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European CF Society Neonatal Screening Working Group (ECFS NSWG): Sabine Renner , Max Zeyda , Elke de Wachter , Luc Regal , Felix Votava, Andrea Holubova, Marianne Skov , Tessa Morgan, Paul Bregeaut , Loretta O'Grady, Ines Bucci , Stefano Pantano , Simonetta Simonetti, Domenica De Venuto, Donatello Salvatore, Nicola Perrotti, Mimma Caloiero, Giuseppe Castaldo, Antonella Tosco, Francesca Righetti, Giovanna Pisi, Fiorella Battistini, Antonio Angeloni, Giuseppe Cimino, Giovanni Fiocchi, Antonella Angiolillo, Michela Cassanello, Luisella Alberti, Laura E Claut, Raffaele Badolato, Enza Pavanello, Benedetta Fabrizzi, Elisabetta Bignamini, Anna Cardillo, Mariangela Lombardo, Letizia Cocciadiferro, Lisa Termini, Daniela Dolce, Vito Terlizzi, Anna Tamanini, Francesca Pauro, Giancarlo la Marca, Elina Aleksejeva, Dita Gaidule-Logina, Stoika Fustik, Violeta Anastasovska, Marelle Bouva, Alastair Reid, Jennifer Cundick, Emma Lundman, Egil Bakkeheim, Katarzyna Zybert, Mariusz Oltarzewski, Laura Vilarinho, Victoria Sherman, Elena Kondratyeva, Sarah Smith, Gordana Vilotijevic Dautovic, Maria Knapkova, Zuzana Mydlova, Rosa Mª López, Valle Velasco, Felicitas Díaz Flores, Cristóbal Colón Mejeras, Eva Sl Pedersen, Ugur Ozcelik, Bulent Karadag, Halyna Makukh, Moat Stuart. European CF Society Neonatal Screening Working Group (ECFS NSWG): INSA - Laura Vilarinho Background: The aim of this study was to record the current status of newborn bloodspot screening (NBS) for CF across Europe and assess performance. Methods: Survey of representatives of NBS for CF programmes across Europe. Performance was assessed through a framework developed in a previous exercise. Results: In 2022, we identified 22 national and 34 regional programmes in Europe. Barriers to establishing NBS included cost and political inertia. Performance was assessed from 2019 data reported by 21 national and 21 regional programmes. All programmes employed different protocols, with IRT-DNA the most common strategy. Six national and 11 regional programmes did not use DNA analysis. Conclusions: Integrating DNA analysis into the NBS protocol improves PPV, but at the expense of increased carrier and CFSPID recognition. Some programmes employ strategies to mitigate these outcomes. Programmes should constantly strive to improve performance but large datasets are needed to assess outcomes reliably. Highlights: In 2022, newborn bloodspot screening (NBS) for CF is undertaken in 30 European countries, 26 of them are national programmes; Some programmes are still not achieving ECFS standards. Compared to 2014, there is an improvement in sensitivity but a deterioration in achieving a sufficient PPV; There continues to be a wide variety of approaches, but the majority of national programmes are now using DNA analysis as a 2nd tier; This survey demonstrates areas of good practice, but there is considerable scope for improvement in the quality of NBS for CF across Europe; The framework of the 20 parameters to calculate the 8 key outcomes should be part of any annual report of a CF NBS programme, and thus improve future surveys. The survey was funded by the European CF Society info:eu-repo/semantics/publishedVersion

