Browsing by Author "Marcão, Ana"
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- 3- Methylcrotonyl-coa Carboxylase Deficiency: Biochemical and Molecular Studies in 36 PatientsPublication . Fonseca, Helena; Sousa, Carmen; Marcão, Ana; Rocha, Hugo; Lopes, Lurdes; Vilarinho, Laura3-methylcrotonylglycinuria (MCG) is a disease included in the expanded newborn screening that until recently was considered a rare inherited disorder of the metabolism. In the catabolism of leucine, MCG is blocked in the fourth step due to deficiency of the enzyme 3-methylcrotonyl-CoA carboxylase (3-MCC) (Dantas et al). The biochemical diagnosis of disability in 3-MCC is characterized by marked increase of acid 3-hydroxyisovaleric (3-HIVA) and 3-methylcrotonylglycine (3-MCG) in urine and high concentrations of 3-hydroxyisovalerylcarnitine (C5-OH) in the blood. The molecular characterization is the study of genes MCCA and MCCB that encodes the enzyme 3-MCC. The authors report biochemical and mutation data of 36 MCC deficient individuals, one diagnosed due to clinical symptoms, 25 identified by newborn screening and 10 mothers identified following the positive newborn screening of their sons. All patients had an increased value of C5-OH, primary biochemical marker screening for this condition. In this cohort of 36 patients the genetic study intended to identify the pathogenic mutations using an analysis of 19 exons in the MCCA gene and 17 exons in the MCCB gene. A total of 32 mutations were detected of which 24 (75%) have, neither been described in the literature nor in the Human Gene Mutation Database. The results described show that the genotype cannot predict the phenotype or metabolic risk of these cases, but it is capable to confirm the diagnosis in doubtful cases. The clinical phenotype is very heterogeneous, most patients showing different mutations making the phenotype-genotype correlation difficult. The 3-MCC deficiency is a pathology not completely understood described as a genetic condition with low clinic penetrance. However, it can lead to a severe clinic phenotype resembling classic organic acidurias, has it was recently demonstrated by Grunert et al. Dantas, M. F., T. Suormala, et al. (2005). 3-Methylcrotonyl-CoA carboxylase deficiency: mutation analysis in 28 probands, 9 symptomatic and 19 detected by newborn screening.
- 3-Methylcrotonyl CoA Carboxylase Deficiency: Disorder or Just a Biochemical Phenotype?Publication . Fonseca, Helena; Bueno, Maria; Sousa, Carmen; Marcão, Ana; Lopes, Lurdes; Rocha, Hugo; Vilarinho, LauraIntroduction: 3-methylcrotonyl-CoA carboxylase deficiency (MCCD) was considered extremely rare before newborn screening (NBS) was undertaken but is now found in a number of asymptomatic babies or sometimes their mothers. This disorder of leucine metabolism, is the commonest organic aciduria found by screening, with a incidence of about 1:32 392 in our country. The clinical phenotype has been shown to vary considerably, ranging from entirely asymptomatic to death in infancy. A review of the literature on 37 individuals indicates that only 27% developed normally and stayed completely asymptomatic. Approximately 30% were reported to suffer from muscular hypotonia and psychomotor retardation, and almost half suffer from various other neurological symptoms. Even a lethality of 11% was observed. The metabolic phenotype characterizing MCCD is the elevated excretion of the diagnostic compounds 3-methylcrotonylglycine and 3-hydroxyisovaleric acid, and the presence of abnormally elevated blood levels of 3-hydroxyisovalerylcarnitine (C5-OH), as determined by tandem mass spectrometry (MS/MS). Patient and methods: The authors present a symptomatic case with an increase of C5-OH in the acylcarnitine profile who have a developmental delay. Blood spot samples from newborns are collected between day 3 and 6 in Watman 903 filter paper. Acylcarnitines in samples are analysed by MS/MS. Genes MCCA and MCCB that encodes the enzyme 3-MCC were studied by reported methods. Results: The molecular study has allowed the identification of the compound heterozygous in this patient: the frameshift mutation p.S173FfsX25 and the missense mutation p.V339M. Both mutations are described in the literature. Discussion: The newborn screening identification of a patient which developed symptoms seems to indicate that this disease should be included in NBS programs. More studies are needed to find genetic and/or biochemical markers that explain why a relatively small number of individuals are at risk of developing a severe disease phenotype. Another important reason to include MCCD in our panel is that other disorders are also detected by the marker C5OH; for example deficiencies of holocarboxylase synthetase, and 3-hydroxy- 3-methylglutaryl-CoA lyase.
