•Development and evaluation of a highly sensitive NGS-based chimerism assay.•The assay is suitable for early detection of increasing MRD after HSCT.•Limit of detection is 0,1% with excellent ...accuracy.•Suitable for high-throughput chimerism analysis.
The aim of this study was to evaluate the performance of a novel NGS-based assay to monitor mixed chimerism (MC) and compare its technical capacity to established techniques for chimerism analysis. Artificial and clinical samples with increasing amounts of patient DNA were compared using real-time PCR detection of indels and SNP, fragment analysis of short-tandem repeats (STR) and NGS analysis of indels. Real-time PCR displayed excellent sensitivity (>0,01%) but poor accuracy (>20 CV% at MC > 20%), while fragment analysis exhibited good accuracy (<5 CV% at MC > 20%) with limited sensitivity (>2,5%). In contrast, NGS chimerism demonstrated a sensitivity (>0,1%) equal to real-time PCR and an accuracy equal or better than STR analysis throughout an extensive range of mixed chimerism (0,1 – 100%). To evaluate performance of the separate techniques for chimerism determination, 75 retrospective patient monitoring samples (3–7 weeks post-HSCT) with low (<5%), intermediate (5–20%) or high mixed chimerism (>20%) were analyzed. The between run precision for the NGS assay varied from 0,72% (>20% MC) to 7,38% (MC < 5%). In conclusion, NGS displayed a combination of high sensitivity with good accuracy in both artificial and clinical chimerism samples.
We report the findings from 4437 individuals (3219 patients and 1218 relatives) who have been analyzed by whole genome sequencing (WGS) at the Genomic Medicine Center Karolinska-Rare Diseases ...(GMCK-RD) since mid-2015. GMCK-RD represents a long-term collaborative initiative between Karolinska University Hospital and Science for Life Laboratory to establish advanced, genomics-based diagnostics in the Stockholm healthcare setting.
Our analysis covers detection and interpretation of SNVs, INDELs, uniparental disomy, CNVs, balanced structural variants, and short tandem repeat expansions. Visualization of results for clinical interpretation is carried out in Scout-a custom-developed decision support system. Results from both singleton (84%) and trio/family (16%) analyses are reported. Variant interpretation is done by 15 expert teams at the hospital involving staff from three clinics. For patients with complex phenotypes, data is shared between the teams.
Overall, 40% of the patients received a molecular diagnosis ranging from 19 to 54% for specific disease groups. There was heterogeneity regarding causative genes (n = 754) with some of the most common ones being COL2A1 (n = 12; skeletal dysplasia), SCN1A (n = 8; epilepsy), and TNFRSF13B (n = 4; inborn errors of immunity). Some causative variants were recurrent, including previously known founder mutations, some novel mutations, and recurrent de novo mutations. Overall, GMCK-RD has resulted in a large number of patients receiving specific molecular diagnoses. Furthermore, negative cases have been included in research studies that have resulted in the discovery of 17 published, novel disease-causing genes. To facilitate the discovery of new disease genes, GMCK-RD has joined international data sharing initiatives, including ClinVar, UDNI, Beacon, and MatchMaker Exchange.
Clinical WGS at GMCK-RD has provided molecular diagnoses to over 1200 individuals with a broad range of rare diseases. Consolidation and spread of this clinical-academic partnership will enable large-scale national collaboration.
Recipient-donor chimerism is routinely analyzed after allogeneic hematopoietic stem cell transplantation (HSCT) to monitor engraftment and graft rejection. For malignancies, chimerism can also be ...used to screen for disease relapse post-HSCT but methodology and interpretation of results are not standardized and likely depend on underlying diagnosis. We have implemented highly sensitive and accurate methodologies for chimerism analysis for the purpose of improving relapse prediction. Here, we report an exploratory retrospective analysis of clinical routine chimerism results from all 154 HSCTs for acute myeloid leukemia (AML) performed at our center during the years 2015-2020 with the aim of suggesting a clinically useful threshold at which risk of relapse is high. Relapse was not reliably predicted based on single elevated chimerism values obtained before time of overt relapse. However, early complete donor chimerism, here defined as recipient DNA < 0.2% in CD33
cells in any blood or bone marrow sample taken during the first 60 days after HSCT, correlated inversely with relapse during the observation time (log-rank test P = 0.033). We propose that achievement of complete chimerism determined early after HSCT using sensitive methods can be used for risk-stratification of AML patients.
