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Quantitative autistic trait measurements index background genetic risk for ASD in Hispanic families

BackgroundRecent studies have indicated that quantitative autistic traits (QATs) of parents reflect inherited liabilities that may index background genetic risk for clinical autism spectrum disorder (ASD) in their offspring. Moreover, preferential mating for QATs has been observed as a potential factor in concentrating autistic liabilities in some families across generations. Heretofore, intergenerational studies of QATs have focused almost exclusively on Caucasian populations—the present study explored these phenomena in a well-characterized Hispanic population.MethodsThe present study examined QAT scores in siblings and parents of 83 Hispanic probands meeting research diagnostic criteria for ASD, and 64 non-ASD controls, using the Social Responsiveness Scale-2 (SRS-2). Ancestry of the probands was characterized by genotype, using information from 541,929 single nucleotide polymorphic markers.ResultsIn families of Hispanic children with an ASD diagnosis, the pattern of quantitative trait correlations observed between ASD-affected children and their first-degree relatives (ICCs on the order of 0.20), between unaffected first-degree relatives in ASD-affected families (sibling/mother ICC = 0.36; sibling/father ICC = 0.53), and between spouses (mother/father ICC = 0.48) were in keeping with the influence of transmitted background genetic risk and strong preferential mating for variation in quantitative autistic trait burden. Results from analysis of ancestry-informative genetic markers among probands in this sample were consistent with that from other Hispanic populations.ConclusionsQuantitative autistic traits represent measurable indices of inherited liability to ASD in Hispanic families. The accumulation of autistic traits occurs within generations, between spouses, and across generations, among Hispanic families affected by ASD. The occurrence of preferential mating for QATs—the magnitude of which may vary across cultures—constitutes a mechanism by which background genetic liability for ASD can accumulate in a given family in successive generations.

Applying rigor and reproducibility standards to assay donor-derived cell-free DNA as a non-invasive method for detection of acute rejection and graft injury after heart transplantation

BACKGROUND Use of new genomic techniques in clinical settings requires that such methods are rigorous and reproducible. Previous studies have shown that quantitation of donor-derived cell-free DNA (%ddcfDNA) by unbiased shotgun sequencing is a sensitive, non-invasive marker of acute rejection after heart transplantation. The primary goal of this study was to assess the reproducibility of %ddcfDNA measurements across technical replicates, manual vs automated platforms, and rejection phenotypes in distinct patient cohorts. METHODS After developing and validating the %ddcfDNA assay, we subjected the method to a rigorous test of its reproducibility. We measured %ddcfDNA in technical replicates performed by 2 independent laboratories and verified the reproducibility of %ddcfDNA patterns of 2 rejection phenotypes: acute cellular rejection and antibody-mediated rejection in distinct patient cohorts. RESULTS We observed strong concordance of technical-replicate %ddcfDNA measurements across 2 independent laboratories (slope = 1.02, R2 > 0.99, p < 10-6), as well as across manual and automated platforms (slope = 0.80, R2 = 0.92, p < 0.001). The %ddcfDNA measurements in distinct heart transplant cohorts had similar baselines and error rates. The %ddcfDNA temporal patterns associated with rejection phenotypes were similar in both patient cohorts; however, the quantity of ddcfDNA was significantly higher in samples with severe vs mild histologic rejection grade (2.73% vs 0.14%, respectively; p < 0.001). CONCLUSIONS The %ddcfDNA assay is precise and reproducible across laboratories and in samples from 2 distinct types of heart transplant rejection. These findings pave the way for larger studies to assess the clinical utility of %ddcfDNA as a marker of acute rejection after heart transplantation.

Circulating cell-free DNA as a biomarker of tissue injury: Assessment in a cardiac xenotransplantation model

