Browsing by Author "Jorge, Paula"
Now showing 1 - 10 of 19
Results Per Page
Sort Options
- Caracterização molecular do gene TPO em crianças Portuguesas com hipotiroidismo congénito causado por disormonogénesePublication . Nogueira, Célia; Vaz Osório, Rui; Santos, Rosário; Jorge, PaulaCongenital hypothyroidism (CH) affects about 1:4000 infants and is considered one of the main causes of preventable mental retardation in children. Universal screening of CH performed through the Portuguese National Neonatal Screening Programme, implemented in Portugal in 1985, has resulted in normal development of attained children. Birth defects of the thyroid can be divided into several groups that represent either changes in the development of the gland or the consequences observed in the deficient synthesis of thyroid hormones. The defects of hormone synthesis caused by dyshor - monogenesis occur in only 10% to 15% of cases of HC. Defects in the thyroid peroxidase (TPO) gene are reported to be one of the most frequent causes of CH due to dyshormonogenesis. The aim is to review the mutational spectrum of the TPO gene in the portuguese population through the molecular investigation of 69 patients with permanent CH due to dys - hormonogenesis. To complement previous results, published in 2005, this work describes the molecular characterization of a further fourteen children with CH and the methodology applied. Extensive in-silico analysis was carried out for the newly identified sequence changes as well as the formerly published putative splicing variant. The sequence variations identified in the TPO gene comprise ten distinct mutations and 29 polymorphisms, enabling the determination of the molecular etiology of CH in fifteen patients. In conclusion, it was possible to obtain a differential diagnosis in twelve fami - lies with CH, using a non-invasive procedure and without interruption of medication. Identification of these and other mutations in the TPO gene can therefore contribute considerably towards diagnosis, a precise genetic counselling, adequate monitoring in future pregnancies as well as putative personalized therapies.
- Cumulus cell DNA damage linked to fertilization success in females with an ovulatory dysfunction phenotypePublication . Rodrigues, Bárbara; Sousa, Vanessa; Esteves, Filipa; Vale-Fernandes, Emídio; Costa, Solange; Sousa, Daniela; Brandão, Raquel; Leal, Carla; Pires, Joana; Gaivão, Isabel; Teixeira, João Paulo; Nogueira, António J.A.; Jorge, PaulaIntracytoplasmic sperm injection (ICSI) is a widely used technique in fertility centers. ICSI success depends on both nuclear and cytoplasmic oocyte maturation. Cumulus cells, which surround the oocytes, play a pivotal role in oocyte competence. However, the significance of DNA damage in cumulus cells as a marker of fertilization success remains largely unexplored. This study aims to investigate the relationship between DNA damage in cumulus cells of females undergoing ICSI, and oocyte competence, with a focus on in vitro fertilization (IVF) outcomes. We employed the alkaline comet assay to assess DNA damage levels (%TDNA) in cumulus cells and whole blood from 22 potentially fertile females and 35 infertile females, including 20 with an ovulatory disfunction phenotype. Our results revealed significant differences between the levels of %TDNA in cumulus cells and blood. Females with an ovulatory dysfunction phenotype exhibited higher levels of %TDNA in cumulus cells compared to potentially fertile females. Additionally, within the group of females with ovulatory dysfunction, a significant correlation was observed between %TDNA levels and the number of oocytes with two pronuclei. Our findings suggest that blood does not accurately reflect DNA damage in cumulus cells, which was correlated with the fertilization success in females with ovulatory dysfunction. High levels of %TDNA in cumulus cells were associated with a higher likelihood of successful fertilization. Moreover, our results imply that low levels of %TDNA may be linked to oocytes that fail to complete maturation and, consequently, do not fertilize (oocytes with zero pronuclei). Further research with larger cohorts is necessary to validate these findings and to explore potential applications in female fertility. However, our study provides evidence that DNA damage in cumulus cells may serve as a valuable biomarker for predicting fertilization success and oocyte competence.
