Nearly all adults harbor acute myeloid leukemia (AML)-related clonal hematopoietic mutations at a variant allele fraction (VAF) of ≥0.0001, yet relatively few develop hematologic malignancies. We ...conducted a nested analysis in the Nurses' Health Study and Health Professionals Follow-Up Study blood subcohorts, with up to 22 years of follow up to investigate associations of clonal mutations of ≥0.0001 allele frequency with future risk of AML. We identified 35 cases with AML that had pre-diagnosis peripheral blood samples and matched two controls without history of cancer per case by sex, age, and ethnicity. We conducted blinded error-corrected sequencing on all study samples and assessed variant-associated risk using conditional logistic regression. We detected AML-associated mutations in 97% of all participants (598 mutations, 5.8/person). Individuals with mutations ≥0.01 variant allele fraction had a significantly increased AML risk (OR 5.4, 95%CI: 1.8-16.6), as did individuals with higher-frequency clones and those with
R882H/C mutations. The risk of lower-frequency clones was less clear. In the 11 case-control sets with samples banked ten years apart, clonal mutations rarely expanded over time. Our findings are consistent with published evidence that detection of clonal mutations ≥0.01 VAF identifies individuals at increased risk for AML. Further study of larger populations, mutations co-occurring within the same pre-leukemic clone and other risk factors (lifestyle, epigenetics, etc.), are still needed to fully elucidate the risk conferred by low-frequency clonal hematopoiesis in asymptomatic adults.
Xenograft models are invaluable tools in establishing the current paradigms of hematopoiesis and leukemogenesis. The zebrafish has emerged as a robust alternative xenograft model but, like mice, lack ...specific cytokines that mimic the microenvironment found in human patients. To address this critical gap, we generated the first humanized zebrafish that express human hematopoietic-specific cytokines (GM-CSF, SCF, and SDF1α). Termed GSS fish, these zebrafish promote survival, self-renewal and multilineage differentiation of human hematopoietic stem and progenitor cells and result in enhanced proliferation and hematopoietic niche-specific homing of primary human leukemia cells. Using error-corrected RNA sequencing, we determined that patient-derived leukemias transplanted into GSS zebrafish exhibit broader clonal representation compared to transplants into control hosts. GSS zebrafish incorporating error-corrected RNA sequencing establish a new standard for zebrafish xenotransplantation that more accurately recapitulates the human context, providing a more representative cost-effective preclinical model system for evaluating personalized response-based treatment in leukemia and therapies to expand human hematopoietic stem and progenitor cells in the transplant setting.
Background/Aim
Tooth autotransplantation has been advocated for replacement of missing teeth or teeth that are unsuitable for restoration. The aim of this study was to investigate the outcomes and ...prognostic factors that influenced the success of tooth transplantation in a paediatric population.
Materials and Methods
Data were extracted from the records of 75 patients (89 teeth). Demographic and prognostic factors were recorded and analysed for the clinical and radiographic outcomes for periodontal ligament (PDL) and pulp healing of transplanted teeth.
Results
The mean age at transplant was 13.2 years, and the mean follow‐up observation period was 2.6±1.8 years with a range of 12.0 months to 9.9 years. The main reason for transplantation was to replace upper central incisors lost or missing due to dental trauma, hypodontia and dilaceration. Of the 45 teeth that were monitored for pulp revascularization, 75.6% showed clinical and radiographic signs of pulp healing and 24.4% showed signs of pulp necrosis and infection. Pulp healing was significantly related to the stage of root development of the transplant. Favourable PDL healing was observed in 87.6% of the transplants, while 13.5% showed signs of replacement resorption. PDL healing was significantly related to the stage of root formation of the transplanted tooth at the time of the surgery, the ease of handling and placement of the tooth, and the status of the alveolar bone at the recipient site at the time of the surgery. Overall success of tooth transplantation was 87.6%, and the survival rate was 94.4%.
Conclusions
Tooth transplantation carried out in children and adolescents demonstrated high success and survival, with the stage of root development influencing both the pulp and PDL healing of the transplanted teeth.
Xenograft models are invaluable tools in establishing the current paradigms of hematopoiesis and leukemogenesis. The zebrafish has emerged as a robust alternative xenograft model but, like mice, lack ...specific cytokines that mimic the microenvironment found in human patients. To address this critical gap, we generated the first humanized zebrafish that express human hematopoietic-specific cytokines (GM-CSF, SCF, and SDF1α). Termed GSS fish, these zebrafish promote survival, self-renewal and multilineage differentiation of human hematopoietic stem and progenitor cells and result in enhanced proliferation and hematopoietic niche-specific homing of primary human leukemia cells. Using error-corrected RNA sequencing, we determined that patient-derived leukemias transplanted into GSS zebrafish exhibit broader clonal representation compared to transplants into control hosts. GSS zebrafish incorporating error-corrected RNA sequencing establish a new standard for zebrafish xenotransplantation that more accurately recapitulates the human context, providing a more representative cost-effective preclinical model system for evaluating personalized response-based treatment in leukemia and therapies to expand human hematopoietic stem and progenitor cells in the transplant setting.
Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal ...mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5-2.0%. Thus, standard NGS has a limit of detection for mutations that are >0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is <0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.
For neurodevelopmental disorders (NDDs), etiological evaluation can be a diagnostic odyssey involving numerous genetic tests, underscoring the need to develop a streamlined algorithm maximizing ...molecular diagnostic yield for this clinical indication. Our objective was to compare the yield of exome sequencing (ES) with that of chromosomal microarray (CMA), the current first-tier test for NDDs.
