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- Molecular analysis of the NR0B1 in three Portuguese families with X-linked Adrenal Hypoplasia CongenitaPublication . Pereira-Caetano, Iris; Toste Rêgo, Ana; Pereira, Bernardo; Portugal, Jorge; Raimundo, Luísa; Gonçalves, JoãoX-linked Adrenal Hypoplasia Congenita (X-linked AHC) is a rare disorder associated with acute adrenal insufficiency in the newborn age that typically cause vomiting, feeding difficulty, dehydration, and shock due to a salt-wasting episode. Hypoglycemia, frequently presenting with seizures, may be the first symptom. If untreated, adrenal insufficiency is lethal. Affected males, despite hormonal treatment, typically have delayed puberty (onset after age 14) caused by hypogonadotropic hypogonadism, and most of them are infertile at adult age. Carrier females may occasionally have symptoms of adrenal insufficiency or hypogonadotropic hypogonadism, possibly caused by skewed X-chromosome inactivation. X-linked AHC is caused by mutations in NR0B1 gene, a critical gene involved in the development of adrenals and hypothalamic-pituitary-gonadal axis. Since the identification of the NR0B1 gene, numerous mutations have been discovered including deletions, alterations of splice-sites, missense, nonsense and frameshift mutations. Here we present the molecular results obtained in three Portuguese families with NR0B1 mutations. Mutation analysis was performed by PCR followed by SSCP analysis and sequencing of DNA fragments showing abnormal patterns on a second PCR product, or by direct DNA cycle sequencing of PCR products. Molecular analysis of the NR0B1 gene in proband A revealed a nonsense mutation, c.1084A>T, p.Lys362*, in exon 1, not previously described. His mother and sister were asymptomatic carriers; in family B a nonsense mutation, c.243C>G; p.Tyr81*, also in exon 1, was identified in two affected males and their mother and sister were also asymptomatic carriers; in family C a frameshift mutation, c.1292delG, p.Ser431Ilefs*6, in exon 2, was detected in a 7 years old affected male and his mother. The maternal origin of mutations was confirmed in the three families studied. The identification of a NR0B1 mutation in a family has important implications: a correct clinical diagnosis can be established, appropriate clinical management of affected members and suitable genetic counselling can be offered, female carriers can be identified and disease can be prevented.
- Detection of somatic mutations in Wilms tumours using gene panel sequencingPublication . Silva, Catarina; Carpinteiro, Dina; Ataíde Sampaio, Daniel; Vieira, LuísIntroduction: Wilms tumour (WT) is an embryonal kidney neoplasia in which the causative mutations are largely unknown. However, approximately one third of patients display somatic mutations in WT1, CTNNB1, TP53 and/or WTX genes, prompting the design of molecular tests to determine the mutational profile of each patient. In this work we describe a novel molecular assay based on next-generation sequencing (NGS) technology which we used to identify mutations in 36 Portuguese WT patients. Methods: Design Studio (Illumina) was used to create a sequencing panel of 83 PCR amplicons covering 12.306 bases of exonic sequences of WT1, CTNNB1, TP53 and WTX genes. Amplicons were prepared from tumour and matched peripheral blood DNA samples (n=73) using a TruSeq Custom Amplicon kit (Illumina). Libraries were sequenced on a MiSeq instrument using paired-end 250 bp reads. Sequence reads were aligned to hg19 human genome reference sequence using MiSeq Reporter software (Illumina). Variants were annotated using publicly available databases. Results: Data analysis of the constitutional DNA of WT patients showed the existence of 31 germline variants, including 9 variants not described in the human dbSNP database. Comparison of matched tumour samples revealed the presence of 14 putative mutations in 12 patients. The mutations included WT1 (n=3), CTNNB1 (n=4), WTX (n=5) and TP53 (n=2). In one patient, concomitant WT1 and CTNNB1 mutations were found. Comparison of results with previous Sanger sequencing data for WT1 and CTNNB1 in the same samples confirmed 5 out of 7 mutations detected by NGS in which the mutated allele frequency was above 20%. Discussion: We conclude that gene panel sequencing is a fast and sensitive molecular assay for identification of recurrent somatic mutations in WT. However, because two thirds of patients lack known mutations, other NGS-based approaches such as exome sequencing may be fruitful to identify novel mutations in WT.