- Cumulus cells damage can help to indirectly predict oocyte quality in infertile females undergoing ICSIPublication . Rodrigues, Bárbara; Sousa, Vanessa; Esteves, Filipa; Pires, Joana; Sousa, Daniela; Brandão, Raquel; Leal, Carla; Santos, Rosário; Vale-Fernandes, Emídio; Nogueira, António; Costa, Solange; Jorge, PaulaIntroduction: Intracytoplasmic sperm injection (ICSI) is currently used in clinical practice for couples with fertility issues. Some studies have shown an association between male reproductive ability and sperm DNA damage levels, assessed by comet assay. However, little is known regarding this endpoint and female fertility, mostly due to tissue accessibility. To overcome this, we used cumulus cells (CC) to analyze DNA damage in search of correlation with clinical parameters evaluated in the context of infertility. Methodology: DNA damage was assessed via comet assay, in two different tissues, blood and CC, from females undergoing ICSI: 22 potentially fertile and 35 infertile. DNA damage levels (%TDNA) were compared between the two groups (fertile vs infertile), and correlated, within each group, with hormone levels, stimulation days, number of cumulus-oocyte complexes (COCs) retrieved, and oocytes injected. All analysis were performed using SigmaPlot version 14.0 (Systat Software®Inc., Chicago, IL, United States). Results: No significant differences were found in %TDNA levels between the 2 groups. However, the DNA damage observed in CC was notably increased in the infertile females when compared to the potentially fertile, although it did not reach statistical significance. Interestingly, %TDNA in CC was significantly correlated with the number of oocytes injected, in both groups. This finding was not observed in the blood. Moreover, the difference between number of COCs retrieved and oocytes injected was significantly higher in the infertile females group and showed a correlation with the damage observed in CC. Discussion: Our results established a correlation between DNA damage in CC and oocyte quality. CC support and nurture oocytes during development, but DNA damage in CC can predict a reduced oocyte quality and availability for injection. This finding underscores the importance of CC in oocyte development and emphasizes the need to consider tissue-specific effects in DNA damage studies, particularly those related to fertility and reproductive health. Nevertheless, further studies are needed to confirm our results.
- Desenvolvimento Neuropsicológico na Síndrome de X-Frágil: interpretar os perfis de desenvolvimento.Publication . Carmona, Carla; Marques, Isabel; Santos, Rosário; Jorge, PaulaIntrodução: A Síndrome de X Frágil (SXF) é uma doença genética causada por uma mutação dinâmica da região repetitiva constituída por tripletos CGG no gene FMR1, que leva à ausência da FMRP. Na população normal o número de CGGs oscila entre os 5 e os 45. A maioria dos doentes com SXF apresenta para além da expansão > 200 CGG, uma inativação do gene FMR1 por metilação do seu promotor (mutação completa). A principal manifestação da SXF é o atraso mental ou défice intelectual que varia de ligeiro a grave. Outros sinais incluem o atraso de desenvolvimento psicomotor, alterações de comportamento, a hiperatividade, o défice de atenção, dificuldades de aprendizagem e comportamento autista. Objetivo e métodos: Existe um grupo de doentes, menos comum, que revela ausência (total ou parcial) de metilação do FMR1 e níveis reduzidos da FMRP, denominados high-functioning males (HFM). Um outro conjunto engloba doentes, designados mosaicos (MoPMMC), que para além da mutação completa apresentam, em algumas células, um número de repetições inferior aos 200 (pré-mutação). Estes subgrupos são, sob o ponto de vista investigacional, particularmente interessantes pois apresentam características fenotípicas e genéticas imprevisíveis. O objetivo deste trabalho é analisar e comparar os níveis de desenvolvimento neuropsicológico, avaliados a partir da escala de desenvolvimento psicomotor e o perfil cognitivo, avaliado com as escalas de inteligência, de portadores da SXF e MoPMMC, de ambos os sexos. Resultados e Discussão: A análise detalhada dos perfis dos testes permite compreender a forma como as diferentes mutações podem influenciar no desenvolvimento psicomotor e cognitivo das crianças com SXF. Esta investigação neurogenética terá um importante impacto na seleção de uma futura aproximação terapêutica, no funcionamento e na qualidade de vida destes indivíduos, bem como ajudar reduzir os encargos para as famílias, seus cuidadores e a sociedade.
