Percorrer por autor "Marques, Isabel"
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- 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.
- 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.
- Fragile X syndrome: intergenerational allele instability and associated phenotypes in familiesPublication . Joana, Loureiro; 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. This disease is caused by a (CGG)n expansion in the 5’UTR of the FMR1 gene, which as a result is methylated and gene silenced. Four classes of alleles can be found based on CGG repeat length: normal (5-44), intermediate (45-54), premutation (55-200) and full mutation (>200). In premutation carriers, both FMR1-related primary ovarian insufficiency (FXPOI) and fragile-X associated tremor/ataxia syndrome (FXTAS) have been described. To gain insights into instability of FMR1 CGG repeats and associated phenotypes, we studied 541 individuals from 128 FXS Portuguese families. DNA samples were genotyped by PCR and Southern blot analysis. Additional clinical evaluation was performed in premutation carriers. Among FXS families, 5.3% intermediate, 29.9% premutation and 26.6% full mutation alleles were found. Normal and intermediate alleles were stable upon transmission. For 115 maternal premutation transmissions, 26 (23%) with alleles ranging 60-98 CGGs remained in premutation size with an average expansion of 17 repeat units, whereas 89 (77%) with alleles ranging from 66-199 CGGs expanded to full mutation. In 44 transmissions of maternal full mutation, the offspring inherited alleles in the full mutation range. For 10 paternal transmissions of premutations, ranging 56-120 CGGs, all daughters inherited a premutation allele, with an average expansion of 7 repeat units. After clinical evaluation of 7 premutation carriers, 1 male with FXTAS and 2 females with FXPOI were identified; however the remaining premutation individuals were not yet examined. In Portuguese FXS families, allele instability upon transmission is in agreement with previous reports. The risk of premutation to full mutation expansion increases with maternal premutation size.
- FRAXE molecular diagnosis in individuals referred for FRAXA screeningPublication . Javed, Ali; G., Ali; Caicedo, Lina; Marques, Isabel; Santos, Rosário; Jorge, PaulaIntroduction: FRAXE mental retardation is a form of mild to moderate intellectual disability generally associated with learning difficulties, communication deficits, attention problems, hyperactivity and autistic behavior. The folate-sensitive fragile site FRAXE (AFF2/ FMR2 gene) is ~600 kb distal to FRAXA (FMR1 gene), which is the most common cause of inherited mental retardation. Normal individuals present 4–39 copies of the polymorphic CCG repeat in AFF2, while individuals expressing the fragile site have >200 copies and the CpG island is fully methylated. Goal and methods: Reports of full expansions and pre-mutations in AFF2 are rarely documented. In this respect, it has been very difficult to determine to what extent the alleles with CCG repeats in the range of 36 to 199, have a pathogenic effect. Intellectually disabled individuals are primarily referred for FRAXA screening and individuals who are negative for FRAXA are possible candidates for FRAXE screening. In order to complement our PCR analysis with Southern blot and hybridization, we cloned a segment of the AFF2 gene that could be used as a labeled probe to determine more accurately the extent of expansion of the CCG repeats. Results and discussion: We have developed a probe to be used for Southern blot analysis that reliably detects the AFF2 CCG triple repeat amplification. We present validation data and results of AFF2 molecular analysis in a subpopulation of 5000 individuals originally referred for FRAXA screening. The presence of pre-mutated and fully expanded alleles in either gender was confirmed by Southern blot analysis, which also enabled evaluation of methylation status and exclusion of repeat number mosaics or PCR failure. We recommend the use of this probe as a method of choice for the detection of AFF2 pre-mutations, full mutations and mosaics, specifically in individuals found to be negative upon FRAXA screening.
- FRAXE molecular diagnosis in individuals referred for FRAXA screeningPublication . Ali, Javed; Ali, G.; Caicedo, Lina; Marques, Isabel; Santos, Rosário; Jorge, PaulaFRAXE mental retardation is a form of mild to moderate intellectual disability generally associated with learning difficulties, communication deficits, attention problems, hyperactivity and autistic behavior. FRAXE (AFF2/ FMR2 gene) a folate-sensitive fragile site in Xq28 ~600 kb distal to the FRAXA (FMR1 gene) site is the most common form of inherited mental retardation. Molecular characterization revealed that individuals expressing FRAXE had amplifications of a CCG repeat adjacent to a CpG island. Normal individuals showed 4–39 copies of the polymorphic FRAXE CCG repeat, while individuals expressing the fragile site had >200 copies and their CpG island was fully methylated. These findings are similar to those found for folate-sensitive fragile X site FRAXA. Reports of FRAXE full expansions and pre-mutations are rarely documented. In this respect, it has been very difficult to determine to what extent the alleles, with CCG repeats in the range of 36 and 199, have a pathogenic effect. Intellectually disabled individuals are primarily referred for FRAXA screening and individuals who are negative for FRAXA are possible candidates for FRAXE screening. Traditionally in some laboratories AFF2 molecular analysis is performed by PCR, it is known that CCG repeats in the range of ~80 and above are not reliably amplified. We embarked on an effort to supplement our PCR analysis by Southern blot and cloned a segment of the AFF2 gene that can be used by appropriate labeling as a probe to determine expansion of the CCG repeats in the AFF2 gene. We have developed a probe to be used for Southern blot analysis that reliably detects the AFF2 CCG triple repeat amplification. We present data of AFF2 molecular analysis in a subpopulation of 5,000 individuals referred for FRAXA screening. The presence of pre-mutated and fully expanded alleles in either gender, were confirmed by Southern blot analysis, which also enabled exclusion of methylation or repeat number mosaics as well as PCR failure. We recommend the use of this probe as suitable for genotyping of pre-mutations, full mutations, and mosaics specifically for individuals presented for FRAXA screening with negative results to determine FRAXE status.
- FXTAS is rare among Portuguese patients with movement disorders: FMR1 premutations may be associated with a wider spectrum of phenotypesPublication . Seixas, Ana; Vale, José; Jorge, Paula; Marques, Isabel; Santos, Rosário; Alonso, Isabel; Fortuna, Ana; Pinto-Basto, Jorge; 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. A total of 86 subjects were genotyped for the CGG repeat in the FMR1 gene. We detected one patient with an expansion in the premutation range. The frequency of FMR1 premutations was 1.9% (1/54) in our group of patients with ataxia as the primary clinical feature, and 1.2% (1/86) in the larger movement disorders group. In the family of the FXTAS case, premutation-transmitting females presented a history of psychiatric symptoms, suggesting that, given the wide phenotypical expression of the premutation in females, neuropsychiatric surveillance is necessary. In conclusion, genetic testing for FXTAS should be made available to patients with adult-onset movement disorders to enable adequate genetic counseling to family members.