Browsing by Author "Loureiro, Joana"
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- 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.