Browsing by Author "Santos, R."
Now showing 1 - 10 of 16
Results Per Page
Sort Options
- A 26-Year Experience in Chorionic Villus Sampling Prenatal Genetic DiagnosisPublication . Jorge, P.; Mota-Freitas, M.; Santos, R.; Silva, M.; Soares, G.; Fortuna, A.Abstract: This report describes the trends of chorionic villus sampling (CVS) referred for prenatal genetic diagnosis in the past two and a half decades in a Portuguese Center. Our cohort of 491 CVS was mostly performed by the transcervical method at the 12th gestational week. Data collected within the framework of this study relate to the following: sampling method, referral reason versus abnormality and incidence of procedure-related pregnancy loss, that declined to about 0.5% over the last 15 years. The year 2000 represented a change in referral reasons for chorionic tissue collection, shifting from almost exclusively for cytogenetic testing to an increasing number of molecular tests for monogenic disorders. Herein, success rates as well as cytogenetic and/or molecular DNA results are presented. These latter include not only tests for several monogenic disorders, but also aneuploidy and maternal cell contamination screening. This retrospective analysis reiterates that CVS is a safe and reliable first trimester technique for prenatal diagnosis in high genetic risk pregnancies.
- Arx-related disorders: several distinct phenotypes, one mutated genePublication . Sá, M.J.; Soares, G.; Silva, J.; Fortuna, A.; Santos, R.; Marques, I.; Jorge, P.
- Classical fragile-X phenotype in a female infant disclosed by comprehensive genomic studiesPublication . Jorge, P.; Garcia, E.; Gonçalves, A.; Marques, I.; Maia, N.; Rodrigues, B.; Santos, H.; Fonseca, J.; Soares, G.; Correia, C.; Reis-Lima, M.; Cirigliano, V.; Santos, R.BACKGROUND: We describe a female infant with Fragile-X syndrome, with a fully expanded FMR1 allele and preferential inactivation of the homologous X-chromosome carrying a de novo deletion. This unusual and rare case demonstrates the importance of a detailed genomic approach, the absence of which could be misguiding, and calls for reflection on the current clinical and diagnostic workup for developmental disabilities. CASE PRESENTATION: We present a female infant, referred for genetic testing due to psychomotor developmental delay without specific dysmorphic features or relevant family history. FMR1 mutation screening revealed a methylated full mutation and a normal but inactive FMR1 allele, which led to further investigation. Complete skewing of X-chromosome inactivation towards the paternally-inherited normal-sized FMR1 allele was found. No pathogenic variants were identified in the XIST promoter. Microarray analysis revealed a 439 kb deletion at Xq28, in a region known to be associated with extreme skewing of X-chromosome inactivation. CONCLUSIONS: Overall results enable us to conclude that the developmental delay is the cumulative result of a methylated FMR1 full mutation on the active X-chromosome and the inactivation of the other homologue carrying the de novo 439 kb deletion. Our findings should be taken into consideration in future guidelines for the diagnostic workup on the diagnosis of intellectual disabilities, particularly in female infant cases.
- Clinical and Genetic Analysis of Children with Kartagener SyndromePublication . Pereira, R.; Barbosa, T.; Gales, L.; Oliveira, E.; Santos, R.; Oliveira, J.; Sousa, M.Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder characterized by dysfunction of motile cilia causing ineffective mucus clearance and organ laterality defects. In this study, two unrelated Portuguese children with strong PCD suspicion underwent extensive clinical and genetic assessments by whole-exome sequencing (WES), as well as ultrastructural analysis of cilia by transmission electron microscopy (TEM) to identify their genetic etiology. These analyses confirmed the diagnostic of Kartagener syndrome (KS) (PCD with situs inversus). Patient-1 showed a predominance of the absence of the inner dynein arms with two disease-causing variants in the CCDC40 gene. Patient-2 showed the absence of both dynein arms and WES disclosed two novel high impact variants in the DNAH5 gene and two missense variants in the DNAH7 gene, all possibly deleterious. Moreover, in Patient-2, functional data revealed a reduction of gene expression and protein mislocalization in both genes' products. Our work calls the researcher's attention to the complexity of the PCD and to the possibility of gene interactions modelling the PCD phenotype. Further, it is demonstrated that even for well-known PCD genes, novel pathogenic variants could have importance for a PCD/KS diagnosis, reinforcing the difficulty of providing genetic counselling and prenatal diagnosis to families.
