allelic alteration were identified. Our data suggests that there is a high incidence of the biotinidase defect in the Italian population, most likely due to the high frequency of certain mutations.
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http://dx.doi.org/10.1016/j.ymgmr.2020.100689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7719957PMC
December 2020

Effect of Beta 3 Adrenoreceptor Modulation on Patency of the Ductus Arteriosus.

Cells 2020 12 7;9(12). Epub 2020 Dec 7.

Division of Neonatology and NICU, Department of Clinical and Experimental Medicine, University of Pisa, 56126 Pisa, Italy.

β3-adrenoreceptor (β3-AR), a G-protein coupled receptor, has peculiar regulatory properties in response to oxygen and widespread localization. β3-AR is expressed in the most frequent neoplasms, also occurring in pregnant women, and its blockade reduces tumor growth, indicating β3-AR-blockers as a promising alternative to antineoplastic drugs during pregnancy. However, β3-AR involvement in prenatal morphogenesis and the consequences of its blockade for the fetus remain unknown. In this study, after the demonstrated expression of β3-AR in endothelial and smooth muscle cells of ductus arteriosus (DA), C57BL/6 pregnant mice were acutely treated at 18.5 of gestational day (GD) with indomethacin or with the selective β3-AR antagonist SR59230A, or chronically exposed to SR59230A from 15.5 to 18.5 GD. Six hours after the last treatment, fetuses were collected. Furthermore, newborn mice were treated straight after birth with BRL37344, a β3-AR agonist, and sacrificed after 7 h. SR59230A, at the doses demonstrated effective in reducing cancer progression (10 and 20 mg/kg) in acute and chronic mode, did not induce fetal DA constriction and did not impair the DA ability to close after birth, whereas at the highest dose (40 mg/kg), it was shown to cause DA constriction and preterm-delivery. BRL37344 administered immediately after birth did not alter the physiological DA closure.
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http://dx.doi.org/10.3390/cells9122625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762377PMC
December 2020

Study protocol: treatment with caffeine of the very preterm infant in the delivery room: a feasibility study.

BMJ Open 2020 12 4;10(12):e040105. Epub 2020 Dec 4.

Department of Clinical Sciences and Community Health, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy.

Introduction: Early treatment with caffeine in the delivery room has been proposed to decrease the need for mechanical ventilation (MV) by limiting episodes of apnoea and improving respiratory mechanics in preterm infants. Thus, the purpose of this feasibility study is to verify the hypothesis that intravenous or enteral administration of caffeine can be performed in the preterm infant in the delivery room.

Methods And Analysis: In this multicentre prospective study, infants with 25-29 weeks of gestational age will be enrolled and randomised to receive 20 mg/kg of caffeine citrate intravenously, via the umbilical vein, or enterally, through an orogastric tube, within 10 min of birth. Caffeine plasma level will be measured at 60±15 min after administration and 60±15 min before the next dose (5 mg/kg). The primary endpoint will be evaluation of the success rate of intravenous and enteral administration of caffeine in the delivery room. Secondary endpoints will be the comparison of success rate of intravenous versus oral administration and the evaluation of the need for MV in treated infants. In the absence of previous references, we arbitrarily decided to study 20 infants treated with intravenous caffeine and 20 infants treated with enteral caffeine. Primary endpoint will be evaluated measuring the success rate of intravenous and enteral caffeine administration which will be considered a success when it is followed by the achievement of the caffeine therapeutic level (8-25 µg/mL) 60±15 min before administration of the second dose.

Ethics And Dissemination: The study has been approved by the Italian Medicines Agency (AIFA: AIFA/RSC/P/32755) and by Comitato Etico Pediatrico Regione Toscana. The results will be published in peer-reviewed academic journals.

Trial Registration Number: ClinicalTrials.gov identifier NCT04044976; EudraCT number 2018-003626-91.
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http://dx.doi.org/10.1136/bmjopen-2020-040105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722383PMC
December 2020

Thermal inactivation of SARS COVID-2 virus: Are steam inhalations a potential treatment?

Life Sci 2021 Jan 21;265:118801. Epub 2020 Nov 21.

Section of Pharmacology and Toxicology, Department of Neurosciences, Psychology, Drug Research and Child Health, University of Florence, Florence, Italy; Neurology, Neurogenetics and Neurobiology Unit and Laboratories, Meyer Children's University Hospital, Florence, Italy.

Background: The emergence of SARS-CoV-2 pandemic has upset health systems around the world and caused immeasurable losses and costs. Until a vaccine will become available, the recommended prevention measures remain physical distancing and enhanced hygiene.

Methods And Findings: The proteic structure external to the virus is the main target that may eventually lead to reduce or block its replication in the upper airways. We developed a protocol based of repeated steam inhalation cycles aimed at reducing the risk of progression to full blown infection if performed soon after contagion. The protocol has been used in a single-center open label trial on ten infected asymptomatic or pauci-symptomatic health care professionals.

Conclusions: The promising results we obtained with this easily accessible, non-invasive and inexpensive procedure should prompt controlled trials.
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http://dx.doi.org/10.1016/j.lfs.2020.118801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7680040PMC
January 2021

Children with special health care needs attending emergency department in Italy: analysis of 3479 cases.