The early diagnosis of and intervention in vitamin B12 deficiency in exclusively breastfed infants by mothers with low vitamin B12 is crucial in preventing possible irreversible neurologic damage, megaloblastic anemia, and failure to thrive. We assess the usefulness of the early detection of asymptomatic B12 deficiency related to acquired conditions and highlight the importance of monitoring serum vitamin B12 levels during pregnancy. We describe demographic, clinical, dietary, and biochemical data, including the evolution of a vitamin B12 deficiency's functional biomarkers. We enrolled 12 newborns (5 males) with an age range of 1-2 months old that were exclusively breastfed and asymptomatic. These cases were referred to our metabolic unit due to alterations in expanded newborn screening: high levels of methylmalonic acid and/or total homocysteine (tHcy). All mothers were under a vegetarian diet except three who had abnormal B12 absorption, and all presented low or borderline serum B12 level and high plasma levels of tHcy. Supplementation with oral vitB12 re-established the metabolic homeostasis of the mothers. In infants, therapy with an intramuscular injection of 1.0 mg hydroxocobalamin led to the rapid normalization of the metabolic pattern, and a healthy outcome was observed. Acquired B12 deficiency should be ruled out before proceeding in a differential diagnosis of cobalamin metabolism deficits, methylmalonic acidemia, and homocystinuria. info:eu-repo/semantics/publishedVersion

Exercise training provides several cardiovascular benefits in both physiological and pathological conditions; however, its use as a therapeutic tool for pulmonary arterial hypertension (PAH) has been poorly explored. This study aimed to extend the comprehension of the cardioprotective effects of exercise training in the set of PAH focusing on the metabolic changes promoted by exercise in the right ventricle (RV). The monocrotaline animal model of PAH was used and male Wistar rats were submitted to two weeks of treadmill exercise training (5 days/week, 60 min/day, 25 m/min) following disease establishment. Trained rats showed an improved diastolic function (lower end-diastolic pressure and tau) despite the presence of cardiac overload (increased peak systolic pressure, end-diastolic pressure and arterial elastance). This enhanced hemodynamic response was paralleled by an increased uptake of glucose to cardiomyocytes through glucose transporter type 4 (GLUT4) followed by increased lactate dehydrogenase (LDH) activity. Exercise did not reverse the decrease of fatty acid oxidation related to PAH but increased the content of the transcription factors peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and peroxisome proliferator-activated receptor gamma (PPAR-γ). Two weeks of exercise did not modulate the changes in amino acid metabolism secondary to PAH. Our work suggests that continuous aerobic exercise of moderate intensity, despite its short-term duration and application in a late stage of the disease, supports the RV response to PAH by promoting a shift in the cardiac metabolic phenotype. Highlights: Two weeks of exercise training improved right ventricle diastolic function in MCT rats; Exercise increased glucose uptake through GLUT4 and its oxidation to lactate; Exercise did not reverse MCT-induced decrease reliance of RV on fatty acid oxidation; Exercise moderately impacted amino acid metabolism in the RV of MCT rats; Metabolic adaptations of trained RV are regulated by PGC-1α and PPAR-γ. This work was supported by “Fundação para a Ciência e a Tecnologia” – FCT, European Union, QREN, FEDER and COMPETE for funding the LAQV-REQUIMTE (UIDB/50006/2020), UnIC (UIDB/IC/00051/2020 and UIDP/00051/2020), CIAFEL (UIDB/00617/2020) and UMIB (UIDB/00215/2020 and UIDP/00215/2020) research units, RISE - Health Research Network-From the Lab to the Community (LA/P/0053/ 2020), ITR - Laboratory for Integrative and Translational Research in Population Health (LA/P/0064/2020) and the research projects DOCnet (NORTE-01-0145-FEDER-000003) and NETDIAMOND (SAICT-PAC/ 0047/2015). Rita Nogueira-Ferreira acknowledges FCT for the research contract CEECIND/03935/2021 under the CEEC Individual 2021. info:eu-repo/semantics/publishedVersion

