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- A retrospective study of Down syndrome in prenatal diagnosis. Did chorionic villus sampling allow a better prevention?Publication . Simão, Laurentino; Silva, Marisa; Brito, Filomena; Alves, Cristina; Marques, Bárbara; Ferreira, Cristina; Ambrósio, Paula; Silva, Maria do Céu; Ventura, Catarina; Duarte, Guida; Caetano, Paula; Correia, Joaquim; Melo, AntonietaIntroduction Down syndrome (DS) is the most common single genetic cause of human moderate mental retardation, with an estimated prevalence of 9.2 cases per 10,000 live births. We aimed at analyzing changes in prenatal diagnosis (PND) over time, namely the referral reasons for chromosome analyses and the introduction of chorionic villus sampling (CVS), and its influence on the results obtained in DS cases. Methods We retrospectively evaluated the PND results from samples analyzed between 1987 and 2011 (25 years) in our cytogenetic laboratory taking into account the referral reasons, type of sample, karyotype and reporting time. Results 263 fetuses with a karyotype compatible with DS were identified in a total of 18,107 karyotypes (1.5%). The highest frequencies of DS were found among cases referred because of ultrasonography findigs (namely increased nuchal translucency) or positive first trimester screening and when one parent carries a chromosomal rearrangement. The frequency of recurrence was found to be 1/72. The increasing use of CVS led to an earlier response in terms of gestational age (mean at diagnosis- 13+4 weeks). In addition, an increased percentage of karyotypes with SD was detected (8.4% of CVS samples). On the other hand, implementation of molecular rapid aneuploidy detection in part of the samples allowed a better report time in DS cases, from 23 days in 1987 to 2 days in 2011. Discussion DS detection remains the most important reason for performing PND. The collecting of CVS has been rising over the last years, which has resulted in an increased number of trisomy 21 cases identified in a lower gestational age, allowing a better karyotype-phenotype correlation in earlier pregnancies. Moreover, the use of complementary molecular techniques for the detection of common aneuploidies reduced the mean reporting time and allowed an earlier decision of the couple concerning the future of gestation.
- Clinical, cytogenetic and molecular findings of a “de novo” inv dup del (6q)Publication . Fonseca Silva, M.L.; Mota Freitas, M.; Candeias, C.; Ribeiro, J.; Oliva Teles, N.; Soares, G.; Tkachenko, N.; Marques, B.; Correia, H.Introduction: Complex rearrangements resulting in inverted duplications contiguous to a terminal deletion (inv dup del) were first reported for the short arm of chromosome 8 in1976. Since then this type of structural anomaly has been described for an increasing number of chromosomes. In these rearrangements, the concomitant presence of a deletion and a duplication has important consequences in genotype-phenotype correlations. The authors describe the clinical findings and the cytogenetic characterization of a rare inv dup del involving the long arm of chromosome 6. Material and methods: A girl aged 5 was referred for subtelomeric studies with the indication of psychomotor retardation, autistic features and stereotipies. Chromosome analysis with high resolution GTL-banding was performed on metaphases obtained from cultured peripheral blood lymphocytes. Molecular studies included MLPA (Kits P036 and P070, MRC-Holland), FISH with subtelomeric and whole chromosome painting probes specific for chromosome 6, and cCGH techniques. Results: Initial MLPA studies detected a subtelomeric deletion in the long arm of chromosome 6; the subsequent karyotype revealed a structurally abnormal chromosome 6 with additional material in the end of the long arm. FISH analysis showed the deletion and demonstrated that the extra material was derived from chromosome 6; cCGH tecnhiques defined the extension and confirmed the breakpoints of the duplicated segment. Thus this rearrangement was interpreted as an inv dup del (6q). Since parental karyotypes were normal, this anomaly was considered “de novo”. Discussion: As far as we know this is the first description of a patient presenting with a “de novo” inv dup del (6q). We compare the clinical features in this child with the previously reported cases with either an isolated terminal deletion or a duplication of distal 6q. The authors enhance the importance of the combination of high resolution banding with molecular studies in the characterization of this rare rearrangement.
- 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.
- Interstitial deletion 15q21 and Prader-Willi like syndrome phenotype: Case reportPublication . Pires, S.; Oliva Teles, N.; Ribeiro, J.; Mota Freitas, M.; Marques, B.; Reis, G.; Correia, H.; Fonseca e Silva, M.L.Introduction: Chromosome 15q interstitial deletions not involving the Prader-Willi/Angelman region are uncommon and poorly characterized. Very few cases of different segmental losses involving the 15q21 region have been reported at cytogenetic level. All the described patients present with moderate to several mental retardation and characteristic facial dysmorphic features. Some authors compare the similarity between the phenotype of these patients with some features of Prader-Willi syndrome (PWS). Methods: We report the case of a girl aged 8 referred for conventional cytogenetics and fluorescence in situ hybridization (FISH) for the PWS region, presenting with mental retardation, almond-shaped eyes, obesity, small hands with short fingers and diminished pigmentation of the hair. Results: The chromosomal analysis revealed an interstitial deletion of the long arm of chromosome 15, apparently between 15q21 and 15q22. Deletion at 15q11.2 (Prader-Willi/Angelman critical region) was excluded by FISH. To establish the exact breakpoints molecular studies were performed using bacterial artificial chromosome (BAC) clones spanning the 15q21.3 region. The absence of signal in this region defines the proband’s final karyotype as: 46,XX,del(15)(q21.3q21.3).ish del(15)(q21.3q21.3)(bA74K1-) Discussion: The authors emphasize the importance of complementary FISH and molecular studies in chromosomal abnormalities and compare the proband’s phenotype with similar cases described in the literature.