Ital J Pediatr 2020 Nov 23;46(1):173. Epub 2020 Nov 23.

Department of Pediatrics, ASST-Lariana, Hospital "Sant'Anna", Como, Italy.

Background: Although children with special health care needs (CSHCN) represent a minority of the population, they go through more hospitalizations, more admissions to the Emergency Department (ED), and receive a major number of medical prescriptions, in comparison to general pediatric population. Objectives of the study were to determine the reasons for admission to the ED in Italian CSHCN, and to describe the association between patient's demographic data, clinical history, and health services requirements.

Methods: Ad hoc web site was created to collect retrospective data of 3479 visits of CSHCN to the ED in 58 Italian Hospitals.

Results: Seventy-two percent of patients admitted to ED were affected by a previously defined medical condition. Most of the ED admissions were children with syndromic conditions (54%). 44.2% of the ED admissions were registered during the night-time and/or at the weekends. The hospitalization rate was of 45.6% among patients admitted to the ED. The most common reason for admission to the ED was the presence of respiratory symptoms (26.6%), followed by gastrointestinal problems (21.3%) and neurological disorders (18.2%). 51.4% of the access were classified as 'urgent', with a red/yellow triage code. Considering the type of ED, 61.9% of the visits were conducted at the Pediatric EDs (PedEDs), 33.5% at the Functional EDs (FunEDs) and 4.6% at the Dedicated EDs (DedEDs). Patients with more complex clinical presentation were more likely to be evaluated at the PedEDs. CSHCN underwent to a higher number of medical procedures at the PedEDs, more in comparison to other EDs. Children with medical devices were directed to a PedED quite exclusively when in need for medical attention. Subjects under multiple anti-epileptic drug therapy attended to PedEDs or FunEDs generally. Patients affected by metabolic diseases were more likely to look for medical attention at FunEDs. Syndromic patients mostly required medical attention at the DedEDs.

Conclusions: Access of CSHCN to an ED is not infrequent. For this reason, it is fundamental for pediatricians working in any kind of ED to increase their general knowledge about CHSCN and to gain expertise in the management of such patients and their related medical complexity.
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http://dx.doi.org/10.1186/s13052-020-00937-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685641PMC
November 2020

Incessant Automatic Atrial Tachycardia in a Neonate Successfully Treated with Nadolol and Closely Spaced Doses of Flecainide: A Case Report.

Pediatr Rep 2020 Nov 11;12(3):108-113. Epub 2020 Nov 11.

Department of Pediatric Cardiology, Meyer Children's University Hospital, Viale Pieraccini 24, 50139 Florence, Italy.

Supraventricular tachyarrhythmia (SVT) is the most common type of arrhythmia in childhood. Management can be challenging with an associated risk of mortality. A female neonate was diagnosed with episodes of SVT, controlled antenatally with digoxin. Flecainide was commenced prophylactically at birth. Despite treatment, the infant developed a narrow complex tachycardia at 5 days of age. The electrocardiogram features were suggestive of either re-entry tachycardia or of automatic atrial tachycardia (AAT). Following several unsuccessful treatments, a wide complex tachycardia developed. A transesophageal electrophysiological study led to a diagnosis of AAT. Stable sinus rhythm was finally achieved through increasing daily administrations of flecainide up to six times a day, in association with nadolol. The shortening of intervals to this extent has never been reported before and supports the evidence of a personal, age-specific variability in pharmacokinetics of flecainide. Larger studies are needed to better define the appropriate dose and timing of administration.
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http://dx.doi.org/10.3390/pediatric12030024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717654PMC
November 2020

Development of Strategies to Decrease False Positive Results in Newborn Screening.

Int J Neonatal Screen 2020 Nov 2;6(4). Epub 2020 Nov 2.

Newborn Screening, Biochemistry and Pharmacology Laboratory, Pediatric Neurology, Unit and Laboratories, Meyer Children's University Hospital, 50139 Florence, Italy.

The expansion of national newborn screening (NBS) programmes has provided significant benefits in the diagnosis and early treatment of several rare, heritable conditions, preventing adverse health outcomes for most affected infants. New technological developments have enabled the implementation of testing panel covering over 50 disorders. Consequently, the increment of false positive rate has led to a high number of healthy infants recalled for expensive and often invasive additional testing, opening a debate about the harm-benefit ratio of the expanded newborn screening. The false-positive rate represents a challenge for healthcare providers working in NBS systems. Here, we give an overview on the most commonly used strategies for decreasing the adverse effects due to inconclusive screening results. The focus is on NBS performance improvement through the implementation of analytical methods, the application of new and more informative biomarkers, and by using post-analytical interpretive tools. These strategies, used as part of the NBS process, can to enhance the positive predictive value of the test and reduce the parental anxiety and healthcare costs related to the unnecessary tests and procedures.
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http://dx.doi.org/10.3390/ijns6040084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712114PMC
November 2020

Regulatory landscape of providing information on newborn screening to parents across Europe.