BACKGROUND Observational studies suggest that cell-free DNA (cfDNA) is a biomarker of tissue injury in a range of conditions including organ transplantation. However, the lack of model systems to study cfDNA and its relevance to tissue injury has limited the advancements in this field. We hypothesized that the predictable course of acute humoral xenograft rejection (AHXR) in organ transplants from genetically engineered donors provides an ideal system for assessing circulating cfDNA as a marker of tissue injury. METHODS Genetically modified pig donor hearts were heterotopically transplanted into baboons (n = 7). Cell-free DNA was extracted from pre-transplant and post-transplant baboon plasma samples for shotgun sequencing. After alignment of sequence reads to pig and baboon reference sequences, we computed the percentage of xenograft-derived cfDNA (xdcfDNA) relative to recipient by counting uniquely aligned pig and baboon sequence reads. RESULTS The xdcfDNA percentage was high early post-transplantation and decayed exponentially to low stable levels (baseline); the decay half-life was 3.0 days. Post-transplantation baseline xdcfDNA levels were higher for transplant recipients that subsequently developed graft loss than in the 1 animal that did not reject the graft (3.2% vs 0.5%). Elevations in xdcfDNA percentage coincided with increased troponin and clinical evidence of rejection. Importantly, elevations in xdcfDNA percentage preceded clinical signs of rejection or increases in troponin levels. CONCLUSION Cross-species xdcfDNA kinetics in relation to acute rejection are similar to the patterns in human allografts. These observations in a xenotransplantation model support the body of evidence suggesting that circulating cfDNA is a marker of tissue injury.

Complement receptor CD46 co-stimulates optimal human CD8 + T cell effector function via fatty acid metabolism

The induction of human CD4+ Th1 cells requires autocrine stimulation of the complement receptor CD46 in direct crosstalk with a CD4+ T cell-intrinsic NLRP3 inflammasome. However, it is unclear whether human cytotoxic CD8+ T cell (CTL) responses also rely on an intrinsic complement-inflammasome axis. Here we show, using CTLs from patients with CD46 deficiency or with constitutively-active NLRP3, that CD46 delivers co-stimulatory signals for optimal CTL activity by augmenting nutrient-influx and fatty acid synthesis. Surprisingly, although CTLs express NLRP3, a canonical NLRP3 inflammasome is not required for normal human CTL activity, as CTLs from patients with hyperactive NLRP3 activity function normally. These findings establish autocrine complement and CD46 activity as integral components of normal human CTL biology, and, since CD46 is only present in humans, emphasize the divergent roles of innate immune sensors between mice and men.Complement, while serving to remove pathogens in the circulation, is also important for synergizing with inflammasomes to modulate CD4 T cell activation. Here the authors show that CD46, a complement receptor expressed only in humans, is essential for inducing optimal activation and effector functions of human CD8 T cells.

Late manifestation of alloantibody-associated injury and clinical pulmonary antibody-mediated rejection: Evidence from cell-free DNA analysis

BACKGROUND Antibody-mediated rejection (AMR) often progresses to poor health outcomes in lung transplant recipients (LTRs). This, combined with the relatively insensitive clinical tools used for its diagnosis (spirometry, histopathology) led us to determine whether clinical AMR is diagnosed significantly later than its pathologic onset. In this study, we leveraged the high sensitivity of donor-derived cell-free DNA (ddcfDNA), a novel genomic tool, to detect early graft injury after lung transplantation. METHODS We adjudicated AMR and acute cellular rejection (ACR) in 157 LTRs using the consensus criteria of the International Society for Heart and Lung Transplantation (ISHLT). We assessed the kinetics of allograft injury in relation to ACR or AMR using both clinical criteria (decline in spirometry from baseline) and molecular criteria (ddcfDNA); percent ddcfDNA was quantitated via shotgun sequencing. We used a mixed-linear model to assess the relationship between and ddcfDNA levels and donor-specific antibodies (DSA) in AMR+ LTRs. RESULTS Compared with ACR, AMR episodes (n = 42) were associated with significantly greater allograft injury when assessed by both spirometric (0.1 liter vs -0.6 liter, p < 0.01) and molecular (ddcfDNA) analysis (1.1% vs 5.4%, p < 0.001). Allograft injury detected by ddcfDNA preceded clinical AMR diagnosis by a median of 2.8 months. Within the same interval, spirometry or histopathology did not reveal findings of allograft injury or dysfunction. Elevated levels of ddcfDNA before clinical diagnosis of AMR were associated with a concurrent rise in DSA levels. CONCLUSION Diagnosis of clinical AMR in LTRs lags behind DSA-associated molecular allograft injury as assessed by ddcfDNA.