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies. This research was supported using resources of the Core Facility Cell Imaging and Ultrastructure Research, University of Vienna, a member of the Vienna Life-Science Instruments (VLSI) and the VetCore Facility (Imaging) of the University of Veterinary Medicine Vienna. We acknowledge International Centre for Genomic Medicine in Neuromuscular Diseases. This research was funded in part, by the Wellcome Trust (WT093205MA, WT104033AIA, and the Synaptopathies Strategic Award, 165908). This study was funded by the Medical Research Council (MR/S01165X/1, MR/S005021/1, G0601943), The National Institute for Health Research University College London Hospitals Biomedical Research Centre, Rosetrees Trust, Ataxia UK, Multiple System Atrophy Trust, Brain Research United Kingdom, Sparks Great Ormond Street Hospital Charity, Muscular Dystrophy United Kingdom (MDUK), Muscular Dystrophy Association (MDA USA) and Senior Non-Clinical Fellow ship to A. Spinazzola, (MC_PC_13029). K.N. and S.E.M.M. were supported by the Austrian Science Funds FWF-P29077 and P31471. A. Spinazzola receives support also from The Lily Foun dation and Brain Research UK. R.K. was supported by European Academy of Neurology Research Training Fellowship and Rosetrees Trust PhD Plus award (PhD2022\100042). info:eu-repo/semantics/publishedVersion

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/34981750/ Objectives: Over 1.9 billion people worldwide are living in areas estimated to be iodine insufficient. Strategies for iodine supplementation include campaigns targeting vulnerable groups, such as women in pre-conception, pregnancy and lactation. Portuguese women of childbearing age and pregnant women were shown to be mildly-to-moderately iodine deficient. As a response, in 2013, the National Health Authority (NHA) issued a recommendation that all women considering pregnancy, pregnant or breastfeeding, take a daily supplement of 150-200 μg iodine. This study explored how the iodine supplementation recommendation has been fulfilled among pregnant and lactating women in Portugal, and whether the reported iodine supplements intake impacted on adverse obstetric and neonatal outcomes. Design and methods: Observational retrospective study on pregnant women who delivered or had a fetal loss in the Braga Hospital and had their pregnancies followed in Family Health Units. Results: The use of iodine supplements increased from 25% before the recommendation to 81% after the recommendation. This was mostly due to an increase in the use of supplements containing iodine only. Iodine supplementation was protective for the number of adverse obstetric outcomes (odds ratio (OR) = 0.791, P = 0.018) and for neonatal morbidities (OR = 0.528, P = 0.024) after controlling for relevant confounding variables. Conclusion: The recommendation seems to have succeeded in implementing iodine supplementation during pregnancy. National prospective studies are now needed to evaluate the impact of iodine supplementation on maternal thyroid homeostasis and offspring psychomotor development and on whether the time of the beginning of iodine supplementation (how early during preconception or pregnancy) is relevant to consider. This work has been funded by National funds, through the Foundation for Science and Technology (FCT) – project UIDB/50026/2020 and UIDP/50026/2020 and by the project NORTE-01-0145-FEDER-000039, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Funding agencies did not participate in any part of the research or in data or manuscript preparation info:eu-repo/semantics/publishedVersion

L-2-hydroxiglutaric aciduria (L2HGA) is a rare, childhood-onset, organic aciduria, with characteristic clinical (cerebellar ataxia) and neuroimaging (subcortical leukodystrophy) features. Movement disorders in this condition are usually of hyperkinetic type. Herein is reported the case of two adult siblings with recent L2HGA diagnosis, presenting with dopa-responsive parkinsonism and MRI iron deposition.