Eur J Hum Genet 2021 Jan 10;29(1):67-78. Epub 2020 Oct 10.

Department of Paediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria.

Newborn screening (NBS) is an important part of public healthcare systems in many countries. The provision of information to parents about NBS is now recognised as an integral part of the screening process. Informing parents on all aspects of screening helps to achieve the benefits, promote trust and foster support for NBS. Therefore, policies and guidelines should exist to govern how the information about NBS is provided to parents, taking into account evidence-based best practices. The purpose of our survey was to explore whether any legally binding provisions, guidelines or recommendations existed pertaining to the provision of information about NBS to parents across Europe. Questions were designed to determine the regulatory process of when, by whom and how parents should be informed about screening. Twenty-seven countries participated in the survey. The results indicated that most countries had some sort of legal framework or guidelines for the provision of information to parents. However, only 37% indicated that the provision of information was required prenatally. The majority of countries were verbally informing parents with the aid of written materials postnatally, just prior to sample collection. Information was provided by a neonatologist, midwife or nurse. A website dedicated to NBS was available for 67% of countries and 89% had written materials about NBS for parents. The survey showed that there is a lack of harmonisation among European countries in the provision of information about NBS and emphasised the need for more comprehensive guidelines at the European level.
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http://dx.doi.org/10.1038/s41431-020-00716-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7853088PMC
January 2021

Point of view of the Italians pediatric scientific societies about the pediatric care during the COVID-19 lockdown: what has changed and future prospects for restarting.

Ital J Pediatr 2020 Oct 2;46(1):142. Epub 2020 Oct 2.

Federazione Italiana delle Associazioni e Società Scientifiche dell'Area Pediatrica e Società Italiana di Emergenza Urgenza Pediatrica, Rome, Italy.

Background: The coronavirus disease 2019 (COVID-19) is currently rare in children and they seem to have a milder disease course and better prognosis than adults. However, SARS-Cov-2 pandemic has indirectly caused problems in pediatric medical assistance. In view of this we wanted to draw a picture of what happened during health emergency and analyze future prospects for restarting.

Methods: We involved the Italian pediatric scientific societies institutionally collected in the Italian Federation of Associations and Scientific Societies of the Pediatric Area (FIARPED); We sent a questionnaire to all scientific societies about the pediatric care activity during the COVID-19 emergency and future perspectives for the phase of post-containment.

Results: The analysis of the questionnaires showed significant decrease of:admission, outpatient visits and specialist consultancy activities during the COVID-19 emergency, primarily linked to the fear of infection. Instead it was increased the serious degree of diseases admitted. Most of scientific societies maintained the relationship with chronic patients through some form of telemedicine, reporting a strong positive opinion about this modality. Finally showed the need to give life a new approach for hospitalizations and outpatient visits through a greater use of telemedicine, educational programs on families and a more decisive role of family pediatricians.

Conclusions: Our study highlighted many aspects that can be improved in pediatric care. We think that It will be necessary a new shared strategy to improve the management and continuity of care for pediatric patients, primarily developing a network of collaboration between families, family pediatrician and hospitals and by enhancing the use of new methods of telecommunications.
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http://dx.doi.org/10.1186/s13052-020-00907-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531060PMC
October 2020

A new strategy implementing mass spectrometry in the diagnosis of congenital disorders of N-glycosylation (CDG).

Clin Chem Lab Med 2020 Aug 10;59(1):165-171. Epub 2020 Aug 10.

Newborn Screening, Biochemistry and Pharmacology Laboratory, Pediatric Neurology Unit and Laboratories, Meyer Children's University Hospital, Florence, Italy.

Objectives: Congenital disorders of N-glycosylation (CDG) are a large group of rare metabolic disorders caused by defects in the most common post-translational modification of proteins. CDGs are often difficult to diagnose as they are manifested with non-specific symptoms and signs. Analysis of serum transferrin (TRF) isoforms, as the classical procedure used to identify a CDG patient, enables to predict pathological steps in the N-linked glycosylation process.

Methods: We devised a new strategy based on liquid chromatography-mass spectrometry (LC-MS) for the analysis of TRF isoforms by combining a simple and fast sample preparation with a specific chromatographic cleanup/separation step followed by mass-spectrometric measurement. Single TRF isoform masses were obtained through reconstruction of multiply charged electrospray data collected by quadrupole-MS technology. Hereby, we report the first analyzed serum samples obtained from 20 CDG patients and 100 controls.

Results: The ratio of desialylated isoforms to total TRF was calculated for patients and controls. CDG-Type I patients showed higher amounts of bi-sialo isoform (range: 6.7-29.6%) compared to controls (<5.5%, mean percentage 3.9%). CDG-Type II pattern showed an increased peak of tri-sialo isoforms. The mean percentage of tri-sialo-TRF was 9.3% (range: 2.9-12.9%) in controls, which was lower than that obtained from two patients with COG5-CDG and MAN1B1-CDG (18.5 and 24.5%). Intraday and between-day imprecisions were less than 9 and 16%, respectively, for bi-sialo- and less than 3 and 6% for tri-sialo-TRF.

