Biochemical and clinical diagnostic aspects of circulating nucleic acids

We investigated the size distribution of fetal DNA using a combination of agorose gel electrophoresis, Southern blot and real time PCR assay. Our results showed plasma DNA presents apoptotic characteristics and the cell free fetal DNA is generally smaller than maternal plasma DNA. Therefore, the fetal DNA can be enriched by size separation. The enriched fetal DNA in turn can be used for the detection of paternally inherited STR sequence that is masked by maternal sequences. Next, we examined paternally inherited single gene point mutations in FGFR3 gene and β-globin gene respectively. Our analysis showed we could be able to detect the fetal single gene mutations in maternal plasma by using size separation. These studies indicated the size-fractionation of plasma DNA is very useful for non-invasive prenatal determination of fetal point mutations. Several reports indicated that cell free fetal DAN can be detected in the urine of pregnant women. We attempted to reproduce those data. We examined the urinary DNA from normal pregnant women, as well as from pregnancies complicated by HELLP syndrome. In no instance were we able to detect fetal DNA in maternal urine. Our data suggest cell free fetal DNA is not readily detectable in maternal urine. We also examined paternal RhD genotype by real time PCR assay since it is important to counsel a couple about the risk of HDN. We tested 39 samples obtained from males who had been serologically typed to be RhD. We designed two multiplex real-time quantitative PCR assays to simulataneously detect the RhD gene in relation to the SRY sequence and the GAPDH sequence, respectively. Our study showed the two assays were in complete concordance. Therefore, the result indicated that real time PCR can potentially be used for the determination of RhD zygosity

Comparison of methods for donor-derived cell-free DNA quantification in plasma and urine from solid organ transplant recipients.

In allograft monitoring of solid organ transplant recipients, liquid biopsy has emerged as a novel approach using quantification of donor-derived cell-free DNA (dd-cfDNA) in plasma. Despite early clinical implementation and analytical validation of techniques, direct comparisons of dd-cfDNA quantification methods are lacking. Furthermore, data on dd-cfDNA in urine is scarce and high-throughput sequencing-based methods so far have not leveraged unique molecular identifiers (UMIs) for absolute dd-cfDNA quantification. Different dd-cfDNA quantification approaches were compared in urine and plasma of kidney and liver recipients: A) Droplet digital PCR (ddPCR) using allele-specific detection of seven common HLA-DRB1 alleles and the Y chromosome; B) high-throughput sequencing (HTS) using a custom QIAseq DNA panel targeting 121 common polymorphisms; and C) a commercial dd-cfDNA quantification method (AlloSeq® cfDNA, CareDx). Dd-cfDNA was quantified as %dd-cfDNA, and for ddPCR and HTS using UMIs additionally as donor copies. In addition, relative and absolute dd-cfDNA levels in urine and plasma were compared in clinically stable recipients. The HTS method presented here showed a strong correlation of the %dd-cfDNA with ddPCR (R 2 = 0.98) and AlloSeq® cfDNA (R 2 = 0.99) displaying only minimal to no proportional bias. Absolute dd-cfDNA copies also correlated strongly (τ = 0.78) between HTS with UMI and ddPCR albeit with substantial proportional bias (slope: 0.25; 95%-CI: 0.19-0.26). Among 30 stable kidney transplant recipients, the median %dd-cfDNA in urine was 39.5% (interquartile range, IQR: 21.8-58.5%) with 36.6 copies/μmol urinary creatinine (IQR: 18.4-109) and 0.19% (IQR: 0.01-0.43%) with 5.0 copies/ml (IQR: 1.8-12.9) in plasma without any correlation between body fluids. The median %dd-cfDNA in plasma from eight stable liver recipients was 2.2% (IQR: 0.72-4.1%) with 120 copies/ml (IQR: 85.0-138) while the median dd-cfDNA copies/ml was below 0.1 in urine. This first head-to-head comparison of methods for absolute and relative quantification of dd-cfDNA in urine and plasma supports a method-independent %dd-cfDNA cutoff and indicates the suitability of the presented HTS method for absolute dd-cfDNA quantification using UMIs. To evaluate the utility of dd-cfDNA in urine for allograft surveillance, absolute levels instead of relative amounts will most likely be required given the extensive variability of %dd-cfDNA in stable kidney recipients

