Publications by authors named "Laura Vilarinho"

86 Publications

Role of RNA in Molecular Diagnosis of MADD Patients.

Biomedicines 2021 May 4;9(5). Epub 2021 May 4.

Research & Development Unit, Human Genetics Department, National Institute of Health Doutor Ricardo Jorge, 4000-055 Porto, Portugal.

The electron-transfer flavoprotein dehydrogenase gene () 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 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 mutations and will be important in providing an accurate genetic counseling and a prenatal diagnosis for the affected families.
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http://dx.doi.org/10.3390/biomedicines9050507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8147995PMC
May 2021

Parkinsonism and iron deposition in two adult patients with L-2-hydroxiglutaric aciduria.

Parkinsonism Relat Disord 2021 May 1;86:45-47. Epub 2021 Apr 1.

Neurology Department, Centro Hospitalar Universitário do Porto, Porto, Portugal.

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.
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http://dx.doi.org/10.1016/j.parkreldis.2021.03.025DOI Listing
May 2021

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.

Norwegian National Unit for Newborn Screening, 0424 Oslo, Norway.

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

[Portuguese Newborn Screening Program.]

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

Unidade de Rastreio Neonatal, Metabolismo e Genética. Departamento de Genética Humana. Instituto Nacional de Saúde Doutor Ricardo Jorge. Oporto. Portugal.

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.
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January 2021

Phenylketonuria in Portugal: Genotype-phenotype correlations using molecular, biochemical, and haplotypic analyses.

Mol Genet Genomic Med 2021 Mar 19;9(3):e1559. Epub 2021 Jan 19.

Newborn Screening, Metabolic and Genetics Unit, Department of Human Genetics, National Institute of Health Dr Ricardo Jorge, Porto, Portugal.

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.
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http://dx.doi.org/10.1002/mgg3.1559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104178PMC
March 2021

Impact of iodine supplementation during preconception, pregnancy and lactation on maternal thyroid homeostasis and offspring psychomotor development: protocol of the IodineMinho prospective study.

BMC Pregnancy Childbirth 2020 Nov 13;20(1):693. Epub 2020 Nov 13.

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus Gualtar, 4710-057, Braga, Portugal.

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.
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http://dx.doi.org/10.1186/s12884-020-03376-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664061PMC
November 2020

Clinical, biochemical and molecular findings of 24 Brazilian patients with glutaric acidemia type 1: 4 novel mutations in the GCDH gene.

Metab Brain Dis 2021 02 16;36(2):205-212. Epub 2020 Oct 16.

Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, Ramiro Barcelos, 2350, 90035-003, Porto Alegre, RS, Brazil.

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.
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http://dx.doi.org/10.1007/s11011-020-00632-0DOI Listing
February 2021

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

Eur J Hum Genet 2021 01 10;29(1):67-78. Epub 2020 Oct 10.

Centre for inherited metabolic diseases, Karolinska University Hospital, Solna, Sweden.

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

NPC1 silent variant induces skipping of exon 11 (p.V562V) and unfolded protein response was found in a specific Niemann-Pick type C patient.

Mol Genet Genomic Med 2020 11 15;8(11):e1451. Epub 2020 Sep 15.

Research & Development Unit, Human Genetics Department, National Institute of Health Doutor Ricardo Jorge, Porto, Portugal.

Background: Niemann-Pick type C (NPC, MIM #257220) is a neuro-visceral disease, caused predominantly by pathogenic variants in the NPC1 gene. Here we studied patients with clinical diagnosis of NPC but inconclusive results regarding the molecular analysis.

Methods: We used a Next-Generation Sequencing (NGS)-panel followed by cDNA analysis. Latter, we used massively parallel single-cell RNA-seq (MARS-Seq) to address gene profiling changes and finally the effect of different variants on the protein and cellular levels.

Results: We identified novel variants and cDNA analysis allowed us to establish the functional effect of a silent variant, previously reported as a polymorphism. We demonstrated that this variant induces the skipping of exon 11 leading to a premature stop codon and identified it in NPC patients from two unrelated families. MARS-Seq analysis showed that a number of upregulated genes were related to the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in one specific patient. Also, for all analyzed variants, the NPC1 protein was partially retained in the ER.