- Infecção pelo Citomegalovírus durante a Gravidez e no Recém-NascidoPublication . Lopo, SílviaDistribuição, epidemiologia, estudos de prevalência de anticorpos e de frequência de infeção congénita pelo CMV.Importância do rastreio serológico para o CMV em exame antenatal, durante a gravidez e do estudo pós-natal de infeção congénita pelo CMV no recém-nascido.Descrever as metodologias laboratoriais disponíveis para o diagnóstico do CMV, na grávida e no recém-nascido.Considerar o contributo do laboratório na análise dos resultados e a articulação do diagnóstico laboratorial com o diagnóstico clínico.Sugestões para revisão bibliográfica.
- Distribution of limb girdle muscular dystrophy subtypes among Portuguese patientsPublication . Vieira, EmíliaAlthough the limb girdle muscular dystrophies (LGMD) are collectively characterized by progressive muscle weakness, they exhibit wide clinical and genetic heterogeneity. Over 23 loci have been identified; eight are autosomal dominant subtypes (LGMD1A-1H) and fifteen are autosomal recessive (LGMD2A-2O). The prevalence and relative distribution of the different subtypes varies widely between populations. Here we present an overview of the LGMD profile in our population, as ascertained in patients referred from all over the Country over a period of 15 years, with clinical phenotypes ranging from severe childhood-onset LGMD, adult-onset LGMD and distoproximal myopathy, to barely symptomatic presenting only hyperCKemia. Inclusion criteria were essentially compatible clinical presentation and/or muscle biopsy (histology and immunocytochemistry). A total of 425 families were selected, representing 525 genetic tests for different genes: CAPN3 (n=87), DYSF (n=105), SGCG (n=94), SGCA (n=46), SGCB (n=39), SGCD (n=18), TCAP (n=6), FKRP (n=55), TTN (n=4), ANO5 (n=11), MYOT (n=5), LMNA (n=46), and CAV3 (n=9). Differential diagnosis was achieved in 183 unrelated families, where a total of 92 different mutations were identified, 44 of which were novel. The gamma-sarcoglycanopathies were the most frequent subtype (13.4%), clearly as a result of a founder effect among families of gypsy (Roma) ethnicity and the presence of a frequent mutation thought to be of North African origin. This was followed by the dysferlinopathies (10.4%), with no particular hotspot along the 56 exons, except for two private mutations. All other forms detected each represented under 5% of the cases, and 56.9% remained genetically unresolved. The profile of our patients differs slightly from that of other large cohorts in studies reported for the European, North American, Australian and Brazilian populations. In this group of disorders with wide genetic heterogeneity, knowledge of the subtype and mutational spectrum in our population facilitates the diagnostic approach.
