Browsing by Author "Vilarinho, Laura"
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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.
- Acquired Vitamin B12 Deficiency in Newborns: Positive Impact on Newborn Health through Early DetectionPublication . Lipari Pinto, Patrícia; Florindo, Cristina; Janeiro, Patrícia; Santos, Rita Loureiro; Mexia, Sandra; Rocha, Hugo; Tavares de Almeida, Isabel; Vilarinho, Laura; Gaspar, AnaThe 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.
- 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.
- Alterations in lipid profile and enzymes paraoxonase and butyrylcholinesterase in CBS-deficient patientsPublication . Vanzin, Camila; Nogueira, Célia; Vilarinho, Laura; Wajner, Moacir; Wyse, Angela; Vargas, CarmenHomocystinuria is an inborn error of metabolism most frequently caused by cystathionine β-synthase (CBS) deficiency. Homocysteine (Hcy), methionine (Met) and other metabolites of Hcy accumulate in the body of affected patients, leading to clinical manifestations such as dislocation of the optic lents, osteoporosis, mental retardation, and thromboembolism. Despite the fact that thromboembolism represent the major cause of morbidity and the most frequent cause of death in CBS-deficient patients, the cause of cardiovascular changes found in homocystinuria remain unclear. In this work, we evaluated the lipid profile (total cholesterol, HDL cholesterol, LDL cholesterol, oxidized LDL cholesterol, apolipoprotein A-1) and the activities of the enzymes paraoxonase (PON1) and butyrylcholinesterase (BuChE) in plasma of patients with homocystinuria due CBS deficiency, at diagnosis and during the treatment. It was verified a significant decrease in HDL cholesterol and apolipoprotein A1 levels in the both groups of CBS-deficient patients (at diagnosis and under treatment) when compared to controls. PON1 activity was also significantly lower in the both groups of CBS-deficient patients when compared to controls which may be related with an Hcy-dependent oxidation of any group important to catalytic activity of the enzyme that favors the atherogenesis. BuChE activity was significantly increased only in CBS-deficient patients at diagnosis and it is known that this enzymatic activity is positively associated with cardiovascular risk factors. Evaluated together, our results demonstrated that treated or not CBS-deficient patients presented important alterations in lipid metabolism. This work contributes to the understanding of the responsible mechanisms of vascular lesions in CBS-deficient patients.
- 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.
- Assessing Lysosomal Disorders in the NGS Era: Identification of Novel Rare VariantsPublication . Encarnação, Marisa; Coutinho, Maria Francisca; Silva, Lisbeth; Ribeiro, Diogo; Ouesleti, Souad; Campos, Teresa; Santos, Helena; Martins, Esmeralda; Cardoso, Maria Teresa; Vilarinho, Laura; Alves, SandraLysosomal 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.
- Assessing Niemann-Pick Type C (NP-C) through a multi-omics approach genomic and transcriptomic profile of challenging casesPublication . Encarnação, Marisa; Coutinho, Maria Francisca; Cho, Soo-Min; Cardoso, Maria Teresa; Chaves, Paulo; Ribeiro, Isaura; Santos, Juliana Inês; Gaspar, Paulo; Quelhas, Dulce; Lacerda, Lúcia; Leão-Teles, Elisa; Futerman, Anthony H.; Vilarinho, Laura; Alves, SandraNiemann-Pick type C (NP-C) is a neurodegenerative Inherited Metabolic Disease with a heterogeneous clinical presentation, due to mutations in either the NPC1 or NPC2 genes. We studied patients with clinical diagnosis of NP-C but presenting inconclusive results regarding biomarkers testing and molecular analysis. Using NGS- targeted DNA sequencing we have identified some novel putative mutations and subsequent cDNA analysis allowed us to stablish the functional effect of a silent mutation (previously reported as a polymorphism) in the NPC1 splicing process. We demonstrated that this mutation leads to exon skipping, frameshift and premature stop codon and identified it in two NP-C patients from two unrelated Portuguese families. This mutation most likelly leads to a very unstable transcript that was overlooked. Also, to better characterize the pathomechanisms related to specific disease-causing mutations in NP-C patients, we analysed gene expression profiles in cultured skin fibroblasts and compared them to control individuals using Massively Parallel RNA Single-Cell Sequencing (MARS-Seq). The most prominent hits from this transcriptomics analysis were validated by qRT-PCR. The MARS-Seq analysis showed that a number of genes were upregulated and a significant number of the highly enriched genes are related to the unfold protein response (UPR) and Endoplasmic Reticulum (ER) stress, in a specific patient, which deserves further studies. ER stress is a hallmark of many neurodegenerative diseases, including LSD and can be due to misfolded/unfolded proteins as result of a specific missense mutation. Our preliminary results suggest that UPR activation is variable among NP-C patients, and this is likely to depend on the mutation type. Several other factors may contribute to this though, which could explain the heterogeneous presentation of this pathology. Additionally, we have investigated the same patients studied in MARS-Seq at the protein and celular levels. Interestingly, and according to recently published work, we observed that, for the analyzed mutations a significant part of the mutated NPC1 protein was retained/ delayed in the ER.