Cystinosis is a rare autosomal recessive lysosomal storage disorder leading to end-stage renal disease and many extra-renal complications. Seventeen month old girl with normal development until 14 months, when parents noted polydipsia, polyuria and growth stagnation. Investigations performed in a tertiary setting were compatible with Fanconi Syndrome and X-ray with rickets. The hypothesis of diagnosis of infantile nephropathic cystinosis was raised. The high intra-leukocytic cystine and the genetic study confirmed the disease. Treatment with cysteamine was started. At 21 months, cystine eye crystals appeared and topical cysteamine was added. She is currently 3 years old, with a satisfactory weight and height progression, with a stable kidney disease, without progression of rickets and with asymptomatic eye crystals. A high degree of suspicion for this disease allows an early start of targeted therapy and an adequate follow-up, improving the prognosis of a disease with high morbility. Keywords: Cystinosis; Cysteamine; Fanconi Syndrome; Chronic Kidney Diseases; Growth disorders; Pediatrics, Portuguese Journal of Pediatrics, Vol. 52 No. 4 (2021)

Background: Congenital hypothyroidism (CH) has severe and irreversible neurological consequences for the child if treatment is not readily initiated, which justifies fast diagnosis. Screening of CH is a universal procedure well established in Portugal. It is based on measuring the pituitary thyroid stimulating hormone. The physiology of maturation of the pituitary-thyroid axis is incomplete at delivery in preterm and very-low birth weight babies (VLBW), which raises concerns with respect to the standard timing of CH diagnosis.The Portuguese Neonatal Screening Program recommends testing preterm/VLBW babies at three time points. The purpose of this study was to assess compliance of these guidelines.Methods: Questionnaires were sent to neonatal care units (NCU) and to general practitioner community health units in two time periods.Results: All NCU have written guidelines and the great majority follows the recommendations. Few NCU still postpone the first collection for newborns with protein intake restrictions, some retest new-borns in special circumstances, and a great number still use iodine disinfectants.Conclusions: This study shows that performing the metabolic screening is a well standardized procedure that can still be improved: not delaying the first collection, retesting sick and medicated babies, and avoiding iodine disinfectants.

Case Reports Key Points: - Blood ammonia levels should be measured in every encephalopathy of unknown origin, especially when an intoxication cause is thought; - Late-onset inherited metabolic disorders may only manifest in the presence of triggering environmental factors such as some drugs (e.g. anti-epileptics); an undiagnosed metabolic disorder should be considered in such cases. info:eu-repo/semantics/publishedVersion

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/31497476/ In the era of genomics, the number of genes linked to mitochondrial disease has been quickly growing, producing massive knowledge on mitochondrial biochemistry. LYRM4 gene codifies for ISD11, a small protein (11 kDa) acting as an iron-sulfur cluster, that has been recently confirmed as a disease-causing gene for mitochondrial disorders. We present a 4-year-old girl patient, born from non-consanguineous healthy parents, with two episodes of cardiorespiratory arrest after respiratory viral illness with progressive decreased activity and lethargy, at the age of 2 and 3 years. She was asymptomatic between crisis with regular growth and normal development. During acute events of illness, she had hyperlactacidemia (maximum lactate 5.2 mmol/L) and urinary excretion of ketone bodies and 3-methylglutaconic acid, which are normalized after recovery. A Next Generation Sequence approach with a broad gene panel designed for mitochondrial disorders revealed a novel probably pathogenic variant in homozygosity in the LYRM4 gene [p.Tyr31Cys (c.92A>G)] with Mendelian segregation. Functional studies in the skeletal muscle confirmed a combined deficiency of the mitochondrial respiratory chain (I, II, and IV complexes). To our knowledge, this is the third case of LYRM4 deficiency worldwide and the first with 3-methylglutaconic aciduria, not reported in any Fe-S cluster deficiency. Remarkably, it appears to be no neurological involvement so far, only with life-threating acute crisis triggered by expectably benign autolimited illnesses. Respiratory chain cofactors and chaperones are a new field of knowledge and can play a remarkable effect in system homeostasis. Fundação para a Ciência e a Tecnologia, Grant/Award Number: PTDC/DTP‐PIC/2220/2014; NORTE2020, Grant/Award Number: NORTE‐01‐0246‐FEDER‐000014 info:eu-repo/semantics/publishedVersion

