Functional analyses and novel markers for pulmonary arterial hypertension

Abstract

Pulmonary Arterial Hypertension (PAH) is a rare disease characterized by progressive obliteration of the pulmonary precapillary arteries, leading to an increase in pulmonary vascular resistance that causes right heart failure, and subsequently, death. In the last decade, mass sequencing has increased the number of genes involved in PAH, doubling the number of known genes. This quantitative increase in the available data has shown low efficiency in classifying variants, resulting in high levels of variants of uncertain significance, which show as a problem for both diagnosis and genetic counseling. To improve the classification of variants into PAH-related genes, we will perform functional studies in new genes (ABCC8) and others with broad known involvement but no functional analysis (TBX4). The ABCC8 gene encodes for the regulatory subunit of an ATP-dependent potassium channel known as SUR1, which needs binding to the pore-forming protein Kir6, the involvement of this channel in diseases such as diabetes is known, but as it fits exactly into the pathogenesis of PAH it is for now diffuse. ABCC8 is a gene of complex structure, consisting of 39 small exons with broad intronic regions. Due to these characteristics, most of the mutations found in the Spanish HAP registry (REHAP), are found in the vicinity of regions bordering exons and introns. To determine whether these seemingly change-of-direction mutations can induce exon jump events, we will make minigenes for 11 variants detected in REHAP. On the other hand, TBX4 is one of the genes with the greatest involvement in pediatric PAH, although in recent years the proportion of variants in adult forms has increased considerably. The function of TBX4 is well known in embryonic development, but its role in adults is not. For this reason, we collected 51 variants in TBX4 for their functional study, by analyzing their transcriptional activity and cell location, as well as minigenes for the cut and splice variants. At present, the diagnosis of PAH is produced using invasive and expensive means that require the hospitalization of the patient, no marker allows the diagnosis quickly and easily. In the last decade the use of circulating microRNAs (miRNAs) as biomarkers has begun to be proposed, miRNAs are small non-coding RNA molecules of 16-22 base pairs that regulate posttranscriptional activity by binding to the 3 'UTR region of messenger RNA. stable in the blood and their levels in this fluid are affected in pathological processes. Which makes them potential biomarkers for countless diseases, such as PAH, which due to the processes of remodeling, proliferation and inhibition of apoptosis that occurs in endothelial cells and smooth muscle of the pulmonary precapillary arteries, makes it a strong candidate for the development of a model for its diagnosis based on circulating miRNAs. To perform this model, microRNA-Seq will be used, from total RNA from patient plasma and controls. Initially a differential expression analysis will be done, and then the results will be validated using qPCR using specific probes. Once we have a set of miRNAs with differentiated expression between the two groups, a variable number of miRNAs will be proposed for prevalence in a cohort belonging to REHAP, to finally be validated in 100 PAH patients and 100 controls using digital droplet PCR. Machine learning methods will be used to assess different combinations of circulating miRNAs. Besides, the effect of more deregulated miRNAs will be assessed in vitro using arterial endothelial cells.