- Environment and Health in Children Day Care Centres (ENVIRH) - Study rationale and protocolPublication . Araújo-Martins, J.; Carreiro Martins, P.; Viegas, J.; Aelenei, D.; Cano, M.M.; Teixeira, João Paulo; Paixão, P.; Papoila, A.L.; Leiria-Pinto, P.; Pedro, C.; Rosado-Pinto, J.; Annesi-Maesano, I.; Neuparth, N.[PT] Antecedentes: A qualidade do ar interior (IAQ) é considerada um determinante importante da saúde humana. A associação entre a exposição a compostos orgânicos voláteis, partículas, ácaros, bolores e bactérias em creches (DCC) não é perfeitamente clara. O objectivo deste estudo foi estudar esses efeitos. Metodologia - desenho do estudo: Este estudo decorreu em duas Fases. A Fase I incluiu uma avaliação de 45 DCCs (25 em Lisboa e 20 no Porto, visando 5.161 crianças). Nesta Fase, foram avaliadas as características dos edifícios, o CO2 e a temperatura ambiente/humidade relativa no interior. Também foi distribuído um questionário de saúde respiratória das crianças derivado do ISAAC (Estudo Internacional sobre a Asma e Alergias em Crianças). A Fase II englobou duas avaliações e incluiu 20 DCCs seleccionadas da fase I, após uma análise de clusters (11 em Lisboa e 9 no Porto, visando 2.287 crianças). Nesta Fase, foram recolhidos dados sobre a ventilação, IAQ (qualidade do ar interior), parâmetros de conforto térmico, saúde em termos respiratórios e alérgicos, marcadores biológicos de inflamação das vias respiratórias, padrões de infecção de vírus respiratórios e stress dos pais e crianças. Discussão: Este documento destaca a metodologia que pode ser implementada por outros investigadores que realizam estudos sobre a associação entre a saúde respiratória e a qualidade do ar interior, em creches e infantários. J. Araújo-Martins, P. Carreiro Martins, J. Viegas, D. Aelenei, M.M. Cano, J.P. Teixeira, P. Paixão, A.L. Papoila, P. Leiria-Pinto, C. Pedro, J. Rosado-Pinto, I. Annesi-Maesano, N. Neuparth
- Accreditation under the International Standard ISO 15189: Experience of a Genetics Laboratory in DNA SequencingPublication . Silva, Catarina; Cardoso, Ana; A Sampaio, Daniel; Carpinteiro, Dina; Mendonça, Joana; Duarte, Sílvia; Barreiro, Paula; Torgal, Helena; Isidro, Glória; Vieira, LuísIntroduction: Health care is to some extent influenced by the results of laboratory tests. In order to provide the best care for the patient, laboratories must seek to achieve high levels of quality and competence. International Standard ISO 15189 specifies these requirements and may be used by laboratories to perform accredited genetic tests of materials derived from the human body. Here we describe the procedures to establish Accreditation of DNA sequencing in our laboratory and the first Accreditation of its kind in Portugal. Methods: Our laboratory started to prepare to comply with ISO 15189 Accreditation requirements for DNA sequencing in 2010. Documents describing administrative and technical procedures of the sequencing workflow including sample registries, laboratory protocols, operation and maintenance of equipments, as well as preparation and use of reagents were produced. Regular examination of laboratory equipments by an external entity was implemented to confirm compliance with working requirements. Requisites for personnel training and demonstration of competence were also implemented. The laboratory participated regularly in the DNA sequencing scheme organized by the European Molecular Genetics Quality Network (EMQN). Results: The laboratory obtained formal recognition by Instituto Português de Acreditação (IPAC) in May 2014. A maximum genotyping score for DNA sequencing has been obtained in the external quality assessment scheme since 2010. Sequencing quality measured in terms of the quality read overlap metrics is currently of approximately 96% according to the EMQN scheme. The laboratory processes and analyzes an average of 28.750 samples per year. Discussion: Accreditation of a genetic test under ISO 15189 is a highly demanding and laborious task for a genetic laboratory. However, it is an important step in order to guarantee the highest quality and reproducibility of genetic test results.
- Advances in phenolic compounds analysis of aromatic herbs and their potential applications.Publication . Carvalho-Costa, D.; Costa, H.S.; Reis, A.R.; Albuquerque, T.G.; Castilho, M.C.; Ramos, F.; Machado, A.V.; Sanches-Silva, A.; PhenolicIntroduction: Herbs can be considered important sources of antioxidants and have a long history of medicinal and culinary applications. The use of plants as sources of antioxidants for nutritional and preservation purposes in the food industry is currently growing. Materials and Methods: An extensive bibliographic review on the methods for analysis of phenolic compounds present in herbs was carried out. Results, Discussion and Conclusion: Plant derived phenolic compounds can be divided in four groups: phenolic acids, phenolic diterpenes, flavonoids and volatile compounds. Considering the first three groups, the identification and quantification of the compounds in the literature is accomplished using High Performance Liquid Chromatography while for volatile compounds, the identification and quantification is accomplished with Gas Chromatography. Several herbs, such as sage (Salvia officinalis), thyme (Thymus vulgaris), rosemary (Rosmarinus officinalis), oregano (Origanum majorana), dandelion (Taraxacum officinale), peppermint (Mentha piperita) and basil (Ocimum basilicum) were analyzed in different studies regarding their phenolic compounds. The major phenolic acids found in herbs were ferulic, caffeic, neochlorogenic and rosmarinic. In terms of phenolic diterpenes, carnosol, rosmanol and carnosic acid were the most reported. Regarding flavonoids, luteolin, quercetin and apigenin were predominant. Concerning volatile compounds, thymol, carvacrol and eugenol were the most common. The herbs with more antioxidant potential regarding their composition on phenolic compounds were compared and the potential of utilisation of these food matrices for newer applications such as active packaging was discussed.