Conclusion: We showed that the NPC1 silent polymorphism (p.V562V) is a disease-causing variant in NPC and that the UPR is upregulated in an NPC patient.
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http://dx.doi.org/10.1002/mgg3.1451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667330PMC
November 2020

Assessing Lysosomal Disorders in the NGS Era: Identification of Novel Rare Variants.

Int J Mol Sci 2020 Sep 1;21(17). Epub 2020 Sep 1.

Research and Development Unit, Human Genetics Department, National Institute of Health Doutor Ricardo Jorge, 4000-055 Porto, Portugal.

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 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.
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http://dx.doi.org/10.3390/ijms21176355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503609PMC
September 2020

TYROSINEMIA TYPE III: A CASE REPORT OF SIBLINGS AND LITERATURE REVIEW.

Rev Paul Pediatr 2020 5;38:e2018158. Epub 2020 Jun 5.

Centro Hospitalar Universitário do Porto, Porto, Portugal.

Objective: Tyrosinemia type III (HT III) is the rarest form of tyrosinemia, and the full clinical spectrum of this disorder is still unknown. The neurological involvement varies, including intellectual impairment and attention deficit disorder with hyperactivity (ADHD). We report the case of two siblings diagnosed with HT III at different ages.

Case Description: The index case was diagnosed by newborn screening for endocrine and metabolic disorders, starting a low-protein diet immediately, with a consistent decrease in tyrosine levels. By the age of three, the child displayed a hyperactive behavior, starting treatment for ADHD two years later. At seven years of age, he shows a slight improvement in terms of behavior and attention span and has a cognitive performance slightly lower than his peers, despite maintaining acceptable tyrosine levels. His sister, who had a history of ADHD since age five, was diagnosed with HT III after family screening at the age of eight. Despite initiating a dietetic treatment, her behavior did not improve, and she has a mild intellectual impairment.

Comments: This is the first case report describing siblings with HT III who underwent nutritional treatment with a low-protein diet in different phases of life, with a better neurological and behavioral evaluation in the patient who started treatment earlier.
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http://dx.doi.org/10.1590/1984-0462/2020/38/2018158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274528PMC
March 2021

Correction to: Molecular basis of Leigh syndrome: a current look.

Orphanet J Rare Dis 2020 Mar 25;15(1):77. Epub 2020 Mar 25.

Newborn screening, metabolism and genetics unit - human genetics department, Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), Porto, Portugal.

The original version of this article [1] unfortunately included an error to an author's name. Author Manuela Schubert Baldo was erroneously presented as Manuela Baldo Schubert.
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http://dx.doi.org/10.1186/s13023-020-1351-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098111PMC
March 2020

Molecular basis of Leigh syndrome: a current look.

Orphanet J Rare Dis 2020 01 29;15(1):31. Epub 2020 Jan 29.

Newborn screening, metabolism and genetics unit - human genetics department, Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), Porto, Portugal.

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.
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http://dx.doi.org/10.1186/s13023-020-1297-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6990539PMC
January 2020

Molecular Characterization of a Novel Splicing Mutation underlying Mucopolysaccharidosis (MPS) type VI-Indirect Proof of Principle on Its Pathogenicity.

Diagnostics (Basel) 2020 Jan 21;10(2). Epub 2020 Jan 21.

Research and Development Unit, Department of Human Genetics, INSA, 4000-055 Porto, Portugal.

Here, we present the molecular diagnosis of a patient with a general clinical suspicion of Mucopolysaccharidosis, highlighting the different tools used to perform its molecular characterization. In order to decrease the turnaround time for the final report and contribute to reduce the "diagnostic odyssey", which frequently afflicts affected families, the proband's sample was simultaneously screened for mutations in a number of lysosomal function-related genes with targeted next-generation sequencing (NGS) protocol. After variant calling, the most probable cause for disease was a novel intronic variant, c.1213+5G>T [IVS6+5G>T], detected in homozygosity. In general, homozygous or compound heterozygous mutations in the gene, underlie MPS type VI or Maroteaux-Lamy syndrome. Still, even though the novel c.1213+5G>T variant was easy to detect by both NGS and Sanger sequencing, only through indirect studies and functional analyses could we present proof of principle on its pathogenicity. Globally, this case reminds us that whenever a novel variant is detected, its pathogenicity must be carefully assessed before a definitive diagnosis is established, while highlighting alternative approaches that may be used to assess its effect in the absence RNA/cDNA sample(s) from the proband. This is particularly relevant for intronic variants such as the one here reported. Special attention will be given to the use of reporter minigene systems, which may be constructed/designed to dissect the effect of this sort of alterations, providing an insight into their consequences over the normal pre-mRNA splicing process of the affected gene.
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http://dx.doi.org/10.3390/diagnostics10020058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168280PMC
January 2020

Iron-sulfur cluster ISD11 deficiency ( gene) presenting as cardiorespiratory arrest and 3-methylglutaconic aciduria.

