Browsing by Author "David, Dezső"
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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.
- 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.
- Clinical and genetic findings in Hungarian pediatric patients carrying chromosome 16p copy number variants and a review of the literaturePublication . Lengyel, Anna; Pinti, Éva; Pikó, Henriett; Jávorszky, Eszter; David, Dezső; Tihanyi, Mariann; Gönczi, Éva; Kiss, Eszter; Tóth, Zsuzsa; Tory, Kálmán; Fekete, György; Haltrich, IrénThe short arm of chromosome 16 (16p) is enriched for segmental duplications, making it susceptible to recurrent, reciprocal rearrangements implicated in the etiology of several phenotypes, including intellectual disability, speech disorders, developmental coordination disorder, autism spectrum disorders, attention deficit hyperactivity disorders, obesity and congenital skeletal disorders. In our clinical study 73 patients were analyzed by chromosomal microarray, and results were confirmed by fluorescence in situ hybridization or polymerase chain reaction. All patients underwent detailed clinical evaluation, with special emphasis on behavioral symptoms. 16p rearrangements were identified in 10 individuals. We found six pathogenic deletions and duplications of the recurrent regions within 16p11.2: one patient had a deletion of the distal 16p11.2 region associated with obesity, while four individuals had duplications, and one patient a deletion of the proximal 16p11.2 region. The other four patients carried 16p variations as second-site genomic alterations, acting as possible modifying genetic factors. We present the phenotypic and genotypic results of our patients and discuss our findings in relation to the available literature.
- Clinical Severity of PGK1 Deficiency Due To a Novel p.E120K Substitution Is Exacerbated by Co-inheritance of a Subclinical Translocation t(3;14)(q26.33;q12), Disrupting NUBPL GenePublication . David, Dezső; Almeida, Lígia S.; Maggi, Maristella; Carlos, Araújo; Imreh, Stefan; Valentini, Giovanna; Fekete, Gyorgy; Haltrich, IrénCarriers of cytogenetically similar, apparentlybalanced familial chromosome translocations not alwaysexhibit the putative translocation-associated disease phenotype.Additional genetic defects, such as genomic imbalanceat breakpoint regions or elsewhere in the genome,have been reported as the most plausible explanation.By means of comprehensive molecular and functionalanalyses, additional to careful dissection of the t(3;14)(q26.33;q12) breakpoints, we unveil a novel X-linkedPGK1 mutation and examine the contribution of these tothe extremely severe clinical phenotype characterized byhemolytic anemia and neuromyopathy.The 3q26.33 breakpoint is 40 kb from the 50 region oftetratricopeptide repeat domain 14 gene (TTC14), whereasthe 14q12 breakpoint is within IVS6 of nucleotide-bindingprotein-like gene (NUBPL) that encodes a mitochondrialcomplex I assembly factor. Disruption of NUBPL intranslocation carriers leads to a decrease in thecorresponding mRNA accompanied by a decrease inprotein level. Exclusion of pathogenic genomic imbalanceand reassessment of familial clinical history indicate theexistence of an additional causal genetic defect. Consequently,by WES a novel mutation, c.358G>A, p.E120K,in the X-linked phosphoglycerate kinase 1 (PGK1) wasidentified that segregates with the phenotype. Specificactivity, kinetic properties, and thermal stability of thisenzyme variant were severely affected. The novel PGK1mutation is the primary genetic alteration underlying thereported phenotype as the translocation per se only resultsin a subclinical phenotype. Nevertheless, its co-inheritancepresumably exacerbates PGK1-deficient phenotype, mostlikely due to a synergistic interaction of the affected genesboth involved in cell energy supply.
