Browsing by Author "Marques, Mariana"
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- Array and NGS based characterization of translocation breakpoints of the t(2:7)(q23;q32),t(5;6)(q23,q26)dnPublication . Marques, Mariana; Talkowski, Michael E.; Freixo, João; Rui, Gonçalves; Morton, Cynthia C.; David, DezsőIntroduction: Congenital anomalies, namely caused by chromosome rearrangements, are a leading cause of infant mortality in European countries. The elucidation of the causal relationship between rearrangements and clinical phenotypes requires an efficient approach for identification of breakpoints at nucleotide resolution. Methods: In the last decade we went from conventional FISH based positional mapping of chromosomal breakpoints to sorting and amplification of derivative (der) chromosomes followed by array painting based mapping. Currently we are moving towards the application of Next-Generation Sequencing (NGS) for the identification of chromosome rearrangement breakpoints at nucleotide resolution. By means of these comprehensive molecular techniques we unveil the structural chromosomal alterations at nucleotide resolution in a proband with t(2:7)(q23;q32),t(5;6)(q23,q26)dn. Expression profiling of the proband’s LCLs was also carried out. Results: Array painting identified the breakpoints of two balanced chromosome translocations. The disruption of the PRPF40A and SND1 genes by the t(2;7) was identified both by array and NGS analysis. While array analysis identified only t(5;6) breakpoints and the affected PACRG gene, NGS revealed further complexity of the breakpoint region. Indeed, der(6) is a complex chromosomal rearrangement (CCR) with three additional breakpoints resulting from an inversion and a PTPRK gene excision/insertion. Discussion: Because of the complexity of this rearrangement we are not yet able to establish the candidate genes for the observed clinical phenotype. As shown by the CCR, NGS is currently the only methodology able to identify the full spectrum of balanced structural alterations. Thus, the introduction of NGS technology for high-throughput delineation of chromosomal rearrangements is presently underway.
- Characterization of the molecular pathogenesis of a malformation syndrome associated with a complex double chromosome translocationPublication . Marques, Mariana; David, Dezso; Dias, Deodália Maria AntunesCongenital anomalies are devastating conditions responsible for high neonatal mortality, as well as high morbidity of the surviving individuals. Chromosomal rearrangements are a leading cause of severe congenital malformations and are associated with about 25% of perinatal deaths due to congenital anomalies. The aim of this study is the identification of candidate genes responsible for the phenotype characterized by intrauterine growth retardation, severe developmental delay, brain malformations and refractory epilepsy identified in an individual with an apparently balanced de novo double chromosomal translocation t(2;7)(q23;q32),t(5;6)(q23;q26)dn. Identification and mapping of the structural chromosomal aberrations were performed by whole-genome array analysis, array painting with genomic amplicons of the derivative chromosomes and by whole genome sequencing of large-insert jumping libraries (liWGS). Subsequently all junction fragments were amplified and the breakpoints were identified at nucleotide resolution by Sanger sequencing. Genome array analysis identified a 651.76 kb deletion at 14q24.3 (g.76,673,181-77,324,937 [GRCh37/hg19]). Transforming growth factor beta 3 (TGFB3), a gene associated with autosomal dominant arrythmogenic right ventricular dysplasia and Loeys-Dietz syndrome (OMIM #107970 and #615582), is situated 224 kb upstream from the proximal deletion breakpoint.. Translocation breakpoints were identified both by array painting and liWGS. The 2q23.3 breakpoint of the t(2;7)(q23.3;q32.1), disrupts IVS5 of pre-mRNA processing factor 40 homolog A (PRPF40A), a protein coding gene related to Huntington’s disease (OMIM#143100). The calcium channel, voltage-dependent, beta-4 subunit (CACNB4) gene, localized 600 kb upstream of this breakpoint, is associated with three epilepsy related autosomal dominant disorders (OMIM #613855, 607682 and 607682). The Staphylococcal nuclease and tudor domain containing 1 (SND1) gene disrupted by the 7q32.1 breakpoint, is not presently associated with any known phenotype. However, the RNA binding motif protein 28 coding gene (RBM28), situated 300 kb downstream of the 7q32.1 breakpoint, has been associated with progressive neurological defects (OMIM #612079). Concerning the t(5;6)(q23.2;q26) translocation, the 5q23.2 breakpoint is situated in an intergenic region whereas the 6q26 breakpoint disrupts IVS3 of PARK2 co-regulated gene (PACRG). This gene shares a bidirectional promoter with parkin RBR E3 ubiquitin protein ligase (PARK2), which is associated with early onset Parkinson disease. About 300kb downstream of this breakpoint is the homolog of quaking mouse (QKI) gene that also plays a role in brain development. The application of liWGS unveiled the presence of two additional cryptic alterations on der(6), an excision/insertion and an inversion. The cryptic excision at 6q22.33 disrupts protein tyrosine phosphatase receptor type K (PTPRK), a gene from the protein tyrosine phosphatase family which is associated with tumor suppression. As a result of the excision/insertion, the excised 48 kb fragment containing PTPRK exon 7 and flanking intronic sequences is inserted 36 Mb further distal at 6q26. Located 70kb from the PTPRK gene, the laminin 2 (LAMA2) gene was reported has being involved in brain malformations, including polymicrogyria. The inversion breakpoint at 5q23.2 is located within an intergenic region. In conclusion, these findings suggest that disruption of PRPF40A and PACRG genes, in association with misregulation of CACNB4, RBM28, PARK2, QKI