JIMD Rep 2019 Sep 24;49(1):11-16. Epub 2019 Jul 24.

Newborn Screening, Metabolism and Genetics Unit, Human Genetics Department National Institute of Health Doutor Ricardo Jorge Lisboa Portugal.

In the era of genomics, the number of genes linked to mitochondrial disease has been quickly growing, producing massive knowledge on mitochondrial biochemistry. 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 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 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.
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http://dx.doi.org/10.1002/jmd2.12058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718106PMC
September 2019

Molecular and Clinical Investigations on Portuguese Patients with Multiple acyl-CoA Dehydrogenase Deficiency.

Curr Mol Med 2019 ;19(7):487-493

Biosystems and Integrative Sciences Institute, Faculdade de Ciencias, Universidade de Lisboa, and Departamento de Quimica e Bioquimica, Faculdade de Ciencias, 1749-016 Lisboa, Portugal.

Background: Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a congenital rare metabolic disease with broad clinical phenotypes and variable evolution. This inborn error of metabolism is caused by mutations in the ETFA, ETFB or ETFDH genes, which encode for the mitochondrial ETF and ETF:QO proteins. A considerable group of patients has been described to respond positively to riboflavin oral supplementation, which constitutes the prototypic treatment for the pathology.

Objectives: To report mutations in ETFA, ETFB and ETFDH genes identified in Portuguese patients, correlating, whenever possible, biochemical and clinical outcomes with the effects of mutations on the structure and stability of the affected proteins, to better understand MADD pathogenesis at the molecular level.

Methods: MADD patients were identified based on the characteristic urinary profile of organic acids and/or acylcarnitine profiles in blood spots during newborn screening. Genotypic, clinical and biochemical data were collected for all patients. In silico structural analysis was employed using bioinformatic tools carried out in an ETF:QO molecular model for the identified missense mutations.

Results: A survey describing clinical and biochemical features of eight Portuguese MADD patients was made. Genotype analysis identified five ETFDH mutations, including one extension (p.X618QextX*14), two splice mutations (c.34+5G>C and c.405+3A>T) and two missense mutations (ETF:QO-p.Arg155Gly and ETF:QO-p.Pro534Leu), and one ETFB mutation (ETFβ- p.Arg191Cys). Homozygous patients containing the ETFDH mutations p.X618QextX*14, c.34+5G>C and ETF:QO-p.Arg155Gly, all presented severe (lethal) MADD phenotypes. However, when any of these mutations are in heterozygosity with the known ETF:QO-p.Pro534Leu mild variant, the severe clinical effects are partly and temporarily attenuated. Indeed, the latter destabilizes an ETF-interacting loop, with no major functional consequences. However, the position 155 in ETF:QO is localized at the ubiquinone binding and membrane interacting domain, and is thus expected to perturb protein structure and membrane insertion, with severe functional effects. Structural analysis of molecular models is therefore demonstrated to be a valuable tool to rationalize the effects of mutations in the context of the clinical phenotype severity.

Conclusion: Advanced molecular diagnosis, structural analysis and clinical correlations reveal that MADD patients harboring a severe prognosis mutation in one allele can actually revert to a milder phenotype by complementation with a milder mutation in the other allele. However, such patients are nevertheless in a precarious metabolic balance which can revert to severe fatal outcomes during catabolic stress or secondary pathology, thus requiring strict clinical follow-up.
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http://dx.doi.org/10.2174/1566524019666190507114748DOI Listing
August 2020

Targeted next generation sequencing identifies novel pathogenic variants and provides molecular diagnoses in a cohort of pediatric and adult patients with unexplained mitochondrial dysfunction.