The application of microfluidic technologies to microalgal research is particularly appealing since these approaches allow the precise control of the extracellular environment and offer a ...high-throughput approach to studying dynamic cellular processes. To expand the portfolio of applications, here we present a droplet-based microfluidic method for analysis and screening of Phaeodactylum tricornutum and Nannochloropsis gaditana, which can be integrated into a genetic transformation workflow. Following encapsulation of single cells in picolitre-sized droplets, fluorescence signals arising from each cell can be used to assess its phenotypic state. In this work, the chlorophyll fluorescence intensity of each cell was quantified and used to identify populations of P. tricornutum cells grown in different light conditions. Further, individual P. tricornutum or N. gaditana cells engineered to express green fluorescent protein were distinguished and sorted from wild-type cells. This has been exploited as a rapid screen for transformed cells within a population, bypassing a major bottleneck in algal transformation workflows and offering an alternative strategy for the identification of genetically modified strains.
•Droplet-based microfluidic systems are promising tools for algal single cell analysis.•Improved intracellular fluorescence detection allows effective sorting of algae cells.•The physiological status of single encapsulated algae cells can be determined.•Sorting in microdroplets enables faster identification of transformed cells.
When green algae are exposed to physiological stresses such as nutrient deprivation, growth is arrested and the cells channel fixed carbon instead into storage compounds, accumulating first starch ...granules and then lipid bodies containing triacylglycerides. In recent years there has been significant interest in the commercial exploitation of algal lipids as a sustainable source of biodiesel. Since starch and lipid biosynthesis involves the same C3 precursor pool, it has been proposed that mutations blocking starch accumulation should result in increased lipid yields, and indeed several studies have supported this. The fast-growing, thermotolerant alga Chlorella sorokiniana represents an attractive strain for industrial cultivation. We have therefore generated and characterized starch-deficient mutants of C. sorokiniana and determined whether lipid levels are increased in these strains under stress conditions. One mutant (ST68) is shown to lack isoamylase, whilst two others (ST3 and ST12) are defective in starch phosphorylase. However, we find no significant change in the accumulation or profile of fatty acids in these mutants compared to the wild-type, suggesting that a failure to accumulate starch per se is not sufficient for the hyper-accumulation of lipid, and that more subtle regulatory steps underlie the partitioning of carbon to the two storage products.
•We have isolated a collection of starch deficient mutants of Chlorella sorokiniana.•We have characterized mutants defective in isoamylase and starch phosphorylase.•Mutants show no increase in storage lipids as seen for other algal starch mutants.
Background
Lymphocyte neogenesis from primary lymphoid organs is essential for a successful reconstitution of immunity after allogeneic hematopoietic stem cell transplantation (HSCT). This ...single-center retrospective study aimed to evaluate T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) as surrogate markers for T and B cell recovery, as predictors for transplantation-related outcomes in adult acute myeloid leukemia (AML) patients.
Methods
Ninety adult patients diagnosed with AML and treated with HSCT between 2010 and 2015 were included in the study. TREC and KREC levels were measured by quantitative PCR at 1, 3, 6, and 12 months after transplantation.
Results
Overall, excision circle levels increased between 3 and 6 months post-HSCT for TREC (p = 0.005) and 1 and 3 months for KREC (p = 0.0007). In a landmark survival analysis at 12 months post-HSCT, TREC levels were associated with superior overall survival (HR: 0.52, 95% CI: 0.34 - 0.81, p = 0.004). The incidence of viral infections within the first 100 days after transplantation was associated with lower TREC levels at 6 months (p = 0.0002). CMV reactivation was likewise associated with lower TREC levels at 6 months (p = 0.02) post-HSCT. KREC levels were not associated with clinical outcomes in statistical analyzes.
Conclusions
Results from the present study indicate that TREC measurement could be considered as part of the post-HSCT monitoring to identify AML patients with inferior survival after transplantation. Further prospective studies are warranted to validate these findings.
Relapse is a major cause of treatment failure after hematopoietic stem cell transplantation (HSCT) for acute leukemia. Here, we report a monocentric retrospective study of all HSCTs for B cell acute ...lymphoblastic leukemia (ALL) performed during the years 2005–2021 (n = 138, including 51 children), aiming to identify the optimal use of lineage‐specific recipient‐donor chimerism analysis for prediction of relapse. In adults, relapse was associated with increased recipient chimerism in CD3+ bone marrow cells sampled at least 30 days before a relapse. Relapse could be predicted with a sensitivity of 73% and a specificity of 83%. Results were similar for children but with a higher recipient chimerism cutoff. Additionally, adults that had at least one chimerism value <0.12% in CD3+ peripheral blood cells within the first 60 days after HSCT had 89% probability of being relapse‐free after 2‐years compared to 64%. Results were similar for children but again necessitating a higher chimerism cutoff. These results suggest that high‐sensitive lineage‐specific chimerism analysis can be used for (1) early ALL relapse prediction by longitudinal chimerism monitoring in CD3+ bone marrow cells and (2) relapse risk stratification by analyzing CD3+ blood cells early post‐HSCT.