Conclusions: This LC-MS-based approach provides a simple, sensitive and fast analytical tool for characterizing CDG disorders in a routine clinical biochemistry while improving diagnostic accuracy and speeding clinical decision-making.
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http://dx.doi.org/10.1515/cclm-2020-0650DOI Listing
August 2020

Sweat chloride assay by inductively coupled plasma mass spectrometry: a confirmation test for cystic fibrosis diagnosis.

Anal Bioanal Chem 2020 Oct 21;412(25):6909-6916. Epub 2020 Jul 21.

Department of Surgical, Medical and Molecular Pathology and Critical Care Medicine, University of Pisa, Pisa, Italy.

The current guidelines for sweat chloride analysis identify the procedures for sweat collection, but not for chloride assay, which is usually performed by methods originally not aiming at the low concentrations of chloride found in sweat. To overcome this limitation, we set up, characterized, and adopted an original inductively coupled plasma mass spectrometry (ICP-MS) method for sweat chloride determination, which was designed for its easy use in a clinical laboratory. The method was linear in the range 8.5E-3 to 272.0E-3 mM, precision exhibited a relative standard deviation < 6%, and accuracy was in the range 99.7-103.8%. Limit of blank, limit of detection, and limit of quantitation were 2.1 mM, 3.2 mM, and 7.0 mM, respectively, which correspond to real concentrations injected into the mass spectrometer of 3.9E-3 mM for LOD and 8.5E-3 mM for LOQ. At first, the method was tested on 50 healthy volunteers who exhibited a mean chloride concentration of 15.7 mM (25-75th percentile 10.1-19.3 mM, range 2.8-37.4 mM); then, it was used to investigate two patients with suspected cystic fibrosis, who exhibited sweat chloride values of 65.6 mM and 81.2 mM, respectively. Moreover, the method was cross-validated by assaying 50 samples with chloride concentration values in the range 10-131 mM, by both ICP-MS and coulometric titration, which is the technology officially used in Tuscany for cystic fibrosis newborn screening. The reference analytical performances and the relatively low cost of ICP-MS, accompanied by the advantageous cost of a single sweat chloride assay, make this technology the best candidate to provide a top reference method for the quantification of chloride in sweat. The method that we propose was optimized and validated for sweat samples ≥ 75 mg, which is the minimum amount requested by the international protocols. However, the method sensitivity and, in addition, the possibility to reduce the sample dilution factor, make possible the quantification of chloride even in samples weighting < 75 mg that are discarded according to the current guidelines. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-020-02821-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495987PMC
October 2020

β3-Adrenoreceptor Blockade Reduces Hypoxic Myeloid Leukemic Cells Survival and Chemoresistance.

Int J Mol Sci 2020 Jun 12;21(12). Epub 2020 Jun 12.

Division of Pediatric Oncology/Hematology, Meyer University Children's Hospital, 50139 Florence, Italy.

β-adrenergic signaling is known to be involved in cancer progression; in particular, beta3-adrenoreceptor (β3-AR) is associated with different tumor conditions. Currently, there are few data concerning β3-AR in myeloid malignancies. Here, we evaluated β3-AR in myeloid leukemia cell lines and the effect of β3-AR antagonist SR59230A. In addition, we investigated the potential role of β3-AR blockade in doxorubicin resistance. Using flow cytometry, we assessed cell death in different in vitro myeloid leukemia cell lines (K562, KCL22, HEL, HL60) treated with SR59230A in hypoxia and normoxia; furthermore, we analyzed β3-AR expression. We used healthy bone marrow cells (BMCs), peripheral blood mononuclear cells (PBMCs) and cord blood as control samples. Finally, we evaluated the effect of SR59230A plus doxorubicin on K562 and K562/DOX cell lines; K562/DOX cells are resistant to doxorubicin and show P-glycoprotein (P-gp) overexpression. We found that SR59230A increased cancer cell lines apoptosis especially in hypoxia, resulting in selective activity for cancer cells; moreover, β3-AR expression was higher in malignancies, particularly under hypoxic condition. Finally, we observed that SR59230A plus doxorubicin increased doxorubicin resistance reversion mainly in hypoxia, probably acting on P-gp. Together, these data point to β3-AR as a new target and β3-AR blockade as a potential approach in myeloid leukemias.
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http://dx.doi.org/10.3390/ijms21124210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352890PMC
June 2020

Inter-laboratory analytical improvement of succinylacetone and nitisinone quantification from dried blood spot samples.

JIMD Rep 2020 May 4;53(1):90-102. Epub 2020 Apr 4.

Sobi Stockholm Sweden.

Background: Nitisinone is used to treat hereditary tyrosinemia type 1 (HT-1) by preventing accumulation of toxic metabolites, including succinylacetone (SA). Accurate quantification of SA during newborn screening is essential, as is quantification of both SA and nitisinone for disease monitoring and optimization of treatment. Analysis of dried blood spots (DBS) rather than plasma samples is a convenient method, but interlaboratory differences and comparability of DBS to serum/plasma may be issues to consider.