The electron-transfer flavoprotein dehydrogenase gene (ETFDH) encodes the ETF-ubiquinone oxidoreductase (ETF-QO) and has been reported to be the major cause of multiple acyl-CoA dehydrogenase deficiency (MADD). In this study, we present the clinical and molecular diagnostic challenges, at the DNA and RNA levels, involved in establishing the genotype of four MADD patients with novel ETFDH variants: a missense variant, two deep intronic variants and a gross deletion. RNA sequencing allowed the identification of the second causative allele in all studied patients. Simultaneous DNA and RNA investigation can increase the number of MADD patients that can be confirmed following the suggestive data results of an expanded newborn screening program. In clinical practice, accurate identification of pathogenic mutations is fundamental, particularly with regard to diagnostic, prognostic, therapeutic and ethical issues. Our study highlights the importance of RNA studies for a definitive molecular diagnosis of MADD patients, expands the background of ETFDH mutations and will be important in providing an accurate genetic counseling and a prenatal diagnosis for the affected families.

Background The impairment of the hepatic enzyme phenylalanine hydroxylase (PAH) causes elevation of phenylalanine levels in blood and other body fluids resulting in the most common inborn error of amino acid metabolism (phenylketonuria). Persistently high levels of phenylalanine lead to irreversible damage to the nervous system. Therefore, early diagnosis of the affected individuals is important, as it can prevent clinical manifestations of the disease. Methods In this report, the biochemical and genetic findings performed in 223 patients diagnosed through the Portuguese Neonatal Screening Program (PNSP) are presented. Results Overall, the results show that a high overlap exists between different types of variants and phenylalanine levels. Molecular analyses reveal a wide mutational spectrum in our population with a total of 56 previously reported variants, most of them found in compound heterozygosity (74% of the patients). Intragenic polymorphic markers were used to assess the haplotypic structure of mutated chromosomes for the most frequent variants found in homozygosity in our population (p.Ile65Thr, p.Arg158Gln, p.Leu249Phe, p.Arg261Gln, p.Val388Met, and c.1066‐11G>A). Conclusion Our data reveal high heterogeneity at the biochemical and molecular levels and are expected to provide a better understanding of the molecular basis of this disease and to provide clues to elucidate genotype–phenotype correlations., The present study identify and characterize the variants underlying PKU in affected individuals in the Portuguese PKU/HPA cohort. Biochemical data, haplotic analysis and genetic findings performed in 223 patients diagnosed through the Portuguese Neonatal Screening Program (PNSP) is presented. The information obtained will improve the diagnostic applicability of mutational analysis and the capacity to predict the evolution of the disease.

International audience; 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.

Glutaric aciduria type 1 (GA-1) is a rare but treatable inherited disease caused by deficiency of glutaryl-CoA dehydrogenase activity due to GCDH gene mutations. In this study, we report 24 symptomatic GA-1 Brazilian patients, and present their clinical, biochemical, and molecular findings. Patients were diagnosed by high levels of glutaric and/or 3-hydroxyglutaric and glutarylcarnitine. Diagnosis was confirmed by genetic analysis. Most patients had the early-onset severe form of the disease and the main features were neurological deterioration, seizures and dystonia, usually following an episode of metabolic decompensation. Despite the early symptomatology, diagnosis took a long time for most patients. We identified 13 variants in the GCDH gene, four of them were novel: c.91 + 5G > A, c.167T > G, c.257C > T, and c.10A > T. The most common mutation was c.1204C > T (p.R402W). Surprisingly, the second most frequent mutation was the new mutation c.91 + 5G > A (IVS1 ds G-A + 5). Our results allowed a complete characterization of the GA-1 Brazilian patients. Besides, they expand the mutational spectrum of GA-1, with the description of four new mutations. This work reinforces the importance of awareness of GA-1 among doctors in order to allow early diagnosis and treatment in countries like Brazil where the disease has not been included in newborn screening programs. info:eu-repo/semantics/publishedVersion