- Evaluating the influence of four variants detected in the FRAXA and FRAXE lociPublication . Marques, Isabel; Jorge, Paula; Loureiro, Joana; Santos, RosárioOf the seven folate-sensitive fragile sites cloned in the human genome, only two have a proven clinical expression, FRAXA and FRAXE, the former with a well-documented clinical impact. The expansion of over 200 [CGG] triplets in the Fragile Mental Retardation 1 gene (FMR1), FRAXA locus, is associated with the Fragile X Syndrome (FXS), the most common form of familial severe mental retardation/intellectual disability. The prevalence of FRAXE full mutations is much lower, and is frequently associated with non-syndromic X-linked mental retardation (FRAXE-MR). This phenotype is due to the silencing of the Fragile Mental Retardation 2 gene (AFF2), as a consequence of a [CCG] expansion to more than 200 hyper-methylated triplets located upstream of the gene. Molecular diagnosis of FXS and FRAXE-MR typically rely on techniques such as PCR (for pre-screening), Southern blotting and linkage analysis based on microsatellite markers. The latter is of particular interest in atypical or complex cases. Additional molecular tools are also currently available such as fluorescent methylation-specific PCR, Multiplex Methylation-Specific Real-Time PCR and Methylation-specific MLPA (Multiplex Ligation-dependent Probe Amplification). In the course of FXS and FRAXE-MR molecular diagnosis using standard molecular methodologies, four variants were identified. Three of them are in the FRAXA locus, two in the 5’UTR region of the FMR1 gene: NM_002024.5: c.-412G>C and NM_002024.5:c.-68T>G; and one located ~7kb upstream the FMR1 gene: g.146986184_146986185insAAGCAGA in the amplified region of the polymorphic marker FRAXAC1. The remainder is in the FRAXE locus, positioned in AFF2 gene promoter region: NT_011681.16: c.-3101G>A. Herein, we describe the characterization of these four variants and illustrate how, besides increasing genetic diversity, they in fact influence the interpretation of results in the context of FXS or FRAXE-MR diagnosis.
- FMR1 premutations may be associated with a wider spectrum of phenotypesPublication . Seixas, Ana; Vale, José; Martins, Márcia; Loureiro, Joana; Jorge, Paula; Maques, Isabel; Santos, Rosário; Coutinho, Paula; Margolis, Russell; Sequeiros, Jorge; Silveira, IsabelThe fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by expansions of 55-200 CGG repeats in the 5’UTR of the FMR1 gene. These FMR1 premutation expansions have relatively high frequency in the general population. To estimate the frequency of FMR1 premutations among Portuguese males with non-familial, late-onset movement disorders of unknown etiology, we assessed CGG repeat size in males with disease onset after the age of 50 and negative or unknown family history for late-onset movement disorders, who were sent for SCA, HD, or PD genetic testing at a reference laboratory. The selected patients had a primary clinical diagnosis based on one of the following cardinal features of FXTAS: ataxia, tremor, or cognitive decline.
- Fragile X mental retardation 1 (FMR1) premutations: instability and associated phenotypesPublication . Loureiro, Joana; Jorge, Paula; Marques, Isabel; Santos, Rosário; Seixas, Ana; Martins, Márcia; Vale, José; Sequeiros, Jorge; Silveira, IsabelFragile X syndrome (FXS) is the most common hereditary form of intellectual disability with an estimated frequency of 1/4000 males and 1/8000 females. FXS is caused by a (CGG)n expansion of over 200 repeats, in the 5’UTR of the FMR1 gene, which as a result is usually methylated and the gene silenced. Based on CGG repeat length, four classes of alleles can be distinguished: normal (5-44), intermediate (45-54), premutation (55-200; PM) and full mutation (>200; PM) alleles. Premutations expand to full mutation alleles only via maternal transmission and larger premutations have an increased risk of expansion to full mutation. Paternal premutations and full mutations are inherited in the premutation range. The aim of this study is to gain insights into instability of FMR1 CGG repeat alleles and associated phenotypes in 128 Portuguese FXS families.