Methods: Eight laboratories with experience in newborn screening and/or monitoring of patients with HT-1 across Europe participated in this study to assess variability and improve SA and nitisinone concentration measurements from DBS by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Quantification of nitisinone from both DBS and plasma was performed to assess sample comparability. In addition, efforts to harmonize laboratory procedures of SA and nitisinone quantifications during 5 rounds of analysis are described.

Results: Nitisinone levels measured from DBS and plasma strongly correlated ( = 0.93). Due to partitioning of nitisinone to the plasma, levels were higher in plasma by a factor of 2.34. In the initial assessment of laboratory performance, all had linear calibrations of SA and nitisinone although there was large inter-laboratory variability in actual concentration measurements. Subsequent analytical rounds demonstrated markedly improved spread and precision over previous rounds, an outcome confirmed in a final re-test round.

Conclusion: The study provides guidance for the determination of nitisinone and SA from DBS and the interpretation of results in the clinic. Inter-laboratory analytical harmonization was demonstrated through calibration improvements.
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http://dx.doi.org/10.1002/jmd2.12112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7203657PMC
May 2020

Leukocyte and Dried Blood Spot Arylsulfatase A Assay by Tandem Mass Spectrometry.

Anal Chem 2020 05 16;92(9):6341-6348. Epub 2020 Apr 16.

Department of Chemistry, University of Washington, Seattle, Washington 98195, United States.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed to measure arylsulfatase A (ARSA) activity in leukocytes and dried blood spots (DBS) using deuterated natural sulfatide substrate. These new assays were highly specific and sensitive. Patients with metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD) displayed a clear deficit in the enzymatic activity and could be completely distinguished from normal controls. The leukocyte assay reported here will be important for diagnosing MLD and MSD patients and for monitoring the efficacy of therapeutic treatments. ARSA activity was measured in DBS for the first time without an antibody. This new ARSA DBS assay can serve as a second-tier test following the sulfatide measurement in DBS for newborn screening of MLD. This leads to an elimination of most of the false positives identified by the sulfatide assay.
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http://dx.doi.org/10.1021/acs.analchem.9b05274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7203001PMC
May 2020

Propranolol 0.2% Eye Micro-Drops for Retinopathy of Prematurity: A Prospective Phase IIB Study.

Front Pediatr 2019 7;7:180. Epub 2019 May 7.

Neonatal Intensive Care Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

Oral propranolol reduces retinopathy of prematurity (ROP) progression, although not safely. Propranolol 0.1% eye micro-drops administered to newborns with stage 2 ROP are well-tolerated, but not sufficiently effective. A multi-center open-label trial was conducted to assess the safety and efficacy of propranolol 0.2% eye micro-drops in newborns with stage 1 ROP. The progression of the disease was evaluated with serial ophthalmologic examinations. Hemodynamic, respiratory, biochemical parameters, and propranolol plasma levels were monitored. Demographic and perinatal characteristics, co-morbidities and co-intervention incidences, together with ROP progression, were compared with a historical control group in the same centers participating in the trial. Ninety-eight newborns were enrolled and compared with the historical control group. Populations were not perfectly homogeneous (as demonstrated by the differences in the Apgar score and the different incidence rate in surfactant administration and oxygen exposure). The progression to ROP stage 2 or 3 plus was significantly lower than the incidence expected on the basis of historical data (Risk Ratio 0.521, 95% CI 0.297- 0.916). No adverse effects related to propranolol were observed and the mean propranolol plasma level was significantly lower than the safety cut-off of 20 ng/mL. Unexpectedly, three newborns treated with oral propranolol before the appearance of ROP, showed a ROP that was unresponsive to propranolol eye micro-drops and required laser photocoagulation treatment. Propranolol 0.2% eye micro-drops were well-tolerated and appeared to reduce the ROP progression expected on the basis of a comparison with a historical control group. Propranolol administered too early appears to favor a more aggressive ROP, suggesting that a β-adrenoreceptor blockade is only useful during the proliferative phase. Further randomized placebo-controlled trials are required to confirm the current results.   The trial was registered at ClinicalTrials.gov with Identifier NCT02504944 and with EudraCT Number 2014-005472-29.
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http://dx.doi.org/10.3389/fped.2019.00180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514240PMC
May 2019

Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood.

EBioMedicine 2019 May 29;43:114-126. Epub 2019 Apr 29.

Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9, Trento 38123, Italy. Electronic address:

Background: Extracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice.

Methods: We designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility.

Findings: From plasma of 46 healthy donors, we systematically recovered small EV (~250 nm of mean diameter; ~3 × 10/ml) and large EV (~560 nm of mean diameter; ~5 × 10/ml) lineages ranging from 50 to 700 nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, we detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, we identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies.

Interpretation: We propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies. FUND: Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Università e Ricerca (Miur).
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http://dx.doi.org/10.1016/j.ebiom.2019.04.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558028PMC
May 2019

β -Adrenoceptor as a potential immuno-suppressor agent in melanoma.

Br J Pharmacol 2019 07 9;176(14):2509-2524. Epub 2019 May 9.

Neonatal Intensive Care Unit, Medical Surgical Fetal-Neonatal Department, Meyer University Children's Hospital, Florence, Italy.