Mitochondrion 2019 07 1;47:309-317. Epub 2019 Mar 1.

Research & Development Unit, Human Genetics Department, National Institute of Health Doutor Ricardo Jorge, Porto, Portugal; Newborn Screening, Metabolism & Genetics Unit, Human Genetics Department, National Institute of Health Doutor Ricardo Jorge, Porto, Portugal. Electronic address:

Mitochondrial diseases (MD) are a group of rare inherited disorders, characterized by phenotypic heterogeneity, with hitherto no effective therapeutic options. The aim of this study was to develop a next generation sequencing (NGS) strategy, by using a custom gene panel and whole mitochondrial genome, to identify the disease causing pathogenic variants in 146 patients suspicious of MD. The molecular analysis of this cohort revealed six novel and 15 described pathogenic variants, as well as 26 variants of unknown significance. Our findings are expanding the mutational landscape of these disorders and support the use of a NGS strategy for a higher diagnostic yield.
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http://dx.doi.org/10.1016/j.mito.2019.02.006DOI Listing
July 2019

Newborn screening for homocystinurias: Recent recommendations versus current practice.

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

Unidad de Metabolismo, Hospital Infantil Miguel Servet, Zaragoza, Spain.

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

Late-onset Levodopa Responsive Parkinsonism Due to Polymerase γ 1 Mutations.

Mov Disord Clin Pract 2018 Nov-Dec;5(6):645-648. Epub 2018 Oct 17.

Neurology Department Hospital de Santo António, Centro Hospitalar do Porto Portugal.

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http://dx.doi.org/10.1002/mdc3.12668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277373PMC
October 2018

Follow-up of fatty acid β-oxidation disorders in expanded newborn screening era.

Eur J Pediatr 2019 Mar 7;178(3):387-394. Epub 2019 Jan 7.

Centro de Referência de Doenças Hereditárias do Metabolismo, Departamento de Pediatria Médica, Hospital de Santa Maria - CHULN, Av. Prof. Egas Moniz, 1649-035, Lisbon, Portugal.

Fatty acid β-oxidation (FAO) disorders have a wide variety of symptoms, not usually evident between episodes of acute decompensations. Cardiac involvement is frequent, and severe ventricular arrhythmias are suspected of causing sudden death. Expanded newborn screening (ENS) for these disorders, hopefully, contribute to prevent potentially acute life-threatening events. In order to characterize acute decompensations observed in FAO-deficient cases identified by ENS, a retrospective analysis was performed, covering a period of 9 years. Demographic data, number/type of acute decompensations, treatment, and follow-up were considered. Eighty-three clinical charts, including 66 medium-chain acyl-CoA dehydrogenase deficiency (MCADD), 5 carnitine-uptake deficiency (CUD), 3 carnitine palmitoyltransferase I and II (CPT I/II) deficiency, 5 very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), and 4 multiple acyl-CoA dehydrogenase deficiency (MADD) cases were reviewed. Nineteen patients had acute decompensations (1 CPT I, 1 CPT II, 3 MADD, 14 MCADD). Six patients developed symptoms previously to ENS diagnosis. Severe clinical manifestations included multiple organ failure, liver failure, heart failure, and sudden death. Long-chain FAO disorders had the highest number of decompensations per patient.Conclusion: Despite earlier diagnosis by ENS, sudden deaths were not avoided and acute decompensations with severe clinical manifestations still occur as well. What is Known: • Severe ventricular arrhythmias are suspected to cause unexpected death in FAO disorders. • Neonatal screening intends to reduce the incidence of severe metabolic crisis and death. What is New: • Acute severe decompensations occurred in FAO disorders diagnosed through neonatal screening. • Sudden deaths were not avoided by starting treatment precociously.
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http://dx.doi.org/10.1007/s00431-018-03315-2DOI Listing
March 2019

Diagnosis, management, and follow-up of mitochondrial disorders in childhood: a personalized medicine in the new era of genome sequence.

Eur J Pediatr 2019 Jan 7;178(1):21-32. Epub 2018 Dec 7.

Newborn Screening, Metabolism and Genetics Unit, Human Genetics Department, National Institute of Health Dr. Ricardo Jorge, Porto, Portugal.

