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Projeto de investigação
Explorando novas abordagens terapêuticas para a Cardiomiopatia Hipertrófica
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Exploring new biological frontiers in hypertrophic cardiomyopathy
Publication . Almeida, Rita Mingot de Almeida Mendes de; Fonseca, Maria do Carmo Salazar Velez Roque da; Gonçalves, Maria Teresa Tenório de Figueiredo Carvalho, 1964-
Hypertrophic Cardiomyopathy (HCM) is the most common hereditary disease of the heart (1:500 individuals), and a cause of sudden cardiac death in young adults and athletes. The disease is inherited as an autosomal dominant trait caused by mutations in genes of the cardiac sarcomere. It is a disease with a widely variable genotype and phenotype. At present there is no effective treatment for this genetic disorder. The goal of my work was to explore applications of recent molecular genetics tools to improve patient diagnosis and develop potential new treatment strategies. Inspired by recent reports demonstrating the feasibility of performing “molecular RNA surgery” by using a double trans-splicing approach that results in the specific substitution of a given mutated exon, I investigated whether transsplicing could efficiently correct the expression of a mutant TNNT2 gene in cardiac cells. The TNNT2 gene codes for cardiac troponin T, one of the first sarcomeric proteins to be linked to HCM with more than 30 mutations identified to date. Because there is a significant mutational clustering on TNNT2 exon 9 associated with poor prognosis, I designed a strategy to specifically correct this exon. As a model system I used murine HL-1 cardiomyocytes. Given architectural differences between the human TNNT2 and mouse Tnnt2 genes, human exon 9 corresponds to mouse exon 8. The human TNNT2 exon 9 was used to replace the homologous mouse exon 8, which encodes the same amino acid sequence but differs in nucleotide composition, thus creating unique restriction sites and also a unique binding site for a primer that only hybridizes to the human TNNT2 exon 9. These unique restriction sites or the specific primer were further used to check the efficiency of trans-splicing events. Briefly, double trans-splicing molecules were constructed containing the replacing exon flanked by artificial intronic sequences with strong splice sites and splicing enhancers connected by a spacer linker to antisense sequences designed to anneal the two introns flanking exon 8 in the target murine Tnnt2 pre-mRNA. Cells were transfected with the exon exchange constructions cloned under the control of different promoters. The efficiency of trans-splicing was determined by RT-PCR followed by restriction analysis or, alternatively, by RT-PCR using the primer that only hybridizes to the human TNNT2 exon 9 and thus only amplifying trans-spliced transcripts. An RT-PCR assay using a radioactive γ-32P labelled primer indicated the presence of only residual amounts of the trans-splicing product. In order to improve efficiency, I designed an alternative strategy that involves a single 3' trans-splicing reaction. In brief, the goal was to replace mouse exon 8 and all exons downstream (exons 8 to 15) with a human cDNA containing the nucleotide sequence corresponding to exons 9 to 16. I constructed a 3' trans-splicing vector containing the human cDNA and upstream an artificial intronic sequence with a strong splice site and splicing enhancers connected by a spacer linker to an antisense sequence designed to anneal to intron 7 upstream of the mouse Tnnt2 exon 8 in the target pre-mRNA. After transfection, no trans-splicing product was detected. All together, these results argue that trans-splicing does not ensure efficient correction of expression of a TNNT2 gene in cardiac cells. This could be due to inefficiency of trans-splicing reactions in general, or a particular resistance to trans-splicing of the targeted region in the Tnnt2 pre-mRNA. High throughput sequencing technologies have revolutionized the identification of mutations responsible for HCM. Detection of pathogenic mutations has important implications for the medical management of patients and their families. However, approximately 50% of individuals with a clinical diagnosis of HCM have no causal mutation identified. In my host lab, we hypothesized that this may be due to the presence of pathogenic mutations located deep within the introns, which are not detected by conventional sequencing analysis restricted to exons and exonintron boundaries. The aim of my study was to develop a whole-gene sequencing strategy to prioritize deep intronic variants that may play a role in HCM pathogenesis. In collaboration with other members of the host lab, the full genomic DNA sequence of 26 genes previously associated with HCM was analysed in 16 unrelated patients. We identified likely pathogenic deep intronic variants in VCL, PRKAG2 and TTN genes. These variants, which are predicted to act through disruption of either splicing or transcription factor binding sites, were 3-fold more frequent in our cohort of probands than in normal European populations. Moreover, we found a patient that is compound heterozygous for a splice site mutation in MYBPC3 and the deep intronic VCL variant. Analysis of family members revealed that carriers of the MYBPC3 mutation alone do not manifest the disease, while family members that are compound heterozygous are clinically affected. In conclusion, we developed a framework for scrutinizing variation along the complete sequence of HCM-associated genes and our results suggest that deep intronic variation contributes to HCM phenotype. In order to translate the novel genetic information that we found to clinical decision taking requires further functional analysis. To date, mechanistic and functional studies of HCM mutations have been largely restricted to animal models in part due to difficulties in obtaining human tissue from patients. However, the recent emergence of patient-derived induced pluripotent stem cells (iPSCs) that can be differentiated into functional cardiomyocytes that recapitulate HCM-specific characteristics holds great promise as an exciting new approach to study how gene mutations relate to clinical outcomes and might be applied to test our hypothesisgenerating data. Thus, I decided to use the CRISPR-Cas9 genome-editing technology to introduce patient mutations in the genome of embryonic stem (ES) cells that were subsequently differentiated in cardiomyocytes. In collaboration with other members of the host lab, I generated sets of isogenic ES cells that differ exclusively by the presence of HCM-causing mutations in the TNNT2 gene. We used mouse ES cells, which are easier to manipulate and differentiate than human cells. In conclusion, during my PhD training I explored the feasibility of inducing trans-splicing as an RNA-targeted therapy to correct the expression of mutant sarcomeric genes in cardiomyocytes, I contributed to the development of a bioinformatics pipeline to identify novel mutations located within intronic regions of sarcomeric genes that may contribute to HCM pathogenesis, and I constructed new genome-edited cellular models of HCM.
Whole gene sequencing identifies deep-intronic variants with potential functional impact in patients with hypertrophic cardiomyopathy
Publication . Almeida, Rita Mendes de; Tavares, Joana; Martins, Sandra; Carvalho, Teresa; Enguita, Francisco J.; Brito, Dulce; Fonseca, Maria Carmo; Lopes, Luís Rocha
High throughput sequencing technologies have revolutionized the identification of mutations responsible for genetic diseases such as hypertrophic cardiomyopathy (HCM). However, approximately 50% of individuals with a clinical diagnosis of HCM have no causal mutation identified. This may be due to the presence of pathogenic mutations located deep within the introns, which are not detected by conventional sequencing analysis restricted to exons and exon-intron boundaries.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
EXPL/BIM-MEC/0201/2013