- 3-Methylcrotonylglycinuria: a new common mutation in the Portuguese population?Publication . Fonseca, Helena; Sousa, Carmen; Marcão, Ana; Rocha, Hugo; Lopes, Lurdes; Vilarinho, LauraIntroduction: 3-Methylcrotonylglycinuria (MCG) is an inborn error of the leucine catabolism resulting from isolated biotin-insensitive deficiency of 3-methylcrotonyl-CoA carboxylase (3-MCC), the enzyme converting 3-methylcrotonoyl-CoA to 3-methylglutaconyl-CoA. The metabolic phenotype characterizing MCC deficiency is the elevated excretion of the diagnostic compounds 3-methylcrotonylglycine and 3-hydroxyisovaleric acid, and the presence of abnormally elevated blood levels of 3-hydroxyisovalerylcarnitine (C5-OH), as determined by tandem mass spectrometry (MS/MS). Expanded newborn screening for inborn errors of metabolism using MS/MS has demonstrated that 3-MCC deficiency is one of the most commonly detected inherited organic acidurias. Patient and methods: The authors report the results of molecular studies performed in six cases in a universe of thirty patients with an increase of C5-OH in the acylcarnitine profile. Blood spot samples from newborns are collected between day 3 and 6 in Watman 903 filter paper. Acylcarnitines in samples are analysed in tandem mass spectrometer. The molecular characterization is the study of genes MCCA and MCCB that encodes the enzyme 3-MCC. Results: The six cases showed the same novel mutation p.N230D in the MCCB gene, proving that this is the most common new mutation in our population. According to the studies conducted to this new mutation using bioinformatic applications, it is considered a benign mutation, but the alignment of species and the population study conducted, showed that this mutation is responsible for the biochemical phenotype found in these cases. Discussion: Of the thirty MCC cases studied, p.N230D mutation revealed to be the most frequent new mutation. Bioinformatic analysis showed that this mutation is located in a non conserved area but the mutant residue was never present in the homologous proteins analyzed.
- Adult-onset form in VLCAD deficiency: seven casesPublication . Sousa, Carmen; Marcão, Ana; Nogueira, Célia; Fonseca, Helena; Rocha, Hugo; Silva, Carla; Guimas, Arlindo; Evangelista, Teresinha; Maré, Rui; Vilarinho, LauraVery long chain acyl-Co-A dehydrogenase deficiency (VLCADD, MIM 201475) is an autosomal recessive disorder characterized by impaired mitochondrial β-oxidation of fatty acids with a chain length between 14 and 18 carbons. The prevalence of VLCAD deficiency in Portugal is 1/101,613. VLCADD has three forms of clinical presentation: severe early-onset; intermediate with childhood onset and adult-onset, of mild severity, characterized by exercise intolerance, myalgia and recurrent episodes of rhabdomyolysis. The development of electrospray ionization tandem mass spectrometry (MS/MS) has allowed beyond the screening of neonatal forms a marked improvement on diagnosis of the adult onset form. The authors report the acyl-carnitines profile that revealed accumulation of tetradecenoyl carnitine (C14:1) in seven individuals with clinical symptoms with the ages between 11 and 63. The eldest patient was diagnosed at the age of 63 years. These results were confirmed by molecular ACADVL gene analysis. When rhabdomyolysis is present in a patient, and after differential diagnosis, it is important to consider the possibility of a VLCAD deficiency. This late-onset form may be undetectable by acyl-carnitine profile in asymptomatic period, and only in crisis is informative. However, if VLCADD is considered the molecular analysis of ACADVL should be performed in all suspected cases.
