Browsing by Author "Cardoso, Rita"
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- Congenital adrenal hyperplasia in paediatric age: molecular analysis of the CYP21A2 gene and implications for genetic counsellingPublication . Gomes, Susana; Silva, Júlia; Pereira-Caetano, Iris; Lopes, Lurdes; Limbert, Catarina; Amaral, Daniela; Pina, Rosa; Kay, Teresa; Sampaio, Lurdes; Pereira, Carla; Moldovan, Oana; Berta, Ana; Rebelo, Irene; Gaspar, Isabel; Cidade Rodrigues, José; Lina, Ramos; Ramos, Fabiana; Dinis, Isabel; Cardoso, Rita; Mirante, Alice; Gonçalves, JoãoIntroduction: Congenital adrenal hyperplasia(CAH) is due to 21-hidroxilase deficiency(21-OHD) in about 95% of the cases. 21-OH is encoded by CYP21A2 gene, and most frequent mutations occurring in CYP21A2 are due to gene conversions originated from its pseudogene(CYP21A1P). The clinical severity of CAH is associated with the impairment of 21-OH activity, which is directly related with the molecular defect. CAH is classified as classic salt-wasting(SW) and simple virilising(SV) forms, and nonclassic(NC) form of the disease. SW and SV are usually diagnosed after birth or during the first years of life, respectively, while most cases of NC-CAH are diagnosed during infancy, puberty or until adult age. Here we present the molecular results performed in paediatric patients with CAH.
- CYP21A2 gene mutations, its nature and frequency in a paediatric Portuguese cohort with congenital adrenal hyperplasiaPublication . Rosmaninho-Salgado, Joana; Caetano, Joana Serra; Gomes, Susana; Pereira-Caetano, Iris; Cardoso, Rita; Dinis, Isabel; Ramos, Lina; Ramos, Fabiana; Carvalho, Ana Luisa; Garabal, Ana; Sá, Joaquim; Maia, Sofia; Sousa, Sérgio; Saraiva, Jorge; Gonçalves, João; Mirante, AliceIntroduction: The most common cause of congenital adrenal hyperplasia (CAH) is 21-hydroxylase deficiency (21-OHD) caused by alterations in CYP21A2 gene. The clinical phenotypes of this autosomal recessive disease are classified as classic (saltwasting and simple virilizing) and non-classic forms of CAH. The severity of the disease is directly related with the impairment of the 21-OH enzymatic activity. Genetic testing can confirm the disease and is crucial for familial studies and genetic counseling. Aim: The aim of this work was to perform the clinical and molecular characterization of the patients observed at the Hospital Pediátrico de Coimbra (Portugal) with the clinical suspecion of CAH. Methods: Retrospective analysis of patient medical records of all cases observed in our hospital with suspicion of CAH and detailed literature comparison. CYP21A2 molecular analysis had been performed in 81 unrelated Portuguese patients (51 female, 30 males) with clinical and endocrine laboratorial findings suggestive of CAH, using mini-sequencing, restriction enzyme digestion, Sanger sequencing or/and multiplex ligation-dependent probe amplification (MLPA). Results: CYP21A2 variants were identified in 74/81 (91%) of the patients. Homozygosity for CYP21A2 was found in 39.2% (29/74) of the patients while 55.4% (41/74) were compound heterozygous and, in 5.4% of the cases (4/74), only one pathogenic variant was identified. The most frequent alterations were p.Val281Leu, g.655A/C>G (splicing variant) and p.Ile172Asn, that account for more than 50% of the alleles of this patient’s cohort. All variants were already described except a novel missense variant identified in a salt-wasting patient, g.1173T>C(p.Trp201Arg). The rare variant p.Gly424Ser which was detected in one patient had been previously associated with a possible founder effect in Brazil and the splicing variant g.391G>A, only described in the Portuguese population. Conclusion: Our study provides a detailed clinical and molecular characterization of a large cohort of CAH Portuguese patients. The overall concordance between the clinical phenotype and the inferred phenotype (based on genotype) was 90%.