Targeted RNA-sequencing for the quantification of measurable residual disease in acute myeloid leukemia

Great effort is spent on developing therapies to improve the dire outcomes of those diagnosed with acute myeloid leukemia. The methods for quantifying response to therapeutic intervention have however lacked sensitivity. Patients achieving a complete remission as defined by conventional cytomorphological methods therefore remain at risk of subsequent relapse due to disease persistence. Improved risk stratification is possible based on tests designed to detect this residual leukemic burden (measurable residual disease). However, acute myeloid leukemia is a genetically diverse set of diseases, which has made it difficult to develop a single, highly reproducible, and sensitive assay for measurable residual disease. Here we present the development of a digital targeted RNA-sequencing-based approach designed to overcome these limitations by detecting all newly approved European LeukemiaNet molecular targets for measurable residual disease in acute myeloid leukemia in a single standardized assay. Iterative modifications and novel bioinformatics approaches resulted in a greater than 100-fold increase in performance compared with commercially available targeted RNA-sequencing approaches and a limit of detection as low as one leukemic cell in 100,000 cells measured, which is comparable to quantitative polymerase chain reaction analysis, the current gold standard for the detection of measurable residual disease. This assay, which can be customized and expanded, is the first demonstrated use of high-sensitivity RNA-sequencing for measurable residual disease detection in acute myeloid leukemia and could serve as a broadly applicable standardized tool.

Properties of global- and local-ancestry adjustments in genetic association tests in admixed populations

Population substructure can lead to confounding in tests for genetic association, and failure to adjust properly can result in spurious findings. Here we address this issue of confounding by considering the impact of global ancestry (average ancestry across the genome) and local ancestry (ancestry at a specific chromosomal location) on regression parameters and relative power in ancestry‐adjusted and ‐unadjusted models. We examine theoretical expectations under different scenarios for population substructure; applying different regression models, verifying and generalizing using simulations, and exploring the findings in real‐world admixed populations. We show that admixture does not lead to confounding when the trait locus is tested directly in a single admixed population. However, if there is more complex population structure or a marker locus in linkage disequilibrium (LD) with the trait locus is tested, both global and local ancestry can be confounders. Additionally, we show the genotype parameters of adjusted and unadjusted models all provide tests for LD between the marker and trait locus, but in different contexts. The local ancestry adjusted model tests for LD in the ancestral populations, while tests using the unadjusted and the global ancestry adjusted models depend on LD in the admixed population(s), which may be enriched due to different ancestral allele frequencies. Practically, this implies that global‐ancestry adjustment should be used for screening, but local‐ancestry adjustment may better inform fine mapping and provide better effect estimates at trait loci.

CRISPR/Cas9‐Based Safe‐Harbor Gene Editing in Rhesus iPSCs

NHP iPSCs provide a unique opportunity to test safety and efficacy of iPSC-derived therapies in clinically relevant NHP models. To monitor these cells in vivo, there is a need for safe and efficient labeling methods. Gene insertion into genomic safe harbors (GSHs) supports reliable transgene expression while minimizing the risk the modification poses to the host genome or target cell. Specifically, this protocol demonstrates targeting of the adeno-associated virus site 1 (AAVS1), one of the most widely used GSH loci in the human genome, with CRISPR/Cas9, allowing targeted marker or therapeutic gene insertion in rhesus macaque induced pluripotent stem cells (RhiPSCs). Furthermore, detailed instructions for screening targeted clones and a tool for assessing potential off-target nuclease activity are provided.

527. Improvement of Pre-Clinical Non-Human Primate Model for Pluripotent Stem Cell Based Therapies by Introducing Marker Genes in Safe Harbor Locus

Induced pluripotent stem cells (iPSCs) are being developed as sources for clinical cellular regenerative therapies, as well as valuable in vitro human disease models. Derivation of iPSCs from non-human primates (NHP) affords the opportunity to test the safety, feasibility and efficacy of proposed iPSC-derived cellular delivery in species with physiology, immunology and scale similar to humans. However, there is a need for stable and safe labeling methods for iPSCs and their differentiated progeny allowing analysis of survival, proliferation, tissue integration and biodistribution, in vitro and in vivo. Typically, marker genes have been inserted into target cells by transduction with randomly-integrating viral vectors. However, these methods raise concerns regarding genotoxicity and transgene silencing, particularly in pluripotent stem cells, limiting their utility for tracking and eventual clinical applications. Targeted integration into genomic “safe harbors” offer a promising alternative approach to mark target cells, potentially circumventing these issues. The adeno-associated virus integration site 1 (AAVS1) has been proposed as a suitable safe harbor for human cells, and we now investigate its utility in our rhesus macaque NHP iPSC model