Functional Genomics
Genome-wide association studies (GWAS) have revealed a large number of genomic variants or singular nucleotide variants (SNVs) which are located in numerous risk loci (>200 for CAD up to date) distributed throughout the genome. Each of these variants is associated with a modest increase in CAD risk. Most of these SNVs are common non-coding variants with a minor allele frequency (MAF) of >5%. Any of these SNVs have the potential to contribute to the functional link between genotype and phenotype either by acting upon one or several genes within the locus (cis , < 1 MB) or by altering gene expression in distal regions (trans, > 5 MB).
In order to understand the pathomechanisms underlying the initiation and progression of atherosclerosis, we aim to reveal the impact of CAD associated genomic variants at the genetic and cellular level using computational (‘dry lab’) and experimental (‘wet lab’) approaches. In the working group ‘Functional Genomics’, we focus on specific pathomechanisms, such as oxidative stress, mitochondrial dysfunction, vascular remodeling or MRAS signaling pathways.
Projects
We will study in-vitro MYOZ2, a gene that is associated with CAD in women, but not in men. The aim is to define mechanisms underlying this difference between men and women, thereby identifying potential novel therapeutic targets for personalized medicine based on sex differences.
Improved understanding of CAD development and prevention has led to a >60% reduction in deaths from heart attack and stroke over the past four decades. Alarmingly, younger women show the lowest rate of improvement in the past 20 years. Although this sex-gap is increasingly recognized in research, it has not yet been fully addressed, as stratification of data by sex remains uncommon.
Genome-wide association studies (GWAS) have identified over 240 loci associated with susceptibility to CAD. Consistent with other complex diseases, genetic discovery analyses in CAD have enabled insights into disease etiology and causal risk factors, facilitated the development of novel tools for predicting clinical risk, and identified novel therapeutic targets.
The largest CAD meta-analysis to date, performed by the applicant and collaborators, identified the first associations that differed by sex. Of the ten sex-differentiated associations showing genome-wide significance, nine loci had stronger effects in the male-only analysis, whereas the tenth, rs7696877, located near MYOZ2, had a significantly stronger effect in females than males.
For none of these loci a causal link between SNP and specific genes have been established yet. Furthermore, there are no data that can explain the sex-specific effects of these loci. Here, we will focus on MYOZ2, encoding myozenin-2, also called calsarcin-1. MYOZ2 is highly expressed in the circulatory system and has been shown to suppress pathologic cardiac hypertrophy. To our knowledge no study has evaluated the role of MYOZ2 in CAD nor any sex-specific function.
This project directly addresses the core objectives of the focus area ‘Translational Vascular Biomedicine’ of the BIH. Particularly, the aim of our in-vitro study is to assess the impact of MYOZ2, a gene that has been associated in GWAS analyses with an increased cardiovascular risk in females only, in the atherosclerotic process, with a special emphasis on the potential differences in males and females.
The project has already started with a comprehensive bioinformatic analysis to define the most likely causal SNP (rs7696877) at the MYOZ2 locus. Next, we started to introduce this variant, using gene-editing methods, into human induced pluripotent stem cells (hiPSCs) from a female and male healthy donor. Furthermore, we will soon start to knock out the MYOZ2 gene in these hiPSCs. QC will ensure both stemness and successful variant introduction through sequencing of the locus using Oxford Nanopore Technologies, as well as immunofluorescence staining for stemness factors (OCT4, NANOG).
Subsequently, these gene-edited hiPSCs will then be differentiated into vascular smooth-muscle cells (VSMC), endothelial cells (EC), and endothelial-smooth muscle cell organoids in Lübeck for further functional follow-up. Respective protocols are established in the Lübeck group.
Funded by the BIH Excellence Award for Sex and Gender Aspects in Health Research 2021.
Dr. rer. nat. Tobias Reinberger
The functional link between genotype and phenotype is poorly understood for most of the risk loci.
It is believed that mitochondrial damage and increased production of reactive oxygen species (ROS) play a crucial role in atherosclerotic lesion formation.
Therefore, we assume that some of the common genomic variants identified by GWAS promote mitochondrial dysfunction, lead to enhanced ROS production, and/or reduce the antioxidative capacity of vascular cells.
To identify genomic variants associated with mitochondrial dysfunction and ROS imbalance, we perform in-silico scoring of all CAD GWAS variants based on different metrices (e.g. eQTL data).
In a second step, we investigate genes linked to high-ranked variants in human smooth muscle cells and other human cell lines.
Dr. rer. nat. Tobias Reinberger
We hypothesize that the stability of the atherosclerotic plaques depends on the phenotypic transition of vascular smooth muscle cells (VSMCs ) involving the transcription factor Kruppel Like Factor 4 (KLF4) which has been identified to be associated with CAD by GWAS. It has been demonstrated that oxidative stress triggers the expression of KLF4.
Therefore, we hypothesize that some of the novel CAD risk variants promote collectively oxidative stress and hence the KLF4-mediated transition of VSMCs.
To test this hypothesis, we measure the production of ROS in human VSMCs following gene silencing and extracellular stimuli. The phenotypic transition of VSMCs is then assessed using specific biomarkers (e.g. CD68).
Funded by the Leducq Foundation (PlaqOmics)
We know that the genetic architecture plays an essential role in atherosclerosis. Single nucleotide variants (SNVs) and their corresponding genes form a complex network which drives the progression of atherosclerosis.
We aim to identify networks of SNVs using bioinformatic methods such as big data analysis and the integration of in-silico and experimental data.
Such genetic and cellular networks will then be validated in primary smooth muscle cells, endothelial cells as well as iPSC-derived smooth muscle cells and endothelial cells.
Moreover, we utilize CRISPR/Cas-based gene editing in iPSCs, i.e. we introduce SNVs into the genome of cells.
This innovative methodology enables us to investigate the interaction of different variants with respect to their physiological function in a suitable model system.
There are two approaches in the identification of genetic disorders i.e. rare variants present in families and common variants (GWAS). In GWAS Single Nucleotide Polymorphisms (SNPs) are studied in depth.
Our project is about defining the role of MRAS gene in atherosclerosis. The single nucleotide polymorphism (SNP) rs9818870, located in the 3’untranslated region (3′-UTR) of the muscle Ras (MRAS) gene at chromosome 3q22.3, was identified in 2009. In-silico analysis revealed that rs9818870 might modify the structure of MRAS mRNA, which further leads to the microRNA-mediated dysregulation of gene expression.
In this project we will utilize state of art technologies including Next Generation Sequencing (NGS) and Oxford Nanopore Technology (ONT) for further understanding the role of CAD risk genes.
The main purpose of this project is to determine the role of MRAS in blood pressure regulation and plaque stabilization in mice and zebra fish as well as in-depth study of the lead SNP at MRAS risk locus on MRAS gene expression.