and LAMA2 genes from the breakpoint regions are the most likely candidate genes responsible for this complex malformation phenotype. Additionally the modulating effect of TGFB3 gene cannot be excluded. Comparative analysis of this complex chromosome rearrangement by array painting and liWGS demonstrates that currently only liWGS is able to identify the full spectrum of balanced, otherwise cryptic, structural alterations. In this way, liWGS allows high-throughput delineation of chromosomal rearrangements, allowing a better phenotype-genotype association. A major drawback of studying chromosome anomalies is the unavailability of relevant human biological material or of data from such samples. Theoretically, to overcome this issue, animal or induced pluripotent stem cells models can be used. During this study, the obtainment of a proband-specific iPSC model was attempted. Unfortunately, the complexity of the pluripotency induction process, the associated costs and the requisites of using non-viral vectors hinder the development of such cellular models for the study of the molecular pathogenesis of congenital anomalies. Proband derived lymphoblastoid cell line (LCL), non-integrative episomal plasmids containing the four Yamanaka factors – OCT3/4, c-MYC, SOX2 and KLF4 and an electroporation platform were used for the pluripotency induction experiments. Electroporated cells were maintained on a human foreskin fibroblasts (HFF) feeder-layer. While performing the reprogramming experiments, several technical difficulties were identified. A major difficulty is achieving high transfection efficiency of LCL with episomal plasmids without high cell mortality rates. Although no LCL derived iPSC colonies were obtained, the identification of the critical steps in the induction protocol of LCL derived cells will certainly contribute for further development of such cellular models. Furthermore, the availability of individual-derived iPSCs will definitely lead to a robust cellular model for the study of the molecular pathogenesis of chromosome rearrangements associated with congenital anomalies.
- Citogenética de Próxima Geração: Implementação e primeiros resultados em PortugalPublication . David, Dezső; Oliva-Teles, Natália; Freixo, João; Fonseca e Silva, ML; Fortuna, Ana; Tkachenko, Natália; Carvalho, Inês; Marques, Mariana; Cardoso, Manuela; Fino, Joana; Marques, Bárbara; Alves, Ana Cristina; Dória, Sófia; Pinto de Moura, Carla; Marques Carreira, Isabel; Correia, Hildeberto; Gonçalves, Rui Miguel; Lavinha, João; Kay, Teresa; Talkowski, Michael; Morton, CynthiaIntrodução: As alterações cromossómicas estruturais provocam doenças de severidade variável que acarretam sofrimento individual e familiar signifi cativo. Para compreensão da sua etiologia e estabelecimento de um possível prognóstico, uma adequada correlação fenótipo-genótipo é fundamental. O presente estudo faz parte do projeto intitulado àCitogenética de Próxima Geração Irrompe nos Cuidados de Saúde e Contribui para Anotação do Genoma Humanoà, que visa a introdu- ção da sequenciação de próxima geração (NGS) na citogené- tica clínica, tirando partido dessa inovação única na deteção de variantes estruturais, com uma resolução de um nucleótido para a criação de uma citogenética de alto rendimento, catalisadora de notáveis avanços no diagnóstico clínico e resulta da colaboração entre seis Instituições nacionais e a Harvard Medical School. Estima-se que exista um número considerável de indivíduos portadores de diversas patologias, incluindo algumas de início tardio associadas a rearranjos genómicos por identifi car. Assim, é fundamental a identifi cação e a referência destes indivíduos com possíveis rearranjos cromossómicos associados a doenças.
- Comprehensive clinically oriented workflow for nucleotide level resolution and interpretation in prenatal diagnosis of de novo apparently balanced chromosomal translocations in their genomic landscapePublication . David, Dezső; Freixo, João P.; Fino, Joana; Carvalho, Inês; Marques, Mariana; Cardoso, Manuela; Piña-Aguilar, Raul E.; Morton, Cynthia C.We present a comprehensive clinically oriented workflow for large-insert genome sequencing (liGS)-based nucleotide level resolution and interpretation of de novo (dn) apparently balanced chromosomal abnormalities (BCA) in prenatal diagnosis (PND). Retrospective or concomitant with conventional PND and liGS, molecular and newly developed clinically inspired bioinformatic tools (TAD-GConTool and CNV-ConTool) are applied to analyze and assess the functional and phenotypic outcome of dn structural variants (dnSVs). Retrospective analysis of four phenotype-associated dnSVs identified during conventional PND precisely reveal the genomic elements disrupted by the translocation breakpoints. Identification of autosomal dominant disease due to the disruption of ANKS1B and WDR26 by t(12;17)(q23.1;q21.33)dn and t(1;3)(q24.11;p25.3)dn breakpoints, respectively, substantiated the proposed workflow. We then applied this workflow to two ongoing prenatal cases with apparently balanced dnBCAs: 46,XX,t(16;17)(q24;q21.3)dn referred for increased risk on combined first trimester screening and 46,XY,t(2;19)(p13;q13.1)dn referred due to a previous trisomy 21 pregnancy. Translocation breakpoints in the t(16;17) involve ANKRD11 and WNT3 and disruption of ANKRD11 resulted in KBG syndrome confirmed in postnatal follow-up. Breakpoints in the t(2;19) are within ATP6V1B1 and the 3' UTR of CEP89, and are not interpreted to cause disease. Genotype-phenotype correlation confirms the causative role of WDR26 in the Skraban-Deardorff and 1q41q42 microdeletion phenocopy syndromes, and that disruption of ANKS1B causes ANKS1B haploinsufficiency syndrome. In sum, we show that an liGS-based approach can be realized in PND care providing additional information concerning clinical outcomes of dnBCAs in patients with such rearrangements.
