A carregar...
Publicação
Characterization of a dand5 p.r152h control-derived ips cell line to be used as a tool for heart disease modeling
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 30.68 MB | Adobe PDF |
Autores
Orientador(es)
Resumo(s)
RESUMO: As doenças cardíacas congênitas (DCC) e os defeitos de lateralidade associados são uma
das principais preocupações de saúde e 1,35 milhões de pessoas são diagnosticadas com
DCC a cada ano em todo o mundo. A complexidade do desenvolvimento do coração e os
obstáculos para investigar os fenótipos da doença tornam difícil identificar as causas
subjacentes das DCC.
O gene DAND5 é o homólogo humano do Cerl-2 de camundongo que codifica uma
proteína envolvida na regulação da via de sinalização Nodal antagonizando a proteína
Nodal no nódulo e inibindo a sinalização Nodal na mesoderme da placa lateral direita (RLPM).
Em um estudo anterior, uma nova variante não-sinônima c.455G> A da DAND5
heterozigótica foi identificada e vinculada ao risco de doença em dois pacientes com DCC
decorrentes de defeitos de lateralidade. Para modelar o fenótipo dos pacientes com a
variante c.455G> A, uma linha iPSC humana foi previamente gerada e caracterizada
(Cristo et al., 2017).
Neste trabalho, caracterizamos uma linha iPSC de controle DAND5 de um doador do sexo
masculino sadio (sem a variante) para servir de controle para a linha DAND5 c.455G>A.
A caracterização foi baseada na detecção de marcadores de pluripotência a nível de genes
e proteínas, e potencial diferencial in vitro. A análise de repetições curtas em tandem
(STR) usou para provar a identidade genética das células ERE e das iPSCs
reprogramadas. A cariotipagem das iPSCs após ≥ 20 passagens mostrou a estabilidade
das iPSCs altamente proliferativas. Além disso, o teste de detecção de Mycoplasma
mostrou a esterilidade da cultura de células.
Esta linha celular de controlo será comparada com a linhagem celular c.455G> A da
variante DAND5, para explorar ainda mais o nosso conhecimento sobre as consequências
da variante nos fenótipos dos doentes (modelação da doença) e, mais precisamente, na
proliferação de cardiomiócitos (terapia possível).
ABSTRACT: Congenital heart diseases (CHDs) and associated laterality defects are a major health concern and 1,35 million people are diagnosed with CHDs each year worldwide. The complexity of heart development and the hurdles to investigate the disease phenotypes make it challenging to identify the underlying causes of CHDs. DAND5 is the human homologue of mouse Cerl-2 gene that codes for a protein involved in regulating the Nodal signalling pathway by antagonizing the Nodal protein in the node and inhibiting the Nodal signaling in the right lateral plate mesoderm (R-LPM). In a previous study, a new DAND5 heterozygous nonsynonymous variant c.455G>A was identified and linked to the risk of disease in two patients with CHDs arising from laterality defects. In order to model the phenotype of the patients with the variant c.455G>A, a human iPSC line was previously generated and characterized (Cristo et al., 2017). In this work, we characterized a DAND5-control iPSC line from a healthy male donor (without the variant) to serve as control for the DAND5 c.455G>A line. The characterization was based on the detection of pluripotency markers at gene- and protein level, and in vitro differential potential. Short tandem repeats (STR) analysis has used to prove the genetic identity of the ERE cells and the reprogrammed iPSCs. Karyotyping of the iPSCs after ≥20 passages has shown the stability of the highly proliferating iPSCs. Additionally, Mycoplasma detection test showed the sterility of the cell culture. This control cell line will be compared to the DAND5 variant c.455G>A cell line to further exploit our knowledge on the consequences of the variant in the phenotypes of the patients (disease modelling) and more precisely in the modulation of cardiomyocyte proliferation (possible therapy).
ABSTRACT: Congenital heart diseases (CHDs) and associated laterality defects are a major health concern and 1,35 million people are diagnosed with CHDs each year worldwide. The complexity of heart development and the hurdles to investigate the disease phenotypes make it challenging to identify the underlying causes of CHDs. DAND5 is the human homologue of mouse Cerl-2 gene that codes for a protein involved in regulating the Nodal signalling pathway by antagonizing the Nodal protein in the node and inhibiting the Nodal signaling in the right lateral plate mesoderm (R-LPM). In a previous study, a new DAND5 heterozygous nonsynonymous variant c.455G>A was identified and linked to the risk of disease in two patients with CHDs arising from laterality defects. In order to model the phenotype of the patients with the variant c.455G>A, a human iPSC line was previously generated and characterized (Cristo et al., 2017). In this work, we characterized a DAND5-control iPSC line from a healthy male donor (without the variant) to serve as control for the DAND5 c.455G>A line. The characterization was based on the detection of pluripotency markers at gene- and protein level, and in vitro differential potential. Short tandem repeats (STR) analysis has used to prove the genetic identity of the ERE cells and the reprogrammed iPSCs. Karyotyping of the iPSCs after ≥20 passages has shown the stability of the highly proliferating iPSCs. Additionally, Mycoplasma detection test showed the sterility of the cell culture. This control cell line will be compared to the DAND5 variant c.455G>A cell line to further exploit our knowledge on the consequences of the variant in the phenotypes of the patients (disease modelling) and more precisely in the modulation of cardiomyocyte proliferation (possible therapy).
Descrição
Palavras-chave
DCC Variante DAND5 Fenótipos dos doentes Cardiomiócitos CHDs DAND5 variant Phenotypes of the patients (disease modelling) Cardiomyocyte