- Classification of the dup 15q13.3 CNV: A National data collectionPublication . Sousa, A.; Serafim, S.; Santos, R.; Custódio, S.; Ávila, M.; Dupont, J.; Dias P, P.; Moldovan, O.; Melo, J.; Ferreira, S.; Pires, L.; Leão, M.; Sá, S.; Prior, C.; Alves, C.; Barreta, A.; Tarelho, A.; Marques, B.; Pedro, S.; Lopes, F.; Maciel, P.; Correia, H.; Dória, S.; Rendeiro, P.; Castedo, S.; Carreira, I.; Sousa, A.B.Introduction: The proximal region 15q11q14 is one of the most unstable regions in the human genome, with six recognizable break points (BP1-BP6). In 15q13.3 there is a recurrent small CNV (BP4-BP5) consisting of a 350-680 Kb duplication, encompassing the CHRNA7 gene, which encodes the alpha 7 subunit of the neuronal nicotinic acetylcholine receptor. Although microdeletions of CHRNA7 are known to cause intellectual disability and neuropsychiatric phenotypes with high penetrance, the patogenicity of CHRNA7 duplications remains unclear. Microduplication 15q13.3 seems to be associated with a phenotypic spectrum of cognitive impairment and neuropsychiatric/neurobehavioral disorders. However, the penetrance of this CNV is considered incomplete since it is present in clinically unaffected individuals in the general population and it is frequently inherited from apparently clinically normal parents. Nonetheless, some pedigree studies have found a history of neuropsychiatric problems among carrier family members. This study aimed at re-evaluating the dup 15q13.3 CNV in national laboratories. Materials and Methods: Our study collected data on 15q13.3 microduplications in eight Portuguese genetics laboratories, among subjects referred for microarray. Results: Here we present a total of seventeen cases with dup 15q13.3. The subjects had somewhat variable phenotypes, with a bias towards developmental delay and autism spectrum disorders. Inheritance was established for eight of the subjects, and the majority originated from the father. We had no access to clinical data on carrier parents. No de novo CNV was found. All laboratories involved classified this variant as of uncertain significance. Discussion/Conclusion: To better determine whether this CNV is benign or pathogenic, careful characterization of patient and control cohorts must be performed, including detailed patient phenotyping, inheritance, clinical evaluation of carrier parents, prevalence in controls, as well as genetic functional studies. We strongly support the creation of a national database for uncertain CNVs in order to clarify the relevance of these recurrent findings, allowing a definitive classification in either pathogenic or benign.
- Dunnigan-type familial partial lipodystrophy in a large Portuguese kindredPublication . Moldovan, O.; Alves, A.C.; Medeiros, A.M.; Sousa, A.B.; Bourbon, M.INTRODUCTION : The lipodystrophies are a clinically heterogeneous group of acquired or inherited disorders affecting adipose tissue distribution. Dunnigan-type familial partial lipodystrophy (FPLD2, OMIM 151660, the most prevalent subtype) is a rare autosomal dominant disease, characterized by selective absence of adipose tissue in the extremities and trunk and accumulation of fat in the face, neck and supraclavicular fossa. The patients have a muscular hypertrophic appearance, especially in the lower limbs. Affected children are born with normal fat distribution, may present hyperlipidemia in childhood and after puberty start to progressively lose the subcutaneous fat. Later in life, affected adults may experience metabolic disorders including hypertriglyceridemia, insulin resistance, diabetes mellitus, hepatic steatosis and high blood pressure. Acanthosis nigricans, hirsutism, menstrual abnormalities and polycystic ovarian disease can also occur in affected women. The phenotype appears more pronounced in females. The aim of this study was to characterize clinically and molecularly a family with clinical diagnosis of lipodystrophy.