- Early results of next-gen cytogenetics implementation in PortugalPublication . David, Dezső; Freixo, João; Marques, Bárbara; Carvalho, Inês; Tkachenko, Natália; Oliva-Teles, Natália; Marques, Mariana; Cardoso, Manuela; Fino, Joana; Alves, Cristina; Fortuna, Ana; Sófia, Dória; Pinto de Moura, Carla; Correia, Hildeberto; Marques Carreira, Isabel; Sá, Joaquim; Gonçalves, Rui Miguel; Lavinha, João; Kay, Teresa; Talkowski, Michael; Morton, CynthiaBackground: Most approaches are insensitive to the full mutational spectrum of chromosome rearrangements associated with human developmental abnormalities. Therefore, our aim is to introduce next-generation sequencing (NGS) into clinical cytogenetics, creating a sequence-based NextGen Cytogenetics to catalyze a dramatic advancement in clinical diagnostics. Methods: Twenty families with chromosome rearrangement-associated diseases, including two prenatal (PN) cases, have been enrolled. Fourteen of these were also analyzed by NGS using large-insert paired-end libraries. Results: The majority of these cases were confirmed to be balanced reciprocal rearrangements, whereas 4 were complex chromosomal rearrangements including 1 of chromothripsis. Thus far, over 50 breakpoints were identified disrupting protein coding genes, lncRNAs, or intergenic regions, thus revealing candidate genes or genomic loci. These cases are further assessed for pathogenicity from positional effects on genes located within topological domains (TADs) containing the breakpoints using DECIPHER predictions of haploinsufficiency. In one PN case, the 16q24 breakpoint disrupts ANKRD11, etiologic in the autosomal dominant KBG syndrome (OMIM #148050), predicting an abnormal phenotype. The chromothripsis case, submitted as 46,XY,t(7;14)(q22;q32.1),inv(15)(q21.2q26.1), proved by NGS to carry two further deletions, at 3p12 (5.3 Mb) and 15q14 (488 kb), as well as an insertion of 644.4 kb from 15q14 into 3p14. The inv(15) is in fact a complex rearrangement of 15q with eight breakpoints. Conclusions: We demonstrate that NGS-based chromosomal rearrangement characterization leads to major improvements in identification of chromosomal aberrations and in prediction of clinical outcomes of postnatally and prenatally detected genomic rearrangements, and to contributions to human genome annotation.
- A Novel Frameshift CHD4 Variant Leading to Sifrim-Hitz-Weiss Syndrome in a Proband with a Subclinical Familial t(17;19) and a Large dup(2)(q14.3q21.1)Publication . Da Silva, Jorge Diogo; Oliva-Teles, Natália; Tkachenko, Nataliya; Fino, Joana; Marques, Mariana; Fortuna, Ana Maria; David, DezsoThe genetic complexity of neurodevelopmental disorders (NDD), combined with a heterogeneous clinical presentation, makes accurate assessment of their molecular bases and pathogenic mechanisms challenging. Our purpose is to reveal the pathogenic variant underlying a complex NDD through identification of the "full" spectrum of structural genomic and genetic variants. Therefore, clinical phenotyping and identification of variants by genome and exome sequencing, together with comprehensive assessment of these and affected candidate genes, were carried out. A maternally-inherited familial translocation [t(17;19)(p13.1;p13.3)mat] disrupting the GSG1 like 2 gene (GSG1L2), a 3.2 Mb dup(2)(q14.3q21.1) encompassing the autosomal dominant OMIM phenotype-associated PROC and HS6ST1 gene, and a novel frameshift c.4442del, p.(Gly1481Valfs*21) variant within exon 30 of the Chromodomain helicase DNA binding protein 4 (CHD4) have been identified. Considering the pathogenic potential of each variant and the proband's phenotype, we conclude that this case basically fits the Sifrim-Hitz-Weiss syndrome or CHD4-associated neurodevelopmental phenotype. Finally, our data highlight the need for identification of the "full" spectrum of structural genomic and genetic variants and of reverse comparative phenotyping, including unrelated patients with variants in same genes, for improved genomic healthcare of patients with NDD.