- Development and validation of a multiplex-PCR assay for X-linked intellectual disabilityPublication . Jorge, P.; Oliveira, B.; Marques, I.; Santos, R.BACKGROUND: X-linked intellectual disability is a common cause of inherited cognitive deficit affecting mostly males. There are several genetic causes implicated in this condition, which has hampered the establishment of an accurate diagnosis. We developed a multiplex-PCR assay for the mutational hotspot regions of the FMR1, AFF2 and ARX genes. METHODS: The multiplex-PCR was validated in a cohort of 100 males selected to include known alleles for the FMR1 repetitive region: five full mutations (250-650 CGGs), ten premutations (70-165 CGGs) and eighty-five in the normal range (19-42 CGGs). Sequencing or Southern blotting was used to confirm the results, depending on the allele class. In this cohort, with the exception of one sample showing an AFF2 intermediate-sized allele, all other samples were normal (8-34 CCGs). No ARX variant was found besides the c.429_452dup. The validated assay was applied to 5000 samples (64.4% males and 35.6% females). RESULTS: The normal-allelic range of both FMR1 and AFF2 genes as well as the nature of ARX variants identified was similar in both genders. The rate of homozygosity observed in female samples, 27.5% for FMR1 and 17.8% for AFF2 alleles, is comparable to that published by others. Two FMR1 premutations were identified, in a male (58 CGGs) and a female case [(CGG)(47)/(CGG)(61)], as well as several FMR1 or AFF2 intermediate-sized alleles. One AFF2 premutation (68 CCGs) and two putative full expansions were picked up in male subjects, which seems relevant considering the rarity of reported AFF2 mutations found in the absence of a family history. CONCLUSIONS: We developed a robust multiplex-PCR that can be used to screen the mutational hotspot regions of FMR1, AFF2 and ARX genes. Moreover, this strategy led to the identification of variants in all three genes, representing not only an improvement in allele-sizing but also in achieving a differential diagnosis. Although the distinction between females who are truly homozygous and those with a second pre- or full mutation sized allele, as well as a definitive diagnosis, requires a specific downstream technique, the use of this multiplex-PCR for initial screening is a cost-effective approach which widens the scope of detection.
- Exonization of an Intronic LINE-1 Element Causing Becker Muscular Dystrophy as a Novel Mutational Mechanism in Dystrophin GenePublication . Gonçalves, A.; Oliveira, J.; Coelho, T.; Taipa, R.; Melo-Pires, M.; Sousa, M.; Santos, R.A broad mutational spectrum in the dystrophin (DMD) gene, from large deletions/duplications to point mutations, causes Duchenne/Becker muscular dystrophy (D/BMD). Comprehensive genotyping is particularly relevant considering the mutation-centered therapies for dystrophinopathies. We report the genetic characterization of a patient with disease onset at age 13 years, elevated creatine kinase levels and reduced dystrophin labeling, where multiplex-ligation probe amplification (MLPA) and genomic sequencing failed to detect pathogenic variants. Bioinformatic, transcriptomic (real time PCR, RT-PCR), and genomic approaches (Southern blot, long-range PCR, and single molecule real-time sequencing) were used to characterize the mutation. An aberrant transcript was identified, containing a 103-nucleotide insertion between exons 51 and 52, with no similarity with the DMD gene. This corresponded to the partial exonization of a long interspersed nuclear element (LINE-1), disrupting the open reading frame. Further characterization identified a complete LINE-1 (~6 kb with typical hallmarks) deeply inserted in intron 51. Haplotyping and segregation analysis demonstrated that the mutation had a de novo origin. Besides underscoring the importance of mRNA studies in genetically unsolved cases, this is the first report of a disease-causing fully intronic LINE-1 element in DMD, adding to the diversity of mutational events that give rise to D/BMD.
- Expanding the MTM1 mutational spectrum: novel variants including the first multi-exonic duplication and development of a locus-specific databasePublication . Oliveira, J.; Oliveira, M.; Kress, W.; Taipa, R.; Melo-Pires, M.; Hilbert, P.; Baxter, P.; Santos, M.; Buermans, H.; den Dunnen, J.; Santos, R.Myotubular myopathy (MIM#310400), the X-linked form of Centronuclear myopathy (CNM) is mainly characterized by neonatal hypotonia and inability to maintain unassisted respiration. The MTM1 gene, responsible for this disease, encodes myotubularin - a lipidic phosphatase involved in vesicle trafficking regulation and maturation. Recently, it was shown that myotubularin interacts with desmin, being a major regulator of intermediate filaments. We report the development of a locus-specific database for MTM1 using the Leiden Open Variation database software (http://www.lovd.nl/MTM1), with data collated for 474 mutations identified in 472 patients (by June 2012). Among the entries are a total of 25 new mutations, including a large deletion encompassing introns 2-15. During database implementation it was noticed that no large duplications had been reported. We tested a group of eight uncharacterized CNM patients for this specific type of mutation, by multiple ligation-dependent probe amplification (MLPA) analysis. A large duplication spanning exons 1-5 was identified in a boy with a mild phenotype, with results pointing toward possible somatic mosaicism. Further characterization revealed that this duplication causes an in-frame deletion at the mRNA level (r.343_444del). Results obtained with a next generation sequencing approach suggested that the duplication extends into the neighboring MAMLD1 gene and subsequent cDNA analysis detected the presence of a MTM1/MAMLD1 fusion transcript. A complex rearrangement involving the duplication of exon 10 has since been reported, with detection also enabled by MLPA analysis. It is thus conceivable that large duplications in MTM1 may account for a number of CNM cases that have remained genetically unresolved.