The Portuguese Newborn Screening Program is a public health program that started in 1979, screening for PKU, being totally supported by public funds. It's a non-mandatory well implemented program that testes about 99.9% of Portuguese newborns. In the actual screening panel encompasses 26 disorders, including inborn errors of metabolism, congenital hypothyroidism and cystic fibrosis. Sample collection is advised to be made at 3rd day of life and treatment begins in average by the 10th day. Every testes are performed in one single national laboratory, that processes about 88,000 samples/year. In the 41 years of program existence, more than 3,800,000 newborns were screened and 2,130 affected newborns detected, reflecting the positive impact of the Program in the population. Future perspectives include the increase of program value in terms of public health by optimizing the screening of the disorders already screened and evaluation the possibility of adding others.El Programa Portugués de Cribado Neonatal es un programa de Salud Pública a nivel nacional, que tuvo su inicio en 1979 con el cribado de la fenilcetonuria y es financiado totalmente por el Estado portugués. Se trata de un programa no obligatorio, con una tasa de cobertura del 99,8%, en el que hoy en día se criban veintiséis enfermedades, incluyendo metabolopatías, hipotiroidismo congénito y fibrosis quística. La toma de muestra se hace al 3er día de vida y el tratamiento de los neonatos afectos empieza en torno al 10º día. Todos los análisis están centralizados en un único laboratorio, que procesa aproximadamente 88.000 muestras al año. En los últimos cuarenta y un años se cribaron más de 3.800.000 neonatos y se detectaron 2.130 niños afectados, lo que es un indicador del impacto del programa en la población. Los desafíos futuros incluyen la búsqueda de nuevas estrategias para incrementar el valor del programa, donde se evalúen nuevas enfermedades a cribar y la optimización del cribado actual.

Lysosomal storage diseases (LSDs) are a heterogeneous group of genetic disorders with variable degrees of severity and a broad phenotypic spectrum, which may overlap with a number of other conditions. While individually rare, as a group LSDs affect a significant number of patients, placing an important burden on affected individuals and their families but also on national health care systems worldwide. Here, we present our results on the use of an in-house customized next-generation sequencing (NGS) panel of genes related to lysosome function as a first-line molecular test for the diagnosis of LSDs. Ultimately, our goal is to provide a fast and effective tool to screen for virtually all LSDs in a single run, thus contributing to decrease the diagnostic odyssey, accelerating the time to diagnosis. Our study enrolled a group of 23 patients with variable degrees of clinical and/or biochemical suspicion of LSD. Briefly, NGS analysis data workflow, followed by segregation analysis allowed the characterization of approximately 41% of the analyzed patients and the identification of 10 different pathogenic variants, underlying nine LSDs. Importantly, four of those variants were novel, and, when applicable, their effect over protein structure was evaluated through in silico analysis. One of the novel pathogenic variants was identified in the GM2A gene, which is associated with an ultra-rare (or misdiagnosed) LSD, the AB variant of GM2 Gangliosidosis. Overall, this case series highlights not only the major advantages of NGS-based diagnostic approaches but also, to some extent, its limitations ultimately promoting a reflection on the role of targeted panels as a primary tool for the prompt characterization of LSD patients. This research was partially supported by NORTE2020 (NORTE-01-0246-FEDER-000014 DESVENDAR “DEScobrir, VENcer as Doenças Raras”, FCT (Fundação para a Ciência e a Tecnologia—MCTES, Portugal): project PTDC/BBB-BMD/6301/2014 and UIDB/00211/2020. info:eu-repo/semantics/publishedVersion

