Periodic Reporting for period 1 - INTERCEPT-T2D (Early Interception of Inflammatory-mediated Type 2 Diabetes)
Periodo di rendicontazione: 2023-01-01 al 2024-06-30
Individuals with type 2 diabetes (T2D) do not use insulin efficiently and, therefore, their glucose levels rise. T2D is a heterogeneous disease, which is an obstacle to the delivery of an optimal tailored treatment. Consequently, patients’ individual trajectories of progressive hyperglycaemia and risk of chronic complications such as stroke, heart attacks, nephropathy and retinopathy are so far difficult to predict. Chronic systemic inflammation has been suggested to be a major contributor to the onset and progression of T2D complications. INTERCEPT-T2D project will bring a new and clinically relevant dimension in T2D care considering at diagnosis inflammatory parameters that are of importance for the transition to T2D-related complications. Moreover, the project will help deliver optimal treatments tailored to patient needs and conduct a clinical trial to evaluate the efficacy of anti-inflammatory strategies.
WP1
the main objective of WP1 for this 1st period was the quantification of more than 7000 human blood samples of people with type 2 diabetes covering spectrum of the disease from non-diabetic, pre-diabetic, diabetic and advanced diabetic characterised by complications and comorbidities. Integration of theses human data is currently ongoing. Preliminary analyses support that 10 to 15% of newly diagnosed type 2 diabetics are characterised by an atypical blood inflammatory response. The nature and severity of this inflammatory response will be deeply deciphered in the 2nd phase of the project through transcriptomic, genetic and immune cell phenotying.
Biomarker measurements for more comprehensive analyses have been completed (proteomics) or are ongoing (transcriptomics, immune cell phenotyping).
WP2
During the initial phase of the INTERCEPT-T2D project, we implemented a multi-omics strategy aimed at identifying SNPs associated with inflammatory components of T2D within regulatory chromatin regions of circulating blood monocytes in patients with T2D (newly-diagnosed and established). This approach is driven by the need to understand the mechanisms underlying the functional impairment of monocytes in the progression of T2D. Our strategy is based on the hypothesis that genetic variants within these regulatory regions could significantly impact gene expression and, consequently, monocyte function, thus contributing to the pathogenesis of T2D.
The implementation of this multi-omics approach involved several critical steps. Firstly, we performed a comprehensive genetic analysis to define SNPs in high linkage disequilibrium with SNPs (“tagSNPs”) from the GWAS catalog which were found to be significantly associated with T2D risk. This allowed us to identify a number of SNPs that likely increase T2D risk. Secondly, we initiated an epigenetic analysis using the ATAC-seq technique to map open chromatin regions in CD14+ monocytes isolated from blood samples of T2D/newly diagnosed patients. This analysis was crucial for pinpointing the genomic location of chromatin regions where regulatory elements such as enhancers are accessible and active to regulate gene expression in blood monocytes. By integrating these genetic and epigenetic datasets, we were able to identify a limited number of SNPs located within candidate regulatory chromatin regions. The results of this first phase of the project have highlighted 31 potential SNPs of interest that reside in the regulatory chromatin of monocytes from T2D patients. These findings provide a foundation for further functional studies to explore how these SNPs, along with epigenetic histone modifications and transcription factors/cofactors, influence gene expression and monocyte activity.
Furthermore, we have established a robust analysis pipeline that will facilitate a comprehensive examination of the genetic components contributing to the inflammatory processes associated with T2D. This pipeline integrates various bioinformatic tools and methodologies, allowing for an in-depth investigation of the genetic and epigenetic interactions that may drive the disease.
WP3
ahead of the anticipated timeline, we made the following progresses:
- Successful pilot phase for the semi-automated multiplex immunofluorescence (IF) staining (Ventana System, Roche) and imaging (Axioscan, Zeiss) of pancreas tissue sections of living donors in the LIDOPACO cohort for markers of various islets, adipocyte, endothelial and inflammatory cells was already completed in 2023 (M1-12 of INTERCEPT-T2D). First biological results in a cohort of 50 patients will be obtained by mid-2024.
- Extensive testing and optimisation of single-cell Laser-Capture-Microdissection (LCM) and single-cell RNA-sequencing (scRNA-seq). Unfortunately, mRNA coverage is still too low for valid profiles. Shall be replaced by advanced targeted in situ single-cell sequencing. The pilot phase of cell-type-specific proteome analysis was also successful, with the detection of ~ 6,000 proteins in beta or alpha of 3 living donors with normoglycemia or T2D. Pancreatic sections from 40 patients will be processed for single islet cell type laser microdissection, protein extraction, mass spectrometry (MS) and computational analysis by the end of 2024.
WP4
We have developed a full protocol, adding important details in the secondary outcomes. All aspects of the study including development of the electronic case report form, data collection system, informed consent forms in EU languages, organization of a central laboratory, transportation of blood samples, continuous blood monitoring devices, neuropathy devices, safety monitoring, data management plan, etc. have been completed. The protocol has been approved by the Swiss Ethics Committee and Swissmedic, and enrollment of the first patient is planned for June 28th 2024.