- Genes, crianças e pediatras: distrofia miotónicaPublication . Dias, C.; Santos, M.; Vilarinho, M.; Santos, R.; Fortuna, A.; Lima, M.
- Investigação Bioquímica e Molecular na Morte Súbita do Lactente (SIDS)Publication . Cardoso, M.; Pinheiro, J.; Pereira, C.; Sousa, C.; Nogueira, C.; Tesa, A.; Ramos, A.; Balreira, A.; Lima, C.; Valongo, C.; Couto, D.; Quelhas, D.; Fonseca, H.; Rocha, H.; Almeida, L.; Rodrigues, R.; Santos, R.; Santorelli, F.; Vilarinho, L.RESUMO SIDS (Sudden Infant Death Syndrome) é a designação utilizada para definir a morte súbita do lactente, que permanece inexplicada após uma cuidadosa investigação do caso, a qual incluí a realização de autópsia, o exame do local da morte e a análise da história clínica. Devido à rápida deterioração que provocam, as doenças metabólicas constituem potenciais causas de morte súbita, quer pelas crises que ocasionam com intoxicação e comprometimento da sobrevivência do indivíduo, quer por provocarem alterações que aumentam o risco de falência de determinados orgãos. Objectivos: Com este trabalho pretendemos avaliar o contributo relativo das doenças metabólicas e dos défices energéticos nas situações de morte súbita e inexplicada do lactente, na população portuguesa. Pacientes e Métodos: Foram disponibilizadas para investigação amostras biológicas congeladas de 51 lactentes cuja causa de morte era desconhecida. Os produtos foram colhidos durante a autópsia e incluíram: soro, urina, humor vítreo, músculo e fígado. Nos fluídos biológicos fez-se o estudo dos aminoácidos e dos ácidos orgânicos e nos tecidos sólidos o doseamento da actividade dos vários complexos da cadeia respiratória mitocondrial e do teor em glicogénio. Foi ainda efectuada a extracção de DNA total a partir dos tecidos sólidos acima referidos o qual foi utilizado para estudos moleculares. Resultados: Foi possível identificar através dos estudos moleculares um caso de intolerância hereditária à frutose - HFI (homozigotia para a mutação A149P no gene ALDOB). Nos estudos bioquímicos foi encontrado um caso positivo de very long-chain acyl-CoA dehydrogenase - VLCAD e na maioria das determinações efectuadas, desvios em relação aos controlos normais inerentes ao catabolismo e aos processos de cadaverização, sendo de salientar: valores muito elevados dos aminoácidos séricos e a presença sistemática de grande quantidade de ácido láctico na urina. Conclusões: Nas doenças metabólicas o risco de recorrência existe e a identificação de um caso classificado como SIDS, como sendo na realidade um erro inato do metabolismo, é importante para a família, quer em termos de instituição de tratamento adequado e adopção de medidas preventivas, quer em termos de aconselhamento genético. ABSTRACT SIDS (Sudden Infant Death Syndrome) is the sudden and unexpected death including performance of a complete autopsy, examination of death scene, and review of clinical history. Metabolic disorders can lead to sudden dead because they cause crises of intoxication and life threatening, with dysfunction of several organs, that raise the risk of general failure. Objectives: Our main objective with this study, was to evaluate the significance of metabolic disorders and energy deficiencies in sudden infant death syndrome, in Portuguese population. Patients and Methods: We got biological frozen samples from 51 children whose cause of dead was unknown. The products were collected during autopsy and included: serum, urine, humour vitreous, muscle and liver. In the biological fluids samples we analysed amino acids as well as organic acids, and in referred tissues we determinate the activity of the respiratory chain complexes and the amount of glycogen. It was also possible to get total DNA from solid tissues which was used for molecular studies. Results: Based on molecular findings, it was possible to identify one case of fructose intolerance - HFI (patient homozygous for A149P in ALDOB gene). Biochemical studies revealed one case of very long-chain acyl-CoA dehydrogenase - VLCAD deficiency. However, in the majority of the assays performed deviations from normal controls were found, due to catabolism post-morten (plasmatic increase of amino acids and high excretion of lactic acid in urine). Conclusions: In metabolic disorders the risk of recurrence exists. The misdiagnosed inherited errors of metabolism as SIDS, it is important for the family, in terms of treatment, prevention attitude and genetic counselling. of an infant which remains unexplained
- Investigação das causas genéticas de doenças primárias do músculo: a experiência da unidade de genética molecular do CGMJMPublication . Oliveira, J.; Vieira, E.; Maia, N.; Gonçalves, A.; Oliveira, M.; Santos, R.