Background: Iodine deficiency is the most common cause of preventable brain harm and cognitive impairment in children. Portuguese women of childbearing age, pregnant women and their progeny were shown to have inadequate iodine intake. Consequently, the Portuguese Health Authorities have recommended a daily supplementation with 150-200 µg iodine in preconception, pregnancy, and lactation. The IodineMinho study intends to evaluate whether (i) this recommendation impacted on the prevalence of iodine deficiency in pregnant women from the Minho region of Portugal, (ii) the time of initiation of iodine supplementation (if any) influences the serum levels of thyroid hormones at several intervals during pregnancy and (iii) there are serum thyroid-hormone parameters in the 1st trimester of pregnancy that predict psychomotor development of the child at 18 months of age. Methods: Most Portuguese women are followed throughout pregnancy in community Family Health Units, where family physicians may choose to follow the National recommendation or other, concerning iodine sufficiency. This study will recruit women (N = 304) who intend to become pregnant or are already pregnant from 10 representative Units. Physician's approach and prescriptions, sociodemographic, nutrition and clinical information will be obtained at baseline and throughout pregnancy. To evaluate endocrine function, blood and urine samples will be collected at recruitment, once in each trimester of pregnancy, at delivery and 3 months after delivery. Breastmilk samples will be collected for iodine and energy content analysis. Children will be evaluated for psychomotor development at 18 months. Maternal thyroid volume will be evaluated by ultrasound scan at baseline, in the 3rd trimester and at 3 months after delivery. Discussion: Iodine deficiency early during development precludes children from achieving full intellectual capabilities. This protocol describes a study that is innovative and unique in its detailed and comprehensive evaluation of maternal and child endocrine and psychomotor parameters. By evaluating the effectiveness of the iodine supplementation recommendation, it will contribute to the public health systems' efforts to provide excellence in maternal and infant care. Trial registration: ClinicalTrials.gov, NCT04288531 . Registered 28 February 2020-Retrospectively registered. Specific funding for this study is pending. The launching phase of this study was supported by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020; by the projects NORTE-01-0145-FEDER-000013, NORTE-01-0145-FEDER-000023 and NORTE-01-0246-FEDER-000012, supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). info:eu-repo/semantics/publishedVersion

Leigh Syndrome (OMIM 256000) is a heterogeneous neurologic disorder due to damage in mitochondrial energy production that usually starts in early childhood. The first description given by Leigh pointed out neurological symptoms in children under 2 years and premature death. Following cases brought some hypothesis to explain the cause due to similarity to other neurological diseases and led to further investigation for metabolic diseases. Biochemical evaluation and specific metabolic profile suggested impairment in energy production (OXPHOS) in mitochondria. As direct approach to involved tissues is not always possible or safe, molecular analysis is a great cost-effective option and, besides biochemical results, is required to confirm the underlying cause of this syndrome face to clinical suspicion. The Next Generation Sequencing (NGS) advance represented a breakthrough in molecular biology allowing simultaneous gene analysis giving short-time results and increasing the variants underlying this syndrome, counting over 75 monogenic causes related so far. NGS provided confirmation of emerging cases and brought up diagnosis in atypical presentations as late-onset cases, which turned Leigh into a heterogeneous syndrome with variable outcomes. This review highlights clinical presentation in both classic and atypical phenotypes, the investigation pathway throughout confirmation emphasizing the underlying genetic heterogeneity and increasing number of genes assigned to this syndrome as well as available treatment.