Dyskeratosis congenita is a rare inherited disease with classic cutaneous symptoms, sometimes accompanied with more severe extracutaneous manifestations such as bone marrow failure, which can be ...lethal. Eltrombopag is an orally available thrombopoietin receptor agonist in clinical use for increasing platelet levels in patients with immune thrombocytopenia and aplastic anemia. Here, 3 pediatric patients with dyskeratosis congenita are presented with varying disease severity, in which off-label eltrombopag treatment had no clinical effect on bone marrow failure. This, in addition to the negative results in a previous case report, supports the preclusion of eltrombopag use in dyskeratosis congenita.
•Mutational screening after transplantation identifies rare relapse-initiating stem cells while patients remain in complete remission.•Mutational screening of HSPCs after stem cell transplantation ...enhances MRD sensitivity ∼100-fold.
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Relapse after complete remission (CR) remains the main cause of mortality after allogeneic stem cell transplantation for hematological malignancies and, therefore, improved biomarkers for early prediction of relapse remains a critical goal toward development and assessment of preemptive relapse treatment. Because the significance of cancer stem cells as a source of relapses remains unclear, we investigated whether mutational screening for persistence of rare cancer stem cells would enhance measurable residual disease (MRD) and early relapse prediction after transplantation. In a retrospective study of patients who relapsed and patients who achieved continuous-CR with myelodysplastic syndromes and related myeloid malignancies, combined flow cytometric cell sorting and mutational screening for persistence of rare relapse-initiating stem cells was performed in the bone marrow at multiple CR time points after transplantation. In 25 CR samples from 15 patients that later relapsed, only 9 samples were MRD-positive in mononuclear cells (MNCs) whereas flowcytometric-sorted hematopoietic stem and progenitor cells (HSPCs) were MRD-positive in all samples, and always with a higher variant allele frequency than in MNCs (mean, 97-fold). MRD-positivity in HSPCs preceded MNCs in multiple sequential samples, in some cases preceding relapse by >2 years. In contrast, in 13 patients in long-term continuous-CR, HSPCs remained MRD-negative. Enhanced MRD sensitivity was also observed in total CD34+ cells, but HSPCs were always more clonally involved (mean, 8-fold). In conclusion, identification of relapse-initiating cancer stem cells and mutational MRD screening for their persistence consistently enhances MRD sensitivity and earlier prediction of relapse after allogeneic stem cell transplantation.
Relapse following complete remission (CR) is the leading cause of death following allogeneic transplant for myeloid malignancy. In this Plenary Paper, Dimitriou et al tracked measurable residual disease (MRD) to predict relapse following transplant for myelodysplasia and related myeloid malignancies. Using flow cytometry to sort hematopoietic stem and progenitor cells (HSPCs) on sequential samples, the authors found that only 60% of unsorted mononuclear cells from patients in CR who later relapsed had detectable MRD, but HSPCs from the same patients were 100% MRD-positive, suggesting a means to identify patients for whom preemptive therapy may prevent overt relapse.
Inherited deficiency of the RNA lariat–debranching enzyme 1 (DBR1) is a rare etiology of brainstem viral encephalitis. The cellular basis of disease and the range of viral predisposition are unclear. ...We report inherited DBR1 deficiency in a 14-year-old boy who suffered from isolated SARS-CoV-2 brainstem encephalitis. The patient is homozygous for a previously reported hypomorphic and pathogenic DBR1 variant (I120T). Consistently, DBR1 I120T/I120T fibroblasts from affected individuals from this and another unrelated kindred have similarly low levels of DBR1 protein and high levels of RNA lariats. DBR1 I120T/I120T human pluripotent stem cell (hPSC)–derived hindbrain neurons are highly susceptible to SARS-CoV-2 infection. Exogenous WT DBR1 expression in DBR1 I120T/I120T fibroblasts and hindbrain neurons rescued the RNA lariat accumulation phenotype. Moreover, expression of exogenous RNA lariats, mimicking DBR1 deficiency, increased the susceptibility of WT hindbrain neurons to SARS-CoV-2 infection. Inborn errors of DBR1 impair hindbrain neuron–intrinsic antiviral immunity, predisposing to viral infections of the brainstem, including that by SARS-CoV-2.
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