Background And Purpose: Stress-related catecholamines have a role in cancer and β-adrenoceptors; specifically, β -adrenoceptors have been identified as new targets in treating melanoma. Recently, β -adrenoceptors have shown a pleiotropic effect on melanoma micro-environment leading to cancer progression. However, the mechanisms by which β -adrenoceptors promote this progression remain poorly understood. Catecholamines affect the immune system by modulating several factors that can alter immune cell sub-population homeostasis. Understanding the mechanisms of cancer immune-tolerance is one of the most intriguing challenges in modern research. This study investigates the potential role of β -adrenoceptors in immune-tolerance regulation.

Experimental Approach: A mouse model of melanoma in which syngeneic B16-F10 cells were injected in C57BL-6 mice was used to evaluate the effect of β-adrenoceptor blockade on the number and activity of immune cell sub-populations (Treg, NK, CD8, MDSC, macrophages, and neutrophils). Pharmacological and molecular approaches with β-blockers (propranolol and SR59230A) and specific β-adrenoceptor siRNAs targeting β - or β -adrenoceptors were used.

Key Results: Only β -, but not β -adrenoceptors, were up-regulated under hypoxia in peripheral blood mononuclear cells and selectively expressed in immune cell sub-populations including Treg, MDSC, and NK. SR59230A and β -adrenoceptor siRNAs increased NK and CD8 number and cytotoxicity, while they attenuated Treg and MDSC sub-populations in the tumour mass, blood, and spleen. SR59230A and β -adrenoceptor siRNAs increased the ratio of M1/M2 macrophages and N1 granulocytes.

Conclusions And Implications: Our data suggest that β -adrenoceptors are involved in immune-tolerance, which opens the way for new strategic therapies to overcome melanoma growth.

Linked Articles: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.
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http://dx.doi.org/10.1111/bph.14660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592854PMC
July 2019

Newborn screening for homocystinurias: Recent recommendations versus current practice.

J Inherit Metab Dis 2019 01;42(1):128-139

Austrian Newborn Screening, Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria.

Purpose: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations.

Methods: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres.

Results: NBS programmes, algorithms and decision limits varied considerably. Only nine centres used the recommended second-tier marker total homocysteine (tHcy). The median decision limits of all centres were ≥ 2.35 for high and ≤ 0.44 MoM for low methionine, ≥ 1.95 for high and ≤ 0.47 MoM for low methionine/phenylalanine, ≥ 2.54 for high propionylcarnitine and ≥ 2.78 MoM for propionylcarnitine/acetylcarnitine. These decision limits alone had a 100%, 100%, 86% and 84% sensitivity for the detection of CBSD, MATI/IIID, iRMD and cRMD, respectively, but failed to detect six individuals with cRMD. To enhance sensitivity and decrease second-tier testing costs, we further adapted these decision limits using the data of 15 000 healthy newborns.

Conclusions: Due to the favorable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, for example, birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.
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http://dx.doi.org/10.1002/jimd.12034DOI Listing
January 2019

Data in support for the measurement of heparan sulfate and dermatan sulfate by LC-MS/MS analysis.

Data Brief 2018 Dec 26;21:2398-2404. Epub 2018 Nov 26.

Newborn Screening, Biochemistry and Pharmacology Laboratory, Pediatric Neurology Unit and Laboratories, Meyer Children׳s University Hospital, Florence, Italy.

This article provides supplementary data for the paper "LC-MS/MS method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by butanolysis derivatization" (Forni et al., 2018). Several parameters were tested to optimize sample preparation by butanolysis in order to carry out simultaneous quantifications of HS and DS by tandem mass spectrometry. Here we describe step-by-step instructions to perform HS and DS analysis in urine samples using external calibration curves of standards of known concentration. Sample are quantified by interpolation from the calibration curve and reported in µg/mL. Then, HS and DS are normalized to creatinine concentration and reported as mg/g uCr.
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http://dx.doi.org/10.1016/j.dib.2018.11.100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282625PMC
December 2018

Oleuropein, the Main Polyphenol of Leaf Extract, Has an Anti-Cancer Effect on Human BRAF Melanoma Cells and Potentiates the Cytotoxicity of Current Chemotherapies.

Nutrients 2018 Dec 8;10(12). Epub 2018 Dec 8.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, 50134 Florence, Italy.

Oleuropein (Ole), a secoiridoid glucoside present in leaves, gained scientific interest thanks to its several biological properties, including the anticancer one. We verified whether Ole might potentiate the cytotoxicity of conventional drugs used to treat melanoma, disclosing a potentially new therapeutic strategy. We tested the cytotoxic action of Ole alone or in combination with chemotherapeutics on A375 human melanoma cells. We found that Ole was able, at a dose of 500 µM, to stimulate apoptosis, while at a non-toxic dose of 250 µM, it affected cell proliferation and induced the downregulation of the pAKT/pS6 pathway. A dose of 250 µM Ole did not potentiate the effect of Vemurafenib (PLX4032), but it succeeded in increasing the cytotoxic effect of Dacarbazine (DTIC). The major effect was found in the association between Ole and Everolimus (RAD001), also on PLX4032-resistant BRAF melanoma cells, which possibly cooperate in the inhibition of the pAKT/pS6 pathway. Of interest, an olive leaf extract enriched in equimolar Ole was more effective and able to further improve DTIC and RAD001 efficacy on BRAF melanoma cells with respect to Ole alone. Therefore, Ole represents a natural product able to potentiate a wide array of chemotherapeutics against BRAF melanoma cells affecting the pAKT/pS6 pathway.
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http://dx.doi.org/10.3390/nu10121950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316801PMC
December 2018

Development of a fast LC-MS/MS protocol for combined measurement of six LSDs on dried blood spot in a newborn screening program.