- Aplicação dos marcadores IRT/PAP/IRT no rastreio neonatal da fibrose quísticaPublication . Lopes, Lurdes; Marcão, Ana; Carvalho, Ivone; Sousa, Carmen; Fonseca, Helena; Rocha, Hugo; Vilarinho, LauraA Fibrose Quística (FQ) é uma doença genética, com transmissão autossómica recessiva. Bioquimicamente deve-se à deficiência na proteína Cystic Fibrosis Transmembrane Condutance Regulator que é codificada pelo gene CFTR, localizado no cromossoma 7. Estão descritas cerca de 2000 variantes genéticas associadas a esta doença. Iniciou-se no final de 2013 um estudo piloto integrado no Programa Nacional de Diagnóstico Precoce (PNDP), que incluiu 80,000 recém-nascidos (RN). O aumento da concentração sanguínea da tripsina imunoreactiva (IRT) nos primeiros dias de vida dos RN com FQ possibilita o rastreio neonatal desta doença. No entanto, apesar de uma boa sensibilidade (95%), o IRT não é um marcador específico (34-75%) para a FQ, e um rastreio baseado unicamente neste marcador tem um número elevado de falsos positivos. Por esta razão, têm sido propostos vários algoritmos de rastreio, incluindo outros marcadores bioquímicos como a Proteína Associada à Pancreatite (PAP). Neste estudo, o algoritmo de rastreio utilizado baseia-se na determinação do IRT / PAP / IRT em sangue colhido em papel de filtro, sendo a amostra de sangue a mesma colhida para as restantes doenças rastreadas. Neste estudo foram identificadas 680 amostras com valor elevado de IRT ao rastreio, mas apenas em 272 casos foram solicitadas novas amostras por apresentarem também aumento de PAP. Esta estratégia reduziu significativamente os pedidos de segunda amostra de sangue. Foram diagnosticados neste estudo 11 doentes, o que implica uma prevalência ao nascimento de aproximadamente 1:7,200, no entanto este estudo será alargado a mais 80,000 RN para estabelecermos a real prevalência desta patologia na nossa população.
- Aplicação dos marcadores IRT/PAP/IRT no rastreio neonatal da fibrose quísticaPublication . Lopes, Lurdes; Marcão, Ana; Carvalho, Ivone; Sousa, Carmen; Fonseca, Helena; Rocha, Hugo; Vilarinho, LauraA Fibrose Quística (FQ) é uma doença genética, com transmissão autossómica recessiva. Bioquimicamente deve-se à deficiência na proteína Cystic Fibrosis Transmembrane Condutance Regulator que é codificada pelo gene CFTR, localizado no cromossoma 7. Estão descritas cerca de 2000 variantes genéticas associadas a esta doença. Iniciou-se no final de 2013 um estudo piloto integrado no Programa Nacional de Diagnóstico Precoce (PNDP), que incluiu 80,000 recém-nascidos (RN). O aumento da concentração sanguínea da tripsina imunoreactiva (IRT) nos primeiros dias de vida dos RN com FQ possibilita o rastreio neonatal desta doença. No entanto, apesar de uma boa sensibilidade (95%), o IRT não é um marcador específico (34-75%) para a FQ, e um rastreio baseado unicamente neste marcador tem um número elevado de falsos positivos. Por esta razão, têm sido propostos vários algoritmos de rastreio, incluindo outros marcadores bioquímicos como a Proteína Associada à Pancreatite (PAP). Neste estudo, o algoritmo de rastreio utilizado baseia-se na determinação do IRT / PAP / IRT em sangue colhido em papel de filtro, sendo a amostra de sangue a mesma colhida para as restantes doenças rastreadas. Neste estudo foram identificadas 680 amostras com valor elevado de IRT ao rastreio, mas apenas em 272 casos foram solicitadas novas amostras por apresentarem também aumento de PAP. Esta estratégia reduziu significativamente os pedidos de segunda amostra de sangue. Foram diagnosticados neste estudo 11 doentes, o que implica uma prevalência ao nascimento de aproximadamente 1:7,200, no entanto este estudo será alargado a mais 80,000 RN para estabelecermos a real prevalência desta patologia na nossa população.
- Bases moleculares de los defectos en el complejo mitocondrial ETF/ETF-QOPublication . Rocha, Hugo; Nogueira, Célia; Martins, Esmeralda; Rodrigues, Esmeralda; Leão, Miguel; Sousa, Carmen; Fonseca, Helena; Marcão, Ana; Gaspar, Ana; Brandão, Otilia; Santos, Helena; Coelho, Teresa; Ribeiro, J. Miguel; Vilarinho, LauraDefectos en el complejo mitocondrial ETF/ETF-QO (ETF - flavoproteína transferidora de electrones; ETFQO - flavoproteína transferidora de electrones ubiquinona oxidorreductasa), resultan en la disfunción secundaria de 11 deshidrogenasas que utilizan este complejo para transferir los electrones a la cadena de transporte electrónico, bloqueando la β-oxidación de los ácidos grasos, de aminoácidos y de la colina. Defectos en el funcionamiento del complejo resultan en una deficiencia multiple de acil-CoA deshidrogenasas (MADD; aciduria glutárica tipo II; OMIM 231680) que representa un grupo muy heterogéneo de enfermedades metabólicas tanto desde el punto de vista clínico como molecular. Pueden tener como causa mutaciones en los genes que codifican para las subunidades del complejo (ETFA, ETFB y ETFDH), genes que codifican los transportadores de riboflavina, del transporte ó de la síntesis del FAD. Bioquímicamente, las MADD se caracterizan por la acumulación de acilcarnitinas de cadena corta, media y larga, siendo su detección la abordaje primaria al diagnóstico, incluso en cribado neonatal. La multiplicidad de defectos genéticos que pueden cursar por una disfunción del complejo ETF/ETF-QO (creyéndose que siguen sin estar identificados todos), el hecho de que no siempre están presentes los marcadores bioquímicos evidentes (acumulación de las acilcarnitinas características) y la grande variabilidad clínica son factores que conducen a una dificultad aumentada en de diagnóstico de este grupo de errores congénitos del metabolismo. En este panorama el conocimiento de la epidemiologia molecular es fundamental. Los autores presentaran las bases moleculares de los trastornos en el funcionamiento del complejo ETF/ETF-QO en Portugal.