- Microarray in clinical practice – utility vs complexity. Mixed phenotype of duplication 15q11.2q13.1 and deletion 16p11.2Publication . Antunes, Diana; Rodrigues, M.I.; Carvalho, I.; Freixo, J.P.; Marques, B.; Pedro, S.; Kay, T.; Correia, H.; Castedo, S.; Nunes, L.Introduction: There’s a consensus to perform chromosomal microarray technique as first-tier clinical diagnostic test for individuals with developmental disabilities. However, given the complexity of clinical presentations, often several diagnostic methods are held before conducting microarray. Method: We report the case of a 5 year-old boy referred to Medical Genetics due to short stature, developmental disabilities and facial dysmorphic features. He was born from eutocic delivery after an uneventful pregnancy. He had psychomotor milestones delayed like sitting at 9 months and walking at 24 months, holding an immature broad-based gait. There was history of learning difficulties from both parents, and the mother has also short stature. On examination it was noted some facial dysmorphic features like high forehead, conical canines and rarefaction of the distal portion of the eyebrows. Due to the history of an episode of transient ataxia, and suspicion of an inherited metabolic disorder, he had already performed various analytical and imaging screenings, all normal. Results: Chromosomal microarray analysis revealed two pathogenic Copy Number Variants (CNV’s): 16p11.2 deletion and 15q11.2q13.1 duplication. The 15q11q13 microduplication syndrome (OMIM # 608636) is a very rare clinical entity with about 30 reported cases with maternal origin, and it is characterized by neurobehavioral disorder, hypotonia, cognitive impairment, epilepsy and short stature. The 16p11.2 microdeletion syndrome (OMIM # 613444) is also a rare clinical entity, with high penetrance, associated with obesity and developmental disabilities. Discussion: Despite the unquestionable utility of microarray, the correlation of the CNV's with the phenotype is often difficult by the rarity of these new microdeletion/duplication clinical entities. In this case the interpretation has increased difficulty because of the simultaneous existence of two distinct clinical entities. Segregation studies, which in the first step include parental analysis, are essential for genetic counseling and determining the risk of recurrence but also for a more accurate correlation genotype-phenotype.
- Differential diagnosis of Autism Spectrum Disorder (ASD) by CNV detection – can early diagnosis be improved?Publication . Kwiatkowska, K.; Conceição, I.C.; Rodrigues, A.C.; Picanço, I.; Marques, I.; Melo, J.; Ferreira, S.; Café, C.; Almeida, J.; Mouga, S.; Oliveira, G.; Vicente, A.M.Autism Spectum Disorder (ASD) is an impairment in neurodevelopment that can be recognized in the first years of life. Symptoms are diverse and vary in severity, determining prognosis and influencing the integration in the community. ASD is characterized by difficulties in interpersonal interaction, verbal and nonverbal communication, and by uncommon interests, inappropriate and uncontrolled behaviors, and repetitive activities. Specific and early diagnosis allows early and effective intervention that improves learning, communication and social skills of autistic children.
- Alterações do Estado de Saúde Associadas à Alimentação: Contaminação Química - MicotoxinasPublication . Alvito, Paula; Oliveira, Luisa; Calhau, Maria Antónia; Dias, Carlos MatiasCom este pequeno apontamento sobre micotoxinas pretende-se contribuir para divulgar o conhecimento técnico e científico disponível sobre a ocorrência destes contaminantes químicos nos alimentos e sobre as alterações do estado de saúde associadas à sua presença nos mesmos. A bibliografia existente neste domínio encontra-se geralmente disponível em língua inglesa pelo que se justifica este pequeno apontamento em português de fácil acesso a profissionais de saúde, investigadores, estudantes universitários e público em geral. Neste documento incluem-se seis secções cujo objetivo consiste em abordar as questões atuais consideradas mais relevantes no domínio das micotoxinas e suas implicações na saúde humana. Na primeira secção é efetuada uma introdução geral sobre as micotoxinas assim como uma referência a importantes desafios futuros no domínio da micotoxicologia. Na segunda, referem-se as características dos principais grupos de micotoxinas e sintetizam-se os resultados de estudos desenvolvidos em Portugal. Na terceira, referem-se as vias de exposição a estas toxinas naturais assim como os seus biomarcadores detetados nos fluidos biológicos (sangue, urina). Na quarta, descrevem-se os quadros clínicos e a abordagem diagnóstica das micotoxicoses (agudas e crónicas) focando, em particular, os efeitos na saúde das crianças. Nas secções cinco e seis referem-se, respetivamente, os aspetos regulamentares e métodos analíticos de determinação de micotoxinas e os efeitos das alterações climáticas no desenvolvimento de espécies de fungos toxigénicos e na produção de micotoxinas. Para além da divulgação dos conhecimentos científicos, a autora pretende alertar o leitor para a importância do tema e para o impacte que a exposição humana a micotoxinas e as alterações do estado de saúde associadas poderão vir a atingir num futuro próximo, em particular, no que se refere às populações mais vulneráveis como as crianças e os grupos sociais mais pobres. A autora inclui também, ao longo do texto, algumas considerações sobre lacunas de conhecimento nos diferentes domínios abordados salientando, por fim, o importante papel que os profissionais de saúde, investigadores, universitários, e público em geral, poderão desempenhar na vigilância e proteção da saúde pública.