J Pharm Biomed Anal 2019 Feb 3;165:135-140. Epub 2018 Dec 3.

Newborn Screening, Biochemistry and Pharmacology Laboratory, Pediatric Neurology, Unit and Laboratories, Meyer Children's University Hospital, Florence, Italy; Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy. Electronic address:

New treatment options and improved strategies for Lysosomal Storage Disorders (LSDs) diagnosis on dried blood spot (DBS) have led to the development of several pilot newborn screening programs. Building on a previously published protocol, we devised a new 6-plex assay based on a single DBS punch incubated into a buffer containing a combination of substrates (GAA, GLA, ASM, GALC, ABG and IDUA). This new protocol incorporates a new trapping and clean-up procedure using perfusion chromatography connected on-line with an analytical column for analyte separation, after enzymatic reaction. Results are available after 4.5 min. Several incubation times were tested in order to reduce sample preparation times and to improve accuracy and reproducibility, also regarding the quenching of the reaction within the time window of linear product accumulation. The collected data demonstrate that an incubation time of 4 h is enough to achieve good reaction efficiency without any impact on sensitivity. The method proved versatile and robust for various instrument configurations. The fast sample preparation and running times allow a high sample throughput; an advantage in newborn screening procedures. This method can also be used for diagnostic purposes, allowing a rapid diagnosis in a few hours.
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http://dx.doi.org/10.1016/j.jpba.2018.12.002DOI Listing
February 2019

LC-MS/MS method for simultaneous quantification of heparan sulfate and dermatan sulfate in urine by butanolysis derivatization.

Clin Chim Acta 2019 Jan 2;488:98-103. Epub 2018 Nov 2.

Newborn Screening, Biochemistry and Pharmacology Laboratory, Paediatric Neurology, Unit and Laboratories, Meyer Children's University Hospital, Florence, Italy; Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy. Electronic address:

Mucopolysaccharidoses are a group of lysosomal storage disorders (LSDs) characterized by the accumulation of glycosaminoglycans (GAGs). Recently, LC-MS/MS has been widely applied in GAGs analysis combined with different sample preparations for cleaving GAGs to disaccharide units. The aim of the present is paper is to present a new method for the simultaneous quantification of urinary dermatan sulfate (DS) and heparan sulfate (HS) by LC-MS/MS, after butanolysis reaction. Chromatographic separation was achieved with a gradient of acetonitrile and water in 0.1% formic acid on a Kinetex Biphenyl analytical column in 21 min. Calibration curves ranging from 0.78 to 50 μg/mL for HS and from 1.56 to 100 μg/mL for DS were prepared and the coefficient of determination (r) was higher than 0.99 for both analytes. Intra-day and inter-day imprecisions and the bias for both compounds were <10.0%. Up to now, most analytical procedures for quantifying GAGs have not had a high level of reproducibility among laboratories, despite the availability of various techniques. The adoption of a new protocol incorporating the methods outlined in this paper could significantly improve the quality and reproducibility of MS results. A procedure using simple steps for preparing samples and reagents that are easily available on the market could promote the standardization of analytical procedures and increase the use of these measurements in clinical practice.
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http://dx.doi.org/10.1016/j.cca.2018.11.001DOI Listing
January 2019

Haematohidrosis treated with propranolol: a case report.

Arch Dis Child 2019 02 6;104(2):171. Epub 2018 Feb 6.

Neonatal Intensive Care Unit, Medical Surgical Feto-Neonatal Department, Anna Meyer Children's University Hospital, University of Florence, Florence, Italy.

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http://dx.doi.org/10.1136/archdischild-2017-314170DOI Listing
February 2019

Serum creatinine during physiological perinatal dehydration may estimate individual nephron endowment.

Eur J Pediatr 2018 Sep 1;177(9):1383-1388. Epub 2018 Feb 1.

Neonatal Intensive Care Unit, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico, University of Milan, via Commenda 12, 20122, Milan, Italy.