- Fragile X mental retardation 1 (FMR1) premutations: instability and associated phenotypesPublication . Loureiro, Joana; Jorge, Paula; Marques, Isabel; Santos, Rosário; Seixas, Ana; Martins, Márcia; Vale, José; Sequeiros, Jorge; Silveira, IsabelFragile X syndrome (FXS) is the most common hereditary form of intellectual disability with an estimated frequency of 1/4000 males and 1/8000 females. FXS is caused by a (CGG)n expansion of over 200 repeats, in the 5’UTR of the FMR1 gene, which as a result is usually methylated and the gene silenced. Based on CGG repeat length, four classes of alleles can be distinguished: normal (5-44), intermediate (45-54), premutation (55-200; PM) and full mutation (>200; PM) alleles. Premutations expand to full mutation alleles only via maternal transmission and larger premutations have an increased risk of expansion to full mutation. Paternal premutations and full mutations are inherited in the premutation range. The aim of this study is to gain insights into instability of FMR1 CGG repeat alleles and associated phenotypes in 128 Portuguese FXS families.
- Fragile X Mental Retardation Protein: broadening the possibilities for studying Fragile X SyndromePublication . Oliveira, Bárbara; Marques, Isabel; Loureiro, Joana; Santos, Rosário; Jorge, PaulaThe presence of chromosomal fragility in locus FRAXA, located at Xq27.3, is directly related with Fragile X Syndrome (FXS), where the main symptoms include intellectual and emotional disabilities. The main cause of this monogenic disorder is the transcriptional silencing of the FMR1 gene, due to an expansion of more than 200 CGG repeats, found in the 5’-untranslated region, and its consequent hypermethylation which extends to the promoter region. The diagnostic complexity of FXS is proportional to the heterogeneity underlying this disease. In situations that strongly suggest a clinical diagnosis of FXS, but in which the repetitive region is not expanded, studying the presence of the encoded protein has proved to be very helpful as a complement to the molecular diagnosis. The Fragile X Mental Retardation Protein (FMRP), a selective RNA-binding protein that negatively regulates local protein synthesis in neuronal dendrites, may be detected applying specific antibodies either by immunocytochemistry or Western Blot analysis. The aim of the present work was to optimize such techniques, so as to complement the routine molecular procedures employed in prenatal and postnatal FXS diagnosis. In order to test the efficacy of the procedure, different types of biological samples were used, namely leukocytes from peripheral blood, human brain tissue, cultured amniocytes and chorionic villi. Additionally, slide preparation and detection method for immunocytochemistry, as well as protein isolation for Western Blot, were optimized resorting to several approaches. Both immunocytochemistry and Western Blot techniques allowed the detection of FMRP and were equally suitable. The advantages and disadvantages of the implementation of these techniques in terms of laboratory workflow and specimen type as well as in diagnostic and research context are discussed herein.
- Fragile X Syndrome: Genetic BackgroudsPublication . Loureiro, Joana; Marques, Isabel; Oliveira, Bárbara; Amorim, António; Santos, Rosário; Jorge, PaulaFragile X Syndrome (FXS) is the most frequent hereditary form of mental retardation, caused by an expansion of polymorphic [CGG] repeats in the 5’UTR region of the FMR1 gene; the molecular mechanism of this expansion is, however, still unknown. Based on [CGG] triplet number, three allele classes can be distinguished: normal sized-alleles (5-50 repeats); pre-mutation alleles (50-200 repeats) and the full mutation where alleles have an expansion of over 200 CGG repeats. Previous studies using Short Tandem Repeat (STR) haplotypes of mutant chromosomes in diverse populations revealed founder effects based on linkage disequilibrium between CGG repeats and flanking molecular markers.