- Effect of pre-analytical conditions on prothrombin time and partial activated thromboplastin timePublication . Moutinho, Beatriz; Pinto, Beatriz; Cardoso, Rita; Alves, Helena; Botelho, MónicaClinical analysis often involves clotting assays. Although the guidelines suggest the storing and freezing of samples before these assays, there are contradictory results in the literature. The objective of this study was to analyse the effect of the temperature and the storage of plasma sample on Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT) in clinical samples for 65 patients without coagulation disorders.
- Espectro de alterações moleculares detetadas no gene CYP21A2 associadas a deficiência em 21 hidroxilasePublication . Gomes, Susana; Pereira-Caetano, Iris; Lopes, Maria Lurdes; Limbert, Catarina; Amaral, Daniela; Pina, Rosa; Antunes, Diana; Carvalho, Inês; Kay, Teresa; Sampaio, Lurdes; Pereira, Carla; Moldovan, Oana; Berta, Ana; Rebelo, Irene; Gaspar, Isabel; Cardoso, Helena; Rodrigues, José Cidade; Ramos, Lina; Ramos, Fabiana; Dinis, Isabel; Cardoso, Rita; Mirante, Alice; Gonçalves, JoãoA maioria dos doentes com hiperplasia suprarrenal congénita (HSC) apresenta alterações moleculares no gene CYP21A2, o qual codifica a enzima 21-hidroxilase (21-OH). Os doentes com a forma clássica de deficiência em 21-OH (21-OHD) apresentam a síntese de cortisol diminuída no córtex adrenal e, os casos mais graves, também apresentam deficiência de aldosterona. As mulheres com 21-OHD grave apresentam excesso de andrógenos desde a sua vida fetal conduzindo à virilização dos órgãos genitais externos. Tanto homens como mulheres com 21-OHD completa não sintetizam a aldosterona e, consequentemente, logo após o nascimento, podem desenvolver crises de perda de sal se não forem corretamente diagnosticados e tratados. A 21- OHD não clássica é devida à deficiência parcial em 21-OH, os fenótipos clínicos são menos graves, as mulheres não apresentam virilização dos genitais externos ao nascimento, e geralmente os sinais relativos a excesso de androgénios podem surgir durante a infância ou até mais tarde (durante ou após a puberdade). Neste trabalho descrevem-se as alterações e os genótipos mais frequentes encontrados em doentes portugueses não adultos com 21-OHD. As alterações mais frequentes encontradas na forma clássica da HSC são c.293-13C> G, diferentes deleções/quimeras/conversões génicas do gene CYP21A2 e c.518T> A, enquanto na 21-OHD não-clássica a variante c.844G> T é a mais frequente. Estes resultados contribuem para um diagnóstico correto e uma melhor gestão clínica dos doentes, para o seu aconselhamento genético e para oferecer o diagnóstico pré-natal a casais com risco de ter filhos afetados com a forma clássica de 21-OHD.
- In Vitro isolation and seroprevalence of Toxoplasma gondii in stray cats and pigeons in Lisbon, PortugalPublication . Waap, Helga; Cardoso, Rita; Leitão, Alexandre; Nunes, Telmo; Vilares, Anabela; Gargate, Maria João; Meireles, José; Cortes, Helder; Ângelo, HelenaOral contamination with Toxoplasma gondii oocysts shed by cats into the environment has been linked to severe outbreaks of human toxoplasmosis. Pigeons (Columba livia) are highly susceptible to oral infection with oocysts and indirectly indicate soil contamination, since they feed from the ground. A seroprevalence study was performed on cats and pigeons captured in the city of Lisbon. Serum samples collected from 1507 pigeons captured at 64 feeding sites and 423 stray cats were screened for antibodies anti-T. gondii using a commercial direct agglutination test. Seroprevalence in pigeons was 2.6% (39/1507) (95% CI: 1.9-3.5%) and 37.5% (24/64) of pigeon flocks sampled showed to be infected with T. gondii. The proportion of infected pigeons within seropositive flocks ranged between 4.8% and 21.1%. Among cats, seroprevalence was 44.2% (187/423) (95% CI: 39.5-49.1%). Isolation of T. gondii from animal tissues was attempted by in vitro assay. Inoculation of brain homogenates from 20 pigeons and 56 cats into Vero cell cultures allowed isolation of T. gondii from 13 pigeons (65%) and 15 cats (26.8%). Inoculation of muscle homogenates (heart and limbs) prepared by acid-peptic digestion from a subset of 15 cats resulted in the recovery of T. gondii from 10 cats (66.7%). Copyright © 2012 Elsevier B.V. All rights reserved.