Primary mitochondrial disorders are highly variable in clinical presentation, biochemistry, and molecular etiology. Mitochondrial disorders can be caused by genetic defects in the mitochondrial, in nuclear genome, or in the interplay between the two genomes. Biochemical screening tests may be inconclusive or misleading since patients, with confirmed mitochondrial disorders specially in pediatric age, may exhibit normal routine biochemistry, muscle histology, or enzymatic analysis of the mitochondrial respiratory chain. Diagnosis is often challenging even with combination of multiple criteria (clinical, biochemical, histological, and functional), as innumerous conditions cause secondary mitochondrial dysfunction. Nowadays, a definite diagnosis is only possible by genetic confirmation since no single score system is satisfactorily accurate, being sensitive but not specific.Conclusion: Awareness between physicians is of major importance considering that clinical suspicion may not be obvious regarding the heterogenicity in presentation and biochemical features of mitochondrial disorders. In this review, we provide information on diagnosis approach to patients suspected for mitochondrial disorders as well as management on chronic and acute settings. Follow-up should provide comprehensive information on patient's status, since intervention on these diseases is mostly supportive and prognosis is variable and sometimes unpredictable. What is Known: • Mitochondrial disorders are heterogenous and may present at any age, with any symptoms and any type of inheritance. • Mitochondrial disorders may be due to pathogenic variants in mitochondrial DNA (mtDNA) or nuclear genes (nDNA). What is New: • Since no single score system is satisfactorily accurate, a definite diagnosis is only possible with genetic studies with gene panels proving to be a cost-effective approach. • Clinical and biochemical features of patients without a confirmed diagnosis must be reviewed and other diagnosis must be considered. A wider genetic approach may be applied (WES or WGS).
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http://dx.doi.org/10.1007/s00431-018-3292-xDOI Listing
January 2019

LEIGH SYNDROME: A CASE REPORT WITH A MITOCHONDRIAL DNA MUTATION.

Rev Paul Pediatr 2018 Oct-Dec;36(4):519-523. Epub 2018 Oct 29.

Centro Hospitalar do Médio Ave, Nova de Famalicão, Portugal.

Objective: Leigh syndrome is a neurodegenerative disorder with an incidence of 1:40,000 live births. It presents wide clinical, biochemical, and genetic heterogeneity, but with homogenous neuropatoradiological alterations. There is no specific treatment, and the prognosis is reserved. This case report aimed familiarize health professionals with the disease.

Case Description: A 16-month-hold girl who was followed in outpatient clinic due to axial hypotonia and delayed psychomotor development. Karyotype, auditory evoked potentials and ophthalmologic evaluation were normal. Evidence of hyperlactacidemia and hypocitrullinemia was detected in the patient. After performing brain magnetic resonance under anesthesia, hypotonia got worse, and the patient was hospitalized after an episode of cyanosis and apnea. The electroencephalogram showed no epileptiform activity. Neuroimaging revealed bilateral lenticular hyperintensity, especially in the putamen and in the left globus pallidus regions. Molecular analysis revealed an 8993T>G (MT-ATP6) mutation in the mitochondrial DNA.

Comments: Between 10 and 30% of individuals with Leigh syndrome have mitochondrial DNA mutations. The decompensation after anesthetic intercurrences is typically associated with neurological deterioration and, in this case, increased the diagnosis suspicion. It is important to alert for similar cases and to reduce invasive diagnostic tests if the diagnosis is suspected.
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http://dx.doi.org/10.1590/1984-0462/;2018;36;4;00003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322804PMC
June 2019

Newborn screening for sickle cell disease in Europe: recommendations from a Pan-European Consensus Conference.

Br J Haematol 2018 11 18;183(4):648-660. Epub 2018 Oct 18.

Programa de Cribado Neonatal, Sección Errores Congénitos del Metabolismo, Servicio de Bioquímica y Genética Molecular, Hospital Clínic, Barcelona, Spain.