WP5
The data has been rapidly retrieved from the clinical data mart, and we are working on cleaning it. Preliminary analyses demonstrate that the models can be used to build pronostic models. One open question is how much these models will have to employ complex and hard to understand pipelines.
the main objective of WP1 for this 1st period was the quantification of more than 7000 human blood samples of people with type 2 diabetes covering spectrum of the disease from non-diabetic, pre-diabetic, diabetic and advanced diabetic characterised by complications and comorbidities. Integration of theses human data is currently ongoing. Preliminary analyses support that 10 to 15% of newly diagnosed type 2 diabetics are characterised by an atypical blood inflammatory response. The nature and severity of this inflammatory response will be deeply deciphered in the 2nd phase of the project through transcriptomic, genetic and immune cell phenotying.
Biomarker measurements for more comprehensive analyses have been completed (proteomics) or are ongoing (transcriptomics, immune cell phenotyping).
WP2
During the initial phase of the INTERCEPT-T2D project, we implemented a multi-omics strategy aimed at identifying SNPs associated with inflammatory components of T2D within regulatory chromatin regions of circulating blood monocytes in patients with T2D (newly-diagnosed and established). This approach is driven by the need to understand the mechanisms underlying the functional impairment of monocytes in the progression of T2D. Our strategy is based on the hypothesis that genetic variants within these regulatory regions could significantly impact gene expression and, consequently, monocyte function, thus contributing to the pathogenesis of T2D.
The implementation of this multi-omics approach involved several critical steps. Firstly, we performed a comprehensive genetic analysis to define SNPs in high linkage disequilibrium with SNPs (“tagSNPs”) from the GWAS catalog which were found to be significantly associated with T2D risk. This allowed us to identify a number of SNPs that likely increase T2D risk. Secondly, we initiated an epigenetic analysis using the ATAC-seq technique to map open chromatin regions in CD14+ monocytes isolated from blood samples of T2D/newly diagnosed patients. This analysis was crucial for pinpointing the genomic location of chromatin regions where regulatory elements such as enhancers are accessible and active to regulate gene expression in blood monocytes. By integrating these genetic and epigenetic datasets, we were able to identify a limited number of SNPs located within candidate regulatory chromatin regions. The results of this first phase of the project have highlighted 31 potential SNPs of interest that reside in the regulatory chromatin of monocytes from T2D patients. These findings provide a foundation for further functional studies to explore how these SNPs, along with epigenetic histone modifications and transcription factors/cofactors, influence gene expression and monocyte activity.
Furthermore, we have established a robust analysis pipeline that will facilitate a comprehensive examination of the genetic components contributing to the inflammatory processes associated with T2D. This pipeline integrates various bioinformatic tools and methodologies, allowing for an in-depth investigation of the genetic and epigenetic interactions that may drive the disease.
WP3
ahead of the anticipated timeline, we made the following progresses:
- Successful pilot phase for the semi-automated multiplex immunofluorescence (IF) staining (Ventana System, Roche) and imaging (Axioscan, Zeiss) of pancreas tissue sections of living donors in the LIDOPACO cohort for markers of various islets, adipocyte, endothelial and inflammatory cells was already completed in 2023 (M1-12 of INTERCEPT-T2D). First biological results in a cohort of 50 patients will be obtained by mid-2024.
- Extensive testing and optimisation of single-cell Laser-Capture-Microdissection (LCM) and single-cell RNA-sequencing (scRNA-seq). Unfortunately, mRNA coverage is still too low for valid profiles. Shall be replaced by advanced targeted in situ single-cell sequencing. The pilot phase of cell-type-specific proteome analysis was also successful, with the detection of ~ 6,000 proteins in beta or alpha of 3 living donors with normoglycemia or T2D. Pancreatic sections from 40 patients will be processed for single islet cell type laser microdissection, protein extraction, mass spectrometry (MS) and computational analysis by the end of 2024.
WP4
We have developed a full protocol, adding important details in the secondary outcomes. All aspects of the study including development of the electronic case report form, data collection system, informed consent forms in EU languages, organization of a central laboratory, transportation of blood samples, continuous blood monitoring devices, neuropathy devices, safety monitoring, data management plan, etc. have been completed. The protocol has been approved by the Swiss Ethics Committee and Swissmedic, and enrollment of the first patient is planned for June 28th 2024.
WP5
The data has been rapidly retrieved from the clinical data mart, and we are working on cleaning it. Preliminary analyses demonstrate that the models can be used to build pronostic models. One open question is how much these models will have to employ complex and hard to understand pipelines.
Pionneer study from the consortium (Julla et al. Circ. Research 2024) provides evidence that monocyte count and phenotype revealed a subgroup of T2D (20% of our T2D population) with elevated cardiovascular risk. The predictive association between monocyte count and major adverse cardiovascular events was validated through an independent prospective cohort of 757 patients with T2D. Integration of monocyte transcriptome analyses and plasma metabolomes showed a disruption of mitochondrial pathways (tricarboxylic acid cycle, oxidative phosphorylation pathway) that underlined a proatherogenic phenotype. This study support our concept that blood immune cells shiuld be used to stratify the disease transition towards complications.