Thoracic aortic aneurysm is a potentially life-threatening disease with a strong genetic contribution. Despite identification of multiple genes involved in aneurysm formation, little is known about the specific underlying mechanisms that drive the pathological changes in the aortic wall. The aim of our study was to unravel the molecular mechanisms underlying aneurysm formation in Marfan syndrome (MFS). We collected aortic wall samples from FBN1 variant-positive MFS patients (n = 6) and healthy donor hearts (n = 5). Messenger RNA (mRNA) expression levels were measured by RNA sequencing and compared between MFS patients and controls, and between haploinsufficient (HI) and dominant negative (DN) FBN1 variants. Immunohistochemical staining, proteomics and cellular respiration experiments were used to confirm our findings. FBN1 mRNA expression levels were highly variable in MFS patients and did not significantly differ from controls. Moreover, we did not identify a distinctive TGF-β gene expression signature in MFS patients. On the contrary, differential gene and protein expression analysis, as well as vascular smooth muscle cell respiration measurements, pointed toward inflammation and mitochondrial dysfunction. Our findings confirm that inflammatory and mitochondrial pathways play important roles in the pathophysiological processes underlying MFS-related aortic disease, providing new therapeutic options.

Single cell RNAseq has been a big leap in many areas of biology. Rather than investigating gene expression on a whole organism level, this technology enables scientists to get a detailed look at rare single cells or within their cell population of interest. The field is growing, and many new methods appear each year. We compared methods utilized in our core facility: Smart-seq3, PlexWell, FLASH-seq, VASA-seq, SORT-seq, 10X, Evercode, and HIVE. We characterized the equipment requirements for each method. We evaluated the performances of these methods based on detected features, transcriptome diversity, mitochondrial RNA abundance and multiplets, among others and benchmarked them against bulk RNA sequencing. Here, we show that bulk transcriptome detects more unique transcripts than any single cell method. While most methods are comparable in many regards, FLASH-seq and VASA-seq yielded the best metrics, e.g., in number of features. If no equipment for automation is available or many cells are desired, then HIVE or 10X yield good results. In general, more recently developed methods perform better. This also leads to the conclusion that older methods should be phased out, and that the development of single cell RNAseq methods is still progressing considerably.

After kidney transplantation (KT), donor-specific hyporesponsiveness (DSH) of recipient T cells develops over time. Recently, apoptosis was identified as a possible underlying mechanism. In this study, both transcriptomic profiles and complete V(D)J variable regions of TR transcripts from individual alloreactive T cells of kidney transplant recipients were determined with single-cell RNA sequencing. Alloreactive T cells were identified by CD137 expression after stimulation of peripheral blood mononuclear cells obtained from KT recipients (N = 7) prior to and 3–5 years after transplantation with cells of their donor or a third party control. The alloreactive T cells were sorted, sequenced and the transcriptome and T cell receptor profiles were analyzed using unsupervised clustering. Alloreactive T cells retain a highly polyclonal T Cell Receptor Alpha/Beta repertoire over time. Post transplantation, donor-reactive CD4+ T cells had a specific downregulation of genes involved in T cell cytokine-mediated pathways and apoptosis. The CD8+ donor-reactive T cell profile did not change significantly over time. Single-cell expression profiling shows that activated and pro-apoptotic donor-reactive CD4+ T cell clones are preferentially lost after transplantation in stable kidney transplant recipients.

Non-invasive prenatal testing (NIPT) has been widely adopted for the screening of chromosomal abnormalities; however, its adoption for monogenic disorders, such as β-thalassaemia, has proven challenging. Haemoglobinopathies are the most common monogenic disorders globally, with β-thalassaemia being particularly prevalent in Cyprus. This study introduces a non-invasive prenatal haplotyping (NIPH) assay for β-thalassaemia, utilizing cell-free DNA (cfDNA) from maternal plasma. The assay determines paternal inheritance by analyzing highly heterozygous single-nucleotide variants (SNVs) in the β-globin gene cluster. To identify highly heterozygous SNVs in the population, 96 randomly selected samples were processed using Illumina DNA-prep NGS chemistry. A custom, high-density NGS genotyping panel, named HAPLONID, was designed with 169 SNVs, including 15 common pathogenic ones. The AmpliSeq for Illumina assay was then applied to cfDNA to evaluate the panel’s efficiency in performing NIPT for β-thalassaemia. Analysis revealed 219 highly polymorphic SNVs, and the sequencing of 17 families confirmed successful paternal allele determination. The NIPH assay demonstrated 100% success in diagnostic interpretation. This study achieved the advancement of an integrated NGS-NIPT assay for β-thalassaemia, bringing it one step closer to being a diagnostic assay and thereby enabling a reduction in the number of risky invasive prenatal sampling procedures in Cyprus and elsewhere.

The cartilage-bone interface, including the zone of calcified cartilage (ZCC), is a thin layer of calcified tissue with a low cell number, thus making it technically challenging to study. The aim of this study was to develop methods to selectively study the ZCC for gene expression analysis and transcriptomic studies. We set-up a method to isolate ZCC explants comprising the ZCC and a thin layer of subchondral bone from human osteochondral tissue (n=4 patients) for tissue culture. The cellular response to a pro-inflammatory cytokine cocktail or TGF-ß1 (72h) was compared to unstimulated explants. Full-thickness non-calcified cartilage (NCC) was used as control group. We have set-up an optical coherence tomography (OCT)-guided 3D-laser microtomy to isolate the calcified ZCC and used FLASH-seq technology (n=4 patients) to characterize the cells in the ZCC compared to donor-matched deep zone of non-calcified cartilage (dNCC). The culture model revealed metabolically active cells in ZCC explants that responded to inflammatory cytokines and to TGF-ß1. The responses were clearly different from the responses of NCC. With 3D-laser microtomy we isolated sufficient RNA from fresh human ZCC. ZCC had similar expression of cartilage-associated genes, lower expression of angiogenesis-related genes, and higher expression of SOST compared to dNCC. The ZCC explant model holds promise to decipher the role of the cartilage-bone interface in disease progression. OCT-guided 3D-laser microtomy and FLASH-seq are innovative technologies overcoming the limitation of standard approaches to phenotypically characterize chondrocytes in the thin ZCC layer and in other calcified tissues.