We performed a PubMed scoping review and meta-analysis investigating the diagnostic yield of ES for NDDs as the basis of a consensus development conference. We defined NDD as global developmental delay, intellectual disability, and/or autism spectrum disorder. The consensus development conference included input from genetics professionals, pediatric neurologists, and developmental behavioral pediatricians.
After applying strict inclusion/exclusion criteria, we identified 30 articles with data on molecular diagnostic yield in individuals with isolated NDD, or NDD plus associated conditions (such as Rett-like features). Yield of ES was 36% overall, 31% for isolated NDD, and 53% for the NDD plus associated conditions. ES yield for NDDs is markedly greater than previous studies of CMA (15-20%).
Our review demonstrates that ES consistently outperforms CMA for evaluation of unexplained NDDs. We propose a diagnostic algorithm placing ES at the beginning of the evaluation of unexplained NDDs.
Introduction: Children with Down syndrome (DS) have a 150-fold higher risk of developing myeloid leukemia (ML-DS) (Wechsler J, Nat Gen 2002). Ten percent (10%) of DS children are also predisposed to ...developing a preleukemic self-limiting condition, transient myeloproliferative disorder (TMD). While the majority of these children achieve complete remission, 20-30% of TMD patients will eventually develop ML-DS (Gamis AS, Blood 2011). Unique to ML-DS and TMD is the association with mutations in the X-linked hematopoietic transcription factor, GATA1 (Alford KA, Blood 2011), which are pathognomonic, but perhaps not sufficient, for ML-DS. Along with GATA1 mutations, ML-DS has a distinct mutational landscape implicating the cohesin complex, epigenetic regulators, signal transducers and the RAS pathway (Yoshida K, Nat Gen 2013). While these findings have informed our understanding of ML-DS etiology, ~30% of healthy DS neonates - without overt TMD or ML-DS - also harbor GATA1 mutations (Roberts I, Blood 2013), suggesting that ML-DS arises from clonal outgrowth of these progenitors that acquire specific additional mutations. Because the spectrum of physiologic clonal hematopoiesis is unknown in DS, we sought to characterize clonal mutations in healthy DS children as compared to a cohort of ML-DS cases using our highly sensitive and specific error-corrected sequencing (ECS) methodology. A precise understanding of their altered hematopoietic development may inform risk stratification, therapeutic selection and outcomes.
Methods: The Druley lab has developed a custom error-corrected sequencing (ECS) panel targeting 80 genes frequently mutated in both pediatric and adult myeloid malignancies (Young AL, Nat Comm 2016) that is validated to identify clonal mutations as rare as 0.0001 variant allele fraction (VAF), 100X below the error-rate of next-generation sequencing (Wong TN, Nature 2015; Young AL, Leukemia 2015). Using ECS, we surveyed 102 DS children (47 ML-DS enrolled from a current Phase III ML-DS study; 55 healthy DS enrolled at St. Louis Children's Hospital).
Results: We identified 294 total clonal variants in 72/102 children (60.0%, healthy DS; 83.0%, ML-DS cases) at 0.0002-0.82 VAF. On average, we found 1.8 clonal variants per healthy DS control and 4.1 clonal variants per ML-DS case. Consistent with Yoshida et al., we find a spectrum of recurrent mutations in ML-DS cases: GATA1 (53.2%), EZH2 (25.5%), RAD21 (14.9%), and STAG2 (10.6%) etc. Surprisingly, we also identified frequent mutations in FAT1 (14.9%) and SETD2 (10.6%), genes previously unassociated with ML-DS. Moreover, we report a different set of recurrent mutations in healthy DS children: FAT1 (12.7%), BCOR (12.7%), TET2 (10.9%), SETD2 (7.3%), and TRIM24 (5.5%), and TP53 (5.5%) etc.
Discussion: Earlier ML-DS studies could only detect more common mutations >0.02 VAF due to the error-rate of standard next-generation sequencing (NGS). Using ECS, we present the first characterization of the unique clonal hematopoietic spectrum in children with DS and ML-DS. These results reveal that clonal hematopoiesis is common amongst healthy DS children without hematologic conditions, and that children with ML-DS have a higher clonal mutation burden than those without. Notably, we identified two novel genes recurrently mutated in both ML-DS cases and healthy DS controls: FAT1 and SETD2. We suspect that these mutations were not identified in other ML-DS studies as the median VAFs of our detected FAT1 and SETD2 mutations were 0.001 and 0.0008, respectively. Mutations in FAT1 and SETD2 have been shown to lead to dysregulated Wnt signaling in numerous cancers (Morris LG, Nat Gen 2013; Yuan H, J Clin Invest 2017). We and others have previously shown that Wnt signaling is crucial in specifying a primitive or definitive hematopoietic program (Sturgeon CM, Nat Biotech 2014; Creamer JP, Blood 2017). We speculate that these mutant FAT1 and SETD2 clones, like mutant GATA1 clones, may inhibit the definitive hematopoietic potential in DS children. Lastly, we note a reduced incidence of GATA1 mutations in our healthy DS cohort and attribute this to an older average age (8.3 years), as compared to Roberts et al. studying neonates. Other groups have speculated that somatic mutations in GATA1 (with a DS background) may only occur during a restricted developmental window (<4 years) as TMD clones cycle into quiescence afterwards (Zhe L, Nat Gen 2005; Hasle H, Leukemia 2007).
No relevant conflicts of interest to declare.
The synthesis and structure–activity relationship of the potent and selective tryptase inhibitor
13 (
K
i
=
0.0054
μM) is reported.
The synthesis of novel 1,2,4oxadiazoles and their ...structure–activity relationship (SAR) for the inhibition of tryptase and related serine proteases is presented. Elaboration of the P′-side afforded potent, selective, and orally bioavailable tryptase inhibitors.
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