- Parasites in Forensic Science: a historic perspectivePublication . Cardoso, Rita; Alves, Helena; Richter, Joachim; C. Botelho, MonicaParasites show a great potential to Forensic Science. Forensic Science is the application of any science and methodology to the legal system. The forensic scientist collects and analyses the physical evidence and produce a report of the results to the court. A parasite is an organism that lives at the expense of another and they exist in any ecosystem. Parasites are the cause of many important diseases. The forensic scientists can use the parasites to identify a crime scene, to determine the murder weapon or simply identify an individual. The applications for parasites in the Forensic Science can be many and more studies should be made in Forensic Parasitology. The most important parasites in Forensic Science are helminths specifically schistosomes. Through history there are many cases where schistosomes were described in autopsies and it was related to the cause of death. Here we review the applications of parasites in Forensic Science and its importance to the forensic scientist.
- Relevance of Multigene Panels in the Molecular diagnosis of patients with disorders of sexual developmentPublication . Pereira-Caetano, Iris; Mendonça, Joana; Mirante, Alice; Cardoso, Rita; Rodrigues, Márcia; Oliveira, João Paulo; Grangeia, Ana; Barbosa, David; Vieira, Luís; Gonçalves, JoãoIntroduction: Next generation sequencing (NGS) is increasingly used in the molecular diagnosis of rare diseases, such as disorders of sexual development (DSD), allowing the analysis of a greater number of genes in a single assay, providing faster results with reduced costs. DSD patients may present high phenotypic overlap (genital ambiguity, sex reversal, delayed/absent puberty, infertility) posing challenges to clinical diagnosis. NGS technology using multigene panels has higher hypothesis to identify the genetic cause and novel genetic variants in a large number of cases. Methodology: 33 genomic DNA samples from DSD patients were sequenced on MiSeq using the Ampliseq technology (Illumina). A customized gene panel covering 40 genes was used to prepare the libraries of the target sequences. The 40 genes were subdivided into 5 subpanels: primary sex determination, sex differentiation, hypogonadotropic hypogonadism/infertility, steroidogenesis and premature ovarian insufficiency. Variant classification, according to ACMG-AMP, was based on bioinformatics tools (ex. VEP, HSF, VarSome, Alamut) and databases (gnomAD, HGMD, ClinVar, dbSNP). Pathogenic, Likely pathogenic and VUS variants were confirmed by Sanger sequencing. Results: 16 of the 33 patients were previously studied in our laboratory with negative results for the main genes associated with DSD (SRY, AR, ANOS1, GNRHR, CYP21A2). We identified 9 causative variants (8P/1LP) in 7 patients (21.2%) in AR, HSD17B3, LHCGR, FGFR1 and NR5A1. One patient was a compound heterozygous for HSD17B3 and another simultaneously homozygous for LHCGR and hemizygous for AR. The remaining 5 were homozigous, heterozigous or hemizygous for HSD17B3, LHCGR, NR5A1, FGFR1 or AR. One VUS, FGFR1:c.566G>A p.Arg189His, requires familial studies to revaluate its pathogenicity. Discussion: Multigene NGS studies allows to increase the rate of variant detection, mainly in genes not included in a first molecular approach. It also contributes to establishing or confirming the clinical diagnosis, assisting in decisions regarding the treatment and reproductive management of patients and families, as well as in genetic counselling.