Core binding factors are necessary for natural killer cell development, and cooperate with Notch signaling during T cell specification

CBF{beta} is the non-DNA binding subunit of the core binding factors (CBFs). Mice with reduced CBF{beta} levels display profound, early defects in T but not B cell development. Here we show that CBF{beta} is also required at very early stages of natural killer (NK) cell development. We also demonstrate that T cell development aborts during specification, as the expression of Gata3 and Tcf7, which encode key regulators of T lineage specification, is substantially reduced, as are functional thymic progenitors. Constitutively active Notch or IL-7 signaling cannot restore T cell expansion or differentiation of CBF{beta} insufficient cells, nor can overexpression of Runx1 or CBF{beta} overcome a lack of Notch signaling. Therefore the ability of the prethymic cell to respond appropriately to Notch is dependent on CBF{beta}, and both signals converge to activate the T cell developmental program

Circulating cell-free DNA: a powerful biomarker for tumor management and a possible monitor tool in other pathological conditions.

\u2018Liquid biopsy\u2019, i.e. the analysis of cfDNA in blood or body fluids, can give a live, \u2018total\u2019 image representing the entire heterogeneity of the system. The application of high throughput analytical procedures, such NGS or ddPCR, are necessary to obtain reliable data on such a small amount of starting material. Only few applications have achieved a clinical validation, due to the great variability in preanalytical procedures. In our work we evaluated cfDNA with three different aims. \u2022 Presence of mutation in a panel of 16 genes of the HR pathway in genomic and cfDNA in patients affected by breast cancer. We observed mutations in 3 out of 6 samples; in one case variant fraction in cfDNA was higer than in genomic DNA, probably due to limited ability to detect clonal heterogeneity in tissue. \u2022 Monitoring tool for determining septic risk in patients undergoing dialysis. We detected in a sample, in accordance with the emoculture, a Staphylococcus strain together with Propionibacterium and Streptococcus strains. The detection of Burkholderia multivorans in another sample raised the possibility to identify those bacteria that take more than the canonical 5 days of emoculture to growth, or that completely do not grow in emoculture conditions. \u2022 Detection of donor-derived cfDNA in transplanted patients. We identified more than 50% of donor derived polymorphisms just after reperfusion, falling down to 10% one day after surgery and then disappearing. The possibility to cross our data with clinical parameters will help us to better describe the pertinence of our results to the effective status of patients. In all the three settings, we are collecting other samples to have a broader amount of data that will allow us to perform statistical analysis to effectively validate our procedures