- Biochemical and molecular heterogeneity in carnitine palmitoyltransferase ii deficiencyPublication . Sousa, Carmen; Fonseca, Helena; Rocha, Hugo; Marcão, Ana; Vilarinho, Laura; Diogo, Luísa; Sequeira, Sílvia; Costa, Cristina; Leão, Elisa; Conceição, Isabel; Gaspar, AnaIntroduction: Carnitine palmitoytransferase II (CPTII) deficiency is a recessively inherited disorder of lipid metabolism. CPT II deficiency has several clinical presentations: the adult form is characterized by episodes of rhabdomyolysis, usually triggered by extensive exercice, cold, fever or prolonged fasting and the infantile-type CPT II hepatocardiomuscular form presents as severe attacks of hypoketotic, hypoglycemia, occasionally associated with cardiac damage. Molecular analysis of CPT II gene allows not only confirmation of classical forms, with typical biochemical abnormalities, but is also key for the diagnosis of less severe forms.The authors will present biochemical and molecular findings of eight CPT II, patients. Material and methods: Acylcarnitine profiles, on dried blood spots, were done as previously reported (Vilarinho et al) Molecular analysis of CPT II gene was done by direct sequencing as reported elsewhere (Finocchiaro et al.). From the eight patients, three were detected through newborn screening and five with clinical symptoms. Results: More pronounced abnormalities in the acylcarnitine profiles are associated with neonatal forms of the disease, while the patients with late onset forms present smother alterations or completely normal profiles. This biochemical heterogeneity correlates well with genetics data. The molecular analysis revealed the presence of ten different mutations, which seven were never reported. Discussion: Our results support that newborn screening can efficiently detect infantile CPT II deficiency but is less effective in detecting adult forms, were biochemical abnormalities may only be present during acute episodes. In these cases accurate clinical characterization alongside with molecular analysis are the key for diagnosis. Bibliography: J.P. Bonnefont, F. et al (2004) Mol. Aspects Med. 25 495–520. Rashed MS, et al.(1995) Diagnosis of inborn errors of metabolism from blood spots by acylcarnitines and amino acids profiling using automated electrospray tandem mass spectrometry. Pediatr Res.38:324–331 Finocchiaro, G. et al (1991). cDNA cloning, sequence analysis, and chomosomal localization of the gene for human carnitinepalmitoyltransferase. Proc. Natl. Acad. Sci. USA 88, 661–665. K. Gempel1 et al. (2002).Screening for Carnitine Palmitoyltransferase II Deficiency by Tandem Mass Spectrometry Anichini A.et al(2011) Genotype-phenotype correlations in a large series of patients with muscle type CPT II deficiency Vilarinho et al. (2009) Four Years of Expanded Newborn Screening in Portugal with MS/MS
- CTNS Molecular Genetics Profile in a Portuguese Cystinosis PopulationPublication . Ferreira, Filipa; Leal, Inês; Sousa, David; Costa, Teresa; Mota, Conceição; Gomes, Ana Marta; Lopes, Daniela; Carmo Macário, Maria do; Tavares, Isabel; Pinto, Helena; Oliveira, João Paulo; Magriço, Rita; Carmona, Célia; Ramos, Sónia; Neiva, Raquel; Marcão, Ana; Vilarinho, LauraBackground: 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.
- Cystic Fibrosis Newborn Screening in Portugal: PAP Value in Populations with Stringent Rules for Genetic StudiesPublication . Marcão, Ana; Barreto, Celeste; Pereira, Luísa; Vaz, Luísa; Cavaco, José; Casimiro, Ana; Félix, Miguel; Silva, Teresa; Barbosa, Telma; Freitas, Cristina; Nunes, Sidónia; Felício, Verónica; Lopes, Lurdes; Amaral, Margarida; Vilarinho, LauraNewborn 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.