Background: Cystinosis is a multisystemic autosomal recessive deficiency of the lysosomal membrane transporter protein (cystinosin) caused by mutations in CTNS gene. Objective: This study summarizes the Portuguese experience in the diagnosis and management of patients with this rare disease over the past few years and reports recurrent mutations in the CTNS gene. Methods: Unrelated patients from different pediatric and adult hospitals all over Portugal with non-nephrotic proteinuria, hypercalciuria, hypokalemia impaired proximal reabsorption of amino acids, glycosuria and hypophosphatemia, suggestive of a Fanconi syndrome and ocular problems, were studied. Intra-leukocyte cystine levels were determined and molecular analysis was performed, to determine the presence or absence of the 57-kb deletion in CTNS, followed by direct sequencing of the coding exons of CTNS. Results: From 1998 to 2017, twenty-one cystinotic patients were biochemically diagnosed. From the remaining seventeen (four deceased), eleven were studied for CTNS gene. Five out of eleven patients were homozygous for the 57-kb deletion (10/22; 45.5%), and other five were compound heterozygous for this variant (15/22; 68.2%). The other mutations found were p.Q128X (c.721 C>T; 2/22), p.S139F (c.755 C>T; 4/22) and c.18-21delGACT (p.T7FfsX7; 1/22). All of these seventeen cystinotic patients are in treatment. Approximately 84% are adults, 16% are young children, and 54.5% are kidney transplant recipient. Conclusions: The authors would like to emphasize the importance of first screening for the 57-kb deletion since it is very common in our population. This genetic study is the first in our country and it could be the basis for future genetic counseling in Portuguese population.

Comment in: Phenotypic and genotypic features in pediatric and adult mitochondrial disorders. [Muscle Nerve. 2017]; Reply. [Muscle Nerve. 2017] Introduction: Mitochondrial disorders display remarkable genetic and phenotypic heterogeneity. Methods: We performed a retrospective analysis of the clinical, histological, biochemical, and genetic features of 65 patients with molecular diagnoses of mitochondrial disorders. Results: The most common genetic diagnosis was a single large-scale mitochondrial DNA (mtDNA) deletion (41.5%), and the most frequent clinical phenotype was chronic progressive external ophthalmoplegia (CPEO). It occurred in 41.5% of all patients, primarily in those with mtDNA deletions. Histological signs of mitochondrial dysfunction were found in 73.8% of patients, and respiratory chain enzyme assay (RCEA) abnormalities were detected in 51.9%. Conclusions: This study confirms the high relative frequency of single large-scale deletions among mitochondrial disorders as well as its particular association with CPEO. Muscle histology seems to be particularly useful in older patients and those with mtDNA deletions, whereas RCEA might be more helpful in young children or individuals with mtDNA depletion. info:eu-repo/semantics/publishedVersion

Introduction Inherited metabolic disorders due to intoxication provoked by highly toxic endogenous compounds are among the causes of encephalopathy. That is the case of urea cycle disorders in which ammonia’s great neurotoxicity can frequently be lethal. Ornithine Transcarbamylase (OTC) Deficiency is an X-link genetic disorder and the most common disorder of the urea cycle. Presentation in heterozygote females is variable and can go from being asymptomatic to having mild chronic symptoms with severe exacerbations in any age. Case report Fifteen year old female with sporadic headache since the age twelve without other relevant medical history. Due to persistent frontoparietal headache for two weeks, she was given topiramate. Despite medication, the headache worsened and dizziness, blurred vision, dysarthria, aphasia, asterixis, motor incoordination and lethargy developed. Trauma, recent infections or toxic ingestions were denied. Diagnostic workup in a private hospital with laboratory studies including urine toxic screening, complete blood count, hepatic and renal function and cerebral MRI were normal. On account of a suspected iatrogenic cause for encephalopathy, topiramate was suspended after two days and the patient was referred to a Pediatric Emergency Department. On admission CSF study was normal but blood tests showed hyperammonemia (229 µmol/L) with latter increase to (359 µmol/L) despite treating measures (endovenous fluids and discontinuation of protein intake); She was given sodium benzoate, sodium phenylbutyrate and arginine resulting in blood levels of ammonia returning to normal. The demonstration of elevated urinary orotic acid led to the probable diagnosis of OTC deficiency which was later confirmed by molecular diagnosis. Discussion Serum ammonia levels sould be measured in every patient with unexplained altered mental status. Primary (urea clycle disorders) and secondary hyperammonemia alike are considered emergency situations therefore, treatment should never be delayed by diagnostic tests. On the other hand, both treatment and exacerbation preventions have an impact in prognosis regardless of age.