SEROPREVALENCE AND PHYLOGENETIC GENOTYPING OF EPSTEIN BARR VIRUS (EBV) AMONG BLOOD DONORS IN QATAR

Background: EBV is a lymphotropic herpesvirus and the causative agent of infectious mononucleosis. EBV is highly prevalent and has been linked to several malignancies. The virus is generally transferred by oral secretions and it persists as a latent infection in human B-cells. However, it can be also transmitted through blood transfusions and organ transplantations. The goal of this study was to (i) estimate the rate of infection of EBV in Qatar in healthy individuals, using serological testing, and viral load quantification, to (ii) study the correlation of EBV with demographic markers, and to (iii) study the molecular similarity of EBV isolates, using phylogenetic genotyping. Materials and methods: For estimating EBV seroprevalence, qualitative ELISA kits for detecting the following EBV antibodies were used: EBV viral capsid antigen (VCA) IgG & IgM, EBV nuclear antigen (EBNA) IgG & IgM, and early antigen (EA-D) IgG. To study the EBV viremia rate, DNA extracted from the buffy coat was subjected for viral detection and quantification using RT-PCR. For genotyping, nested PCR targeting the EBNA2 gene was used. And for further sub genotyping, the highly variable LMP-1 gene was amplified, cloned, sequenced and used for phylogenetic analysis. Results: Out of 673 analyzed samples (223 Qataris, 450 non-Qatari residents), 659 (97.9%) were EBV seropositive with different infection stages. Interestingly, 14 (2.1%) tested negative for all anti-EBV antibodies indicating no prior exposure to EBV. EBV DNA was detected in 354/673 (52.6%). Both EBV seroprevalence and viremia rate increased significantly with age. Genotyping for 51 randomly selected positive DNA samples showed that Genotype 1 was predominating, found in 36/51 (72.5%), while genotype 2 was found in 12 (23.5%) samples. Mixed infection was found in 2 (3.9%) samples. Surprisingly, Sub genotyping for 30 samples revealed that all tested clones (n=119) have the South Eastern Asia 1 (SEA1) strain. 30-bp and 69 bp deletions in the LMP-1 were also found in 10% and 13.3% samples, respectively. Conclusion: This is the first study investigating the seroprevalence, the viremia rate, and the molecular epidemiology of EBV among blood donors in Qatar. Hence, it should provide the epidemiologists, blood banks’ personnel, researchers and clinicians with EBV prevalence estimates and molecular epidemiology of EBV as a highly prevalent transfusion transmissible oncovirus

Further development and refinement of hematopoietic cell transplantation in zebrafish

Hematopoietic stem cells are a rare but crucial population of cells that are responsible for maintaining hematopoiesis throughout vertebrate life. Clinically, hematopoietic stem cells have been utilised for treatment of hematological disease, autoimmune disorders and cancer through the application of hematopoietic cell transplants. A detailed understanding of the behaviour of hematopoietic stem cells and their post-transplant interaction with the niche can lead to improved transplant outcomes. However, there are many challenges in studying hematopoietic stem cell transplantation in mammalian systems owing to the difficulty of observing transplanted cells in vivo. This thesis aims to refine hematopoietic cell transplantation protocols described in adult zebrafish (Danio rerio). To this end, fluorescent hematopoietic stem and precursor populations were further characterised in transgenic donor fish using a combination of flow cytometry and microscopy techniques. Furthermore, Runx:mCherry positive populations were assessed for stem cell functionality through transplantation. The utility of bloodless cmybt25127 mutant fish to investigate hematopoietic cell transplantation by longitudinal imaging was evaluated. In addition, the stimulatory effects of viral mimetics were assessed in transgenic and cmybt25127 mutant fish. Finally, the effect of antibiotic treatment was investigated in transgenic fish. These experiments revealed two fluorescent cell populations in Tg(Runx:mCherry) transgenic zebrafish kidney marrow. Hematopoietic cell transplant studies and transcript analysis indicated that the Runx:mCherry low population could be enriched for hematopoietic stem cells. Furthermore, experiments revealed that homozygous cmybt25127 mutant fish are capable of regenerating their tail fin following amputation and of initiating a partial anti-viral response to resiquimod stimulation. Finally, a post-transplant scoring system was devised in homozygous cmybt25127 mutant fish and used to assess functional differences between Runx:mCherryhigh and low populations. Overall, this thesis has further developed hematopoietic cell transplantation in zebrafish and demonstrated that in vivo imaging can be used to track the transplant outcome and behaviour of transplanted cells.Open Acces

Current Trends in Applications of Circulatory Microchimerism Detection in Transplantation

Primarily due to recent advances of detection techniques, microchimerism (the proportion of minor variant population is below 1%) has recently gained increasing attention in the field of transplantation. Availability of polymorphic markers, such as deletion insertion or single nucleotide polymorphisms along with a vast array of high sensitivity detection techniques, allow the accurate detection of small quantities of donor- or recipient-related materials. This diagnostic information can improve monitoring of allograft injuries in solid organ transplantations (SOT) as well as facilitate early detection of relapse in allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the present review, genetic marker and detection platform options applicable for microchimerism detection are discussed. Furthermore, current results of relevant clinical studies in the context of microchimerism and SOT or allo-HSCT respectively are also summarized