It is well known that the nephron endowment of healthy subjects is highly variable and that individual nephron mass has potentially important implications both in health and disease. However, nephron count is technically impossible in living subjects. Based on the observation of an increase in serum creatinine (sCr) in otherwise healthy newborns with solitary kidney during the physiological perinatal dehydration, we hypothesized that perinatal sCr might be helpful in identifying healthy subjects with a reduced nephron mass. In the framework of a study on blood pressure in babies (NeoNeph), sCr of normal Caucasian neonates was determined 48-96 h after birth and their association with a family history of arterial hypertension (AH) was analyzed. SCr was determined in 182 normal newborns (90 males) at a mean of 61 ± 8 h after birth (range 46-82). Newborns with paternal AH had a higher mean sCr (0.97 + 0.28 mg/dL) then newborns without paternal AH (0.73 + 0.28 mg/dL; p = 0.006). No differences in mean sCr were found in relation with mother or grandparent's history of AH.

Conclusion: The association between parental AH and high sCr during perinatal dehydration supports the hypothesis that the latter is a promising tool for identifying normal subjects with a reduced nephron mass with potential important implications in prevention and in understanding the individual outcome of renal and extrarenal diseases (including AH). What is Known: • Nephron endowment of healthy subjects is highly variable and individual nephron mass has potentially important implications both in health and disease however nephron count is not feasible in living subjects. What is New: • Serum creatinine during perinatal dehydration is a possible biomarker for identifying normal subjects with a reduced nephron mass.
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http://dx.doi.org/10.1007/s00431-018-3087-0DOI Listing
September 2018

Late-Onset N-Acetylglutamate Synthase Deficiency: Report of a Paradigmatic Adult Case Presenting with Headaches and Review of the Literature.

Int J Mol Sci 2018 Jan 24;19(2). Epub 2018 Jan 24.

Molecular and Cell Biology Laboratory of Neurometabolic Diseases, Neuroscience Department, Meyer Children's Hospital, 50139 Florence, Italy.

-acetylglutamate synthase deficiency (NAGSD) is an extremely rare urea cycle disorder (UCD) with few adult cases so far described. Diagnosis of late-onset presentations is difficult and delayed treatment may increase the risk of severe hyperammonemia. We describe a 52-year-old woman with recurrent headaches who experienced an acute onset of NAGSD. As very few papers focus on headaches in UCDs, we also report a literature review of types and pathophysiologic mechanisms of UCD-related headaches. In our case, headaches had been present since puberty (3-4 days a week) and were often accompanied by nausea, vomiting, or behavioural changes. Despite three previous episodes of altered consciousness, ammonia was measured for the first time at 52 years and levels were increased. Identification of the new homozygous c.344C>T (p.Ala115Val) variant allowed the definite diagnosis of NAGSD. Bioinformatic analysis suggested that an order/disorder alteration of the mutated form could affect the arginine-binding site, resulting in poor enzyme activation and late-onset presentation. After optimized treatment for NAGSD, ammonia and amino acid levels were constantly normal and prevented other headache bouts. The manuscript underlies that headache may be the presenting symptom of UCDs and provides clues for the rapid diagnosis and treatment of late-onset NAGSD.
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http://dx.doi.org/10.3390/ijms19020345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855567PMC
January 2018

Vacuolated PAS-positive lymphocytes as an hallmark of Pompe disease and other myopathies related to impaired autophagy.

J Cell Physiol 2018 08 22;233(8):5829-5837. Epub 2018 Feb 22.

2nd Division of Neurology, Department of Medicine, Surgery, Neurology, Metabolic and Aging Science, Reference Center for Neurological and Neuromuscular Rare Disease & Interuniversity Center for Research in Neurosciences, University of Campania "Luigi Vanvitelli", Naples, Italy.

Autosomal recessive Pompe disease is a lysosomal disorder caused by mutations of the acid-α-glucosidase (GAA) gene. Deficiency of GAA enzyme leads to glycogen accumulation and autophagy impairment in cardiac and skeletal muscles, but also in lymphocytes. Since an effective therapy is available, a rapid, sensitive, and specific test is crucial to early identify affected subjects. Number of lymphocytes containing PAS-positive vacuoles was evaluated on blood films from 72 consecutive adult patients with hyperckemia and/or muscle weakness, 13 genetically confirmed late-onset-Pompe-disease (LOPD) and 13 of their offspring. GAA activity, measured on dried blood spot (DBS) in all patients inversely correlated with number of PAS-positive lymphocytes. More than 4 PAS-positive lymphocytes were found in 11 out of the 72 patients (6 new diagnosis of LOPD, 3 different glycogen storage myopathies, 1 glucose-6-phosphate dehydrogenase deficiency, 1 caveolinopathy), in all 13 LOPD patients and in the 13 LOPD offspring. These latter resulted to have all a single GAA mutation but low GAA levels. Immunostaining with the autophagy markers LC3 and p62 confirmed the autophagic nature of lymphocytes vacuoles. ROC curve assessment of PAS-positive lymphocytes disclosed 100% of sensitivity and 94% of specificity in recognizing both compound heterozygous and heterozygous GAA carriers. The other myopathies with more than 4 PAS-positive lymphocytes appeared to be all related to impaired autophagy, which seems to be responsible of PAS-positive vacuolated lymphocytes formation. Quantification of PAS-positive lymphocytes in blood films is useful to identify autophagic vacuolar myopathies and should be routinely used as first level test for Pompe disease.
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http://dx.doi.org/10.1002/jcp.26365DOI Listing
August 2018