- Co-segregation of trichorhinophalangeal syndrome with a t(8;13)(q23.3;q21.31) familial translocation that appears to increase TRPS1 gene expressionPublication . David, Dezső; Marques, Bárbara; Ferreira, Cristina; Araújo, Carlos; Vieira, Luís; Soares, Gabriela; Dias, Cristina; Pinto, MaximinaTrichorhinophalangeal syndrome type I (TRPS I) is a rare autosomal dominant syndrome caused by haploinsufficiency of TRPS1 due to point mutations or deletions. Here we report the first familial TRPS I due to a t(8;13)(q23.3;q21.31) translocation breakpoint <100 kb from the 5’ end of TRPS1. Based on the additional abnormalities observed exclusively in the index patient that are mainly compatible with clinical features of TRPS, her phenotype was defined as expanded TRPS I including brain malformations and intellectual disability. Initial analyses did not reveal any genetic defect affecting TRPS1 or any genomic alteration within the breakpoint regions or elsewhere in the genome. The pathogenic chromosome 8q23.3 breakpoint is at position g.116,768,309_116,768,310 within a transposon type I element, 87 kb from the TRPS1 5’ end. The 13q21.31 breakpoint is within a tandem repeat region at position g.65,101,509_65,101,510 (genome assembly GRCh37/hg19). This breakpoint is flanked by protocadherin 9 (PCDH9) and protocadherin 20 (PCDH20). As an outcome of the translocation, an evolutionarily conserved non-coding VISTA enhancer element from 13q21.31 is placed within the TRPS1 5’ region, 1,294 bp from the breakpoint. The increased expression of TRPS1 found by three independent methods is most probably translocation allele derived and driven by the translocated enhancer element. The index patient’s expanded phenotype presumably involves the epithelial-to-mesenchymal transition pathway that may be due to TRPS1 overexpression. Together, these findings support that the reported translocation associated phenotypes are “cis-ruption” and TRPS1 overexpression related, the latter most probably caused by the novel enhancer element in the TRPS1 5’ region.
- 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.
- Departamento de Genética Humana do INSA pioneiro na caracterização molecular de patologias hemorrágicas e trombóticas hereditárias na população portuguesaPublication . Moreira, Isabel; Ventura, Célia; Certã, Rita; Gonçalves, João; David, Dezső
- Disruption of NUBPL due to balanced translocation [t(3;14) (q26.33;q14)] increases severity of a family-specific PGK1 mutationPublication . David, Dezső; Haltrich, Iren; Marques, Barbara; Fernandes, Cristina; Malveiro, Sara; Fekete, GyörgyAn intriguing group of familiar translocations are those which not always segregate with the “associated” disorder. Here we report the genetic alterations underlying a clinical phenotype characterized by haemolytic anemia and neuro-myopathy, seemingly associated with the familial translocation [t(3;14)(3q26.33;q14)]. Two affected probands and two unaffected relatives have been identified as carriers of this translocation. The 3q26.33 breakpoint was mapped about 40 kb from the TTC14 5’ end, at position 180.28 Mb and the 14q14 breakpoint within IVS 6 of NUBPL. The latter has been implicated in the aetiology of mitochondrial complex I deficiency (OMIM 252010). The most important additional possible candidate gene identified in this region is DNAJC19 causing an autosomal recessive disorder (OMIM 610198) that partially overlaps the reported phenotype. The recognition that a deceased relative most likely suffered from a similar disorder suggested the possibility of an X-linked disorder. Exclusion of additional genomic alterations within the breakpoint regions or elsewhere in the genome, familial X-chromosome segregation analysis and whole exome sequencing identified a novel missense mutation, c.358G>A, p.Glu120Lys, in exon 4 of phosphoglycerate kinase 1 (PGK1). Segregation analysis confirmed the association of this mutation with the disease phenotype. Re-evaluation of clinical data indicates that myopathy is considerably more severe in PGK1 deficient patients carriers of the translocation. The confirmation of this observation is currently underway. In conclusion, we have identified a novel PGK1 mutation whose clinical phenotype is exacerbated by co-inheritance of the disrupted NUBPL and/or by alterations affecting the genes in the breakpoint regions.
