RESUMO
Genome-wide association studies (GWASs) have identified many genetic risk factors for CKD. However, linking common variants to genes that are causal for CKD etiology remains challenging. By adapting self-transcribing active regulatory region sequencing, we evaluated the effect of genetic variation on DNA regulatory elements (DREs). Variants in linkage with the CKD-associated single-nucleotide polymorphism rs11959928 were shown to affect DRE function, illustrating that genes regulated by DREs colocalizing with CKD-associated variation can be dysregulated and therefore, considered as CKD candidate genes. To identify target genes of these DREs, we used circular chromosome conformation capture (4C) sequencing on glomerular endothelial cells and renal tubular epithelial cells. Our 4C analyses revealed interactions of CKD-associated susceptibility regions with the transcriptional start sites of 304 target genes. Overlap with multiple databases confirmed that many of these target genes are involved in kidney homeostasis. Expression quantitative trait loci analysis revealed that mRNA levels of many target genes are genotype dependent. Pathway analyses showed that target genes were enriched in processes crucial for renal function, identifying dysregulated geranylgeranyl diphosphate biosynthesis as a potential disease mechanism. Overall, our data annotated multiple genes to previously reported CKD-associated single-nucleotide polymorphisms and provided evidence for interaction between these loci and target genes. This pipeline provides a novel technique for hypothesis generation and complements classic GWAS interpretation. Future studies are required to specify the implications of our dataset and further reveal the complex roles that common variants have in complex diseases, such as CKD.
Assuntos
Cromatina/química , DNA/química , Conformação de Ácido Nucleico , Insuficiência Renal Crônica/genética , Animais , Vias Biossintéticas/genética , Células Cultivadas , Bases de Dados Genéticas , Células Endoteliais , Predisposição Genética para Doença/genética , Genótipo , Homeostase/genética , Humanos , Túbulos Renais , Camundongos , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , RNA Mensageiro/metabolismo , Análise de Sequência de DNA/métodosRESUMO
BACKGROUND AND AIMS: Women who develop preeclampsia during pregnancy are at a higher risk for developing cardiovascular disease. As platelets are affected by preeclampsia, we set out to identify whether platelets carry information in their transcriptome on cardiovascular risk in women with former preeclampsia. METHODS: Platelets were isolated from asymptomatic women with previous preeclampsia, who underwent screening with coronary computed tomography angiography. Platelet RNA was isolated and used to construct gene networks using an unbiased approach. Platelet gene modules assembled from the network were related to risk factors and clinical traits of these women, including coronary artery calcium scores (CACS). RESULTS: We found multiple gene modules which correlated with CACS (correlation coefficients: 0.44 to 0.59, pâ¯=â¯0.05 to 0.007). The genes from two clinically relevant modules were expressed at a higher level in the group with calcifications (pâ¯=â¯3.9â¯×â¯10-10 and 0.02) and enriched for platelet-related gene-sets such as platelet activation. The first of these modules was also enriched (ppermutationâ¯=â¯0.0546) for genes mapped to known coronary artery disease susceptibility loci. Additional unbiased network analyses in platelet RNA of patients with overt cardiovascular disease underlined the importance of the identified modules for disease by high preservation. (pâ¯=â¯1.6â¯×â¯10-9 to 1.7â¯×â¯10-47). CONCLUSIONS: We found platelet RNA modules that correlated with CACS in asymptomatic women with previous preeclampsia. Whether or not platelets directly contribute to this disease trajectory, or reflect the underlying plaque substrate remains to be determined, but enrichment for coronary artery disease susceptibility genes emphasizes the importance for the disease.
Assuntos
Plaquetas/metabolismo , Doença da Artéria Coronariana/genética , Redes Reguladoras de Genes , Pré-Eclâmpsia/genética , RNA/genética , Transcriptoma , Calcificação Vascular/genética , Doenças Assintomáticas , Coagulação Sanguínea/genética , Estudos de Casos e Controles , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/diagnóstico , Diagnóstico Precoce , Feminino , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Fenótipo , Ativação Plaquetária/genética , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/diagnóstico , Gravidez , RNA/sangue , Fatores de Risco , Calcificação Vascular/sangue , Calcificação Vascular/diagnósticoRESUMO
To identify novel proteins required for receptor-mediated endocytosis, we have developed an RNAi-based screening method in Drosophila S2 cells, based on uptake of a scavenger receptor ligand. Some known endocytic proteins are essential for endocytosis in this assay, including clathrin and alpha-adaptin; however, other proteins important for synaptic vesicle endocytosis are not required. In a small screen for novel endocytic proteins, we identified the Drosophila homologue of Vps35, a component of the retromer complex, involved in endosome-to-Golgi trafficking. Loss of Vps35 inhibits scavenger receptor ligand endocytosis, and causes mislocalisation of a number of receptors and endocytic proteins. Vps35 has tumour suppressor properties because its loss leads to overproliferation of blood cells in larvae. Its loss also causes signalling defects at the neuromuscular junction, including upregulation of TGFbeta/BMP signalling and excessive formation of synaptic terminals. Vps35 negatively regulates actin polymerisation, and genetic interactions suggest that some of the endocytic and signalling defects of vps35 mutants are due to this function.