- The 5´ Untranslated Region of the mammalian Target of Rapamycin mRNA Harbors an Internal Ribosome Entry SitePublication . Marques-Ramos, Ana; Teixeira, Alexandre; Lacerda, Rafaela; Romão, Luísa
- Identification of a mosaic non-inherited small supernumerary ring chromosome 2: cytogenetic-molecular studies and genotype-phenotype correlationPublication . Oliveira, F.P.; Ribeiro, J.; Mota Freitas, M.; Oliva Teles, N.; Bártolo, A.; Correia, H.; Fonseca e Silva, M.L.Introduction: The identification of supernumerary marker chromosomes (SMCs) derived from all the autosomes is currently possible, but rarely by conventional cytogenetics alone. Supernumerary ring chromosomes (SRCs) account for about 10% of these cases. SRCs derived from chromosome 2 are unusual, and there are only a few cases reported in the literature. The severity of the phenotype depends on the type of the mosaicism, the percentage of cells affected by the genetic change and the chromosome involved. Methods: The authors report the case of a boy aged 8 referred for cytogenetic studies, presenting with behavior and learning problems, mental retardation with uncoordenated speech, attention deficit and hyperactivity (PHDA), as well as small slanting palpebral fissures. The karyotype was obtained from peripheral blood lymphocyte cultures using high resolution GTL banding and standard techniques. Fluorescence in situ hybridization (FISH) was performed using specific probes for the centromeric regions of all chromosomes (Chromoprobe Multiprobe - ISystem). Results: Cytogenetic analysis revealed two cell lines: one with a supernumerary marker ring chromosome, 47,XY,+r (52%), and a normal cell line, 46,XY (48%). The SRC was identified by FISH with the chromosome 2 centromeric probe. Since the parents had normal karyotypes, this abnormality was “de novo”. Final karyotype of the proband was: mos 47,XY,+r[26]/46,XY[24].ish r(2)(D2Z2+)dn. Discussion: The clinical description of this patient is in agreement with other reports of the literature. Molecular characterization by FISH analyses is an useful way of investigating the presence of euchromatin contained in a SMC and establishing new chromosomal syndromes. However, to better characterize this ring, in order to establish a more accurate genotype-phenotype correlation, more studies involving other technologies should be performed, thus allowing suitable genetic counselling
- MLH1 and DCC, two genes involved in colorectal cancer, have different mechanisms of translation initiationPublication . Lacerda, Rafaela; Marques-Ramos, Ana; Teixeira, Alexandre; Luísa Romão, LuísaIntroduction: Colorectal cancer (CRC) is one of the leading causes of death worldwide and its development and progression can be dependent upon regulation of gene expression. MLH1 and DCC are two genes whose expression is altered in CRC. MLH1 is a mismatch repair gene and its expression is repressed in CRC. DCC is a tumor suppressor gene that has one allele deleted in CRC. Regulation of gene expression can occur at translation level, namely at the initial step. Translation initiation can be cap-dependent or internal ribosome entry site (IRES)-dependent. IRESs are structures within the 5’ untranslated region (UTR) of the mRNAs that directly recruit the ribosome without scanning the 5’UTR. This work aims to investigate whether MLH1 and DCC transcripts are translated via IRES or if their translation is exclusively cap-dependent. Methods: The MLH1 and DCC 5’UTRs were cloned in the dicistronic p_Renilla_Firefly vector. NCM460 cells (derived from normal intestinal mucosa) and SW480 cells (derived from stage IV CRC adenocarcinoma) were transfected with such plasmids. Luciferase activity was measured by luminometry assays and compared to the negative control (dicistronic vector without insert). Results and Discussion: The levels of Firefly luciferase observed when DCC 5’UTR is present are similar to those of negative control in both cell lines, which means translation of this transcript is exclusively cap-dependent and not mediated by IRES. Regarding MLH1 5’UTR-containing plasmid, the levels of Firefly luciferase observed are 70-fold greater than that of negative control in NCM460 cells and 18-fold greater in SW480 cells, which might indicate IRES activity. However, when cells were transfected with a promoter-less plasmid containing this 5’UTR, we observed a 482-fold increase in Firefly luciferase levels in NCM460 cells and a 42-fold increase in SW480 cells, which suggests this region works as a cryptic promoter that is much more active in normal cells than in adenocarcinoma cells.
- Deleção cromossómica intersticial em 14q “de novo”: apresentação de um casoPublication . Ribeiro, M.C.; Mota Freitas, M.; Ribeiro, J.; Lopes, M.M.; Oliva Teles, N.; Correia, H.; Fonseca e Silva, M.L.Introdução: As deleções intersticiais são anomalias cromossómicas estruturais, desequilibradas, resultantes de dois pontos de quebra, frequentemente associadas a quadros clínicos anormais devido à perda de material genético ativo (eucromatina). As consequências fenotípicas dependem do segmento cromossómico perdido e do número de genes aí localizados. Material e Métodos: Os autores apresentam o caso de um indivíduo do sexo masculino, de 11 anos de idade, referenciado para estudo citogenético por apresentar um quadro clínico de atraso de desenvolvimento psicomotor, défice cognitivo e problemas de comportamento. Realizaram-se culturas sincronizadas de linfócitos de sangue periférico, bandas GTG de alta resolução e, posteriormente, estudos de hibridação in situ por fluorescência (FISH) com sondas de pintura cromossómica total e subtelomérica, específicas para o cromossoma 14. Resultados: A análise das metafases revelou a presença de uma anomalia estrutural no cromossoma 14, interpretada como uma deleção intersticial do segmento compreendido entre as bandas 14q24.3 e 14q32.1. A análise por FISH permitiu confirmar esta deleção intersticial. Como os cariótipos dos pais foram normais, conclui-se que esta anomalia cromossómica é “de novo”, estabelecendo-se o cariótipo do doente como: 46,XY,del(14)(q24.3q32.1).ish del(14)(wcp 14+,SHGC36156+)dn Discussão: A deleção intersticial encontrada no cromossoma 14 implica uma monossomia do segmento 14q24.3→14q32.1. As alterações descritas mais comuns, associadas a esta deleção, incluem ADPM e algumas malformações minor. Os autores apresentam este caso pela raridade da anomalia citogenética encontrada e comparam-no com a literatura atual.