Sickle Cell Disease (SCD) is an increasing global health problem and presents significant challenges to European health care systems. Newborn screening (NBS) for SCD enables early initiation of preventive measures and has contributed to a reduction in childhood mortality from SCD. Policies and methodologies for NBS vary in different countries, and this might have consequences for the quality of care and clinical outcomes for SCD across Europe. A two-day Pan-European consensus conference was held in Berlin in April 2017 in order to appraise the current status of NBS for SCD and to develop consensus-based statements on indications and methodology for NBS for SCD in Europe. More than 50 SCD experts from 13 European countries participated in the conference. This paper aims to summarise the discussions and present consensus recommendations which can be used to support the development of NBS programmes in European countries where they do not yet exist, and to review existing programmes.
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http://dx.doi.org/10.1111/bjh.15600DOI Listing
November 2018

Cystic Fibrosis Newborn Screening in Portugal: PAP Value in Populations with Stringent Rules for Genetic Studies.

Int J Neonatal Screen 2018 Sep 29;4(3):22. Epub 2018 Jun 29.

National Institute of Health Dr Ricardo Jorge, Human Genetics Department, Newborn Screening, Metabolism and Genetic Unit, Rua Alexandre Herculano 321, 4000-055 Porto, Portugal.

Newborn screening (NBS) for cystic fibrosis (CF) has been shown to be advantageous for children with CF, and has thus been included in most NBS programs using various algorithms. With this study, we intend to establish the most appropriate algorithm for CF-NBS in the Portuguese population, to determine the incidence, and to contribute to elucidating the genetic epidemiology of CF in Portugal. This was a nationwide three-year pilot study including 255,000 newborns (NB) that were also screened for congenital hypothyroidism (CH) and 24 other metabolic disorders included in the Portuguese screening program. Most samples were collected in local health centers spread all over the country, between the 3rd and 6th days of life. The algorithm tested includes immunoreactive trypsinogen (IRT) determination, pancreatitis associated protein (PAP) as a second tier, and genetic study for cases referred to specialized clinical centers. Thirty-four CF cases were confirmed positive, thus indicating an incidence of 1:7500 NB. The p.F508del mutation was found in 79% of the alleles. According to the results presented here, CF-NBS is recommended to be included in the Portuguese NBS panel with a small adjustment regarding the PAP cut-off, which we expect to contribute to the improvement of the CF-NBS performance. According to our results, this algorithm is a valuable alternative for CF-NBS in populations with stringent rules for genetic studies.
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http://dx.doi.org/10.3390/ijns4030022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7548908PMC
September 2018

Mitochondrial Encephalopathy: First Portuguese Report of a VARS2 Causative Variant.

JIMD Rep 2018 25;42:113-119. Epub 2018 Feb 25.

Neurogenetics Unit, Department of Medical Genetics, SJHC, Porto, Portugal.

Introduction: Combined oxidative phosphorylation deficiency 20 (COXPD20) is a mitochondrial respiratory chain complex (RC) disorder, caused by disease-causing variants in the VARS2 gene, which encodes a mitochondrial aminoacyl-tRNA synthetase. Here we describe a patient with fatal mitochondrial encephalopathy caused by a homozygous VARS2 gene missense variant.

Case Report: We report the case of a girl, the first child of non-consanguineous and healthy parents, born from an uneventful term pregnancy, who presented, in the neonatal period, major hypotonia and microcephaly. At 4 months of age she showed poor eye contact, nystagmus, global psychomotor development delay and failure to thrive, without dysmorphic features. Focal seizures started at 24 months which evolved to a severe epileptic encephalopathy and finally to super refractory status epilepticus, leading to her death at 28 months of age. Etiologic investigation encompassing metabolic and genetic causes failed to disclose a diagnosis. Post-mortem exome sequencing allowed the identification of a pathogenic variant in VARS2 gene in the homozygous state (c.1100C > T, p.Thr367Ile) in the patient, inherited from her heterozygous parents, leading to the diagnosis of COXPD2.

Conclusion: To the best of our knowledge, this is the fifth case described in the literature of a child with disease-causing variant in VARS2. With this report we expand the knowledge about the phenotype associated with this very rare mitochondrial defect, further emphasizing the use of exome sequencing as a very powerful diagnostic tool.
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http://dx.doi.org/10.1007/8904_2018_89DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226392PMC
February 2018

Raising Awareness of False Positive Newborn Screening Results Arising from Pivalate-Containing Creams and Antibiotics in Europe When Screening for Isovaleric Acidaemia.

Int J Neonatal Screen 2018 Mar 10;4(1). Epub 2018 Feb 10.

Reference Laboratory for Neonatal Screening, Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), 3720 BA Bilthoven, The Netherlands.