- Disruption of NUBPL due to balanced translocation [t(3;14)(q26.33;q14)] increases severity of a family-specific PGK1 mutationPublication . David, Dezső; Haltrich, Iren; Marques, Barbara; Fernandes, Catarina; Malveiro, Sara; Fekete, GyörgyAn intriguing group of familiar translocations are those which not always segregate with the “associated” disorder. Here we report the genetic alterations underlying a clinical phenotype characterized by haemolytic anemia and neuro-myopathy, seemingly associated with the familial translocation [t(3;14)(3q26.33;q14)]. Two affected probands and two unaffected relatives have been identified as carriers of this translocation. The 3q26.33 breakpoint was mapped about 40 kb from the TTC14 5’ end, at position 180.28 Mb and the 14q14 breakpoint within IVS 6 of NUBPL. The latter has been implicated in the aetiology of mitochondrial complex I deficiency (OMIM 252010). The most important additional possible candidate gene identified in this region is DNAJC19 causing an autosomal recessive disorder (OMIM 610198) that partially overlaps the reported phenotype. The recognition that a deceased relative most likely suffered from a similar disorder suggested the possibility of an X-linked disorder. Exclusion of additional genomic alterations within the breakpoint regions or elsewhere in the genome, familial X-chromosome segregation analysis and whole exome sequencing identified a novel missense mutation, c.358G>A, p.Glu120Lys, in exon 4 of phosphoglycerate kinase 1 (PGK1). Segregation analysis confirmed the association of this mutation with the disease phenotype. Re-evaluation of clinical data indicates that myopathy is considerably more severe in PGK1 deficient patients carriers of the translocation. The confirmation of this observation is currently underway. In conclusion, we have identified a novel PGK1 mutation whose clinical phenotype is exacerbated by co-inheritance of the disrupted NUBPL and/or by alterations affecting the genes in the breakpoint regions.
- Doenças Genómicas e Rearranjos Cromossómicos: folheto de divulgação do Programa de InvestigaçãoPublication . David, DezsőEnquadramento: As anomalias do desenvolvimento no Homem são condições devastadores que contribuem com quase metade da mortalidade em recém-nascidos de termo nos países desenvolvidos. Os indivíduos sobreviventes apresentam anomalias, malformações e/ou outras alterações do desenvolvimento psicomotor que lhes causam uma incapacidade permanente, tendo um profundo impacto na saúde pública. No entanto, a causa genética e as alterações genómicas associadas a rearranjos cromossómicos, na esmagadora maioria destas situações, permanecem desconhecidas. Embora sejam doenças raras, o custo dos cuidados de saúde associados a estes indivíduos é surpreendentemente alto. Paralelamente, a causa genética da infertilidade recorrente, não resultante de rearranjos cromossómicos desequilibrados, permanece por esclarecer. As premissas deste programa de investigação baseiam-se na baixa e média resolução das técnicas de diagnóstico atualmente disponíveis. Tal resulta na (i) insensibilidade à complexidade mutacional destas doenças, (ii) limitação do conhecimento de como os rearranjos genómicos geram alterações moleculares patogénicas, e (iii) dificuldade de efetuar um diagnóstico preditivo devido a lacunas ao nível do conhecimento funcional do genoma humano. Assim, este programa de investigação propõe introduzir a Sequenciação de Próxima-Geração (NGS) na Citogenética Clínica, tirando partido das inovações únicas na deteção de variações estruturais a nível genómico e proporcionando um grande avanço no diagnóstico clínico, possibilitando a criação da Citogenética da Próxima-Geração. Mais, o estudo da patologia molecular das doenças do desenvolvimento associadas a rearranjos cromossómicos e infertilidades irá contribuir para uma melhor capacidade de predição das consequências clínicas (fenotípicas) de translocações e inversões até então desconhecidas, assim como o diagnóstico pré-natal.
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