While the early and asymptomatic recognition of treatable conditions offered by newborn screening confers clear health benefits for the affected child, the clinical referral of patients with screen positive results can cause significant harm for some families. The use of pivalate-containing antibiotics and more recently the inclusion of neopentanoate as a component within moisturising creams used as nipple balms by nursing mothers can result in a significant number of false positive results when screening for isovaleric acidaemia (IVA) by measuring C5 acylcarnitine. A recent survey conducted within centres from nine countries indicated that this form of contamination had been or was a significant confounding factor in the detection of IVA in seven of the nine who responded. In three of these seven the prominent cause was believed to derive from the use of moisturising creams and in another three from antibiotics containing pivalate; one country reported that the cause was mixed. As a result, four of these seven centres routinely perform second tier testing to resolve C5 isobars when an initial C5 result is elevated, and a fifth is considering making this change within their national programme. The use of creams containing neopentanoate by nursing mothers and evolving patterns in the prescription of pivalate-containing antibiotics during pregnancy require those involved in the design and operation of newborn screening programmes used to detect IVA and the doctors who receive clinical referrals from these programmes to maintain an awareness of the potential impact of this form of interference on patient results.
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http://dx.doi.org/10.3390/ijns4010008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510208PMC
March 2018

Atypical adult-onset methylmalonic acidemia and homocystinuria presenting as hemolytic uremic syndrome.

CEN Case Rep 2018 May 2;7(1):73-76. Epub 2018 Jan 2.

Nephrology Department, Centro Hospitalar de Lisboa Central E.P.E., Hospital Curry Cabral, Rua da Beneficência 8, 1069-166, Lisbon, Portugal.

Thrombotic microangiopathy (TMA) syndromes can be secondary to a multitude of different diseases. Most can be identified with a systematic approach and, when excluded, TMA is generally attributed to a dysregulation in the activity of the complement alternative pathways-atypical hemolytic uremic syndrome (aHUS). We present a challenging case of a 19-year-old woman who presented with thrombotic microangiopathy, which was found to be caused by methylmalonic acidemia and homocystinuria, a rare vitamin B12 metabolism deficiency. To our knowledge, this is the first time that an adult-onset methylmalonic acidemia and homocystinuria presents as TMA preceding CNS involvement.
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http://dx.doi.org/10.1007/s13730-017-0298-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886929PMC
May 2018

PCR in the Analysis of Clinical Samples: Prenatal and Postnatal Diagnosis of Inborn Errors of Metabolism.

Methods Mol Biol 2017 ;1620:213-224

Research & Development Unit, Human Genetics Department, National Institute of Health Dr Ricardo Jorge, Rua Alexandre Herculano, 321, 4000-055, Porto, Portugal.

Inborn errors of metabolism (IEMs) are individually rare but collectively common. As more and more genes are cloned and specific disease-causing mutations are identified, the diagnosis of IEMs is becoming increasingly confirmed by mutation analysis. Diagnosis is important not only for treatment and prognosis but also for genetic counselling and prenatal diagnosis in subsequent pregnancies. A wide range of molecular methods is available for the identification of mutations and other DNA variants, most of which are based on the Polymerase Chain Reaction (PCR). In this chapter, we focus on PCR-based methods for the detection of point mutations or small deletions/insertions as these are the most frequent causes of IEMs.
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http://dx.doi.org/10.1007/978-1-4939-7060-5_15DOI Listing
February 2018

Reply.

Muscle Nerve 2017 11 12;56(5):E49. Epub 2017 Apr 12.

Newborn Screening, Metabolism and Genetics Unit, Human Genetics Department, Dr Ricardo Jorge National Health Institute, Porto, Portugal.

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http://dx.doi.org/10.1002/mus.25650DOI Listing
November 2017

Clinical, biochemical, molecular, and histological features of 65 Portuguese patients with mitochondrial disorders.

Muscle Nerve 2017 Nov 4;56(5):868-872. Epub 2017 Apr 4.

Newborn screening, Metabolism and Genetics Unit, Human Genetics Department, Dr. Ricardo Jorge National Health Institute, Porto, Portugal.

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. Muscle Nerve 56: 868-872, 2017.
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http://dx.doi.org/10.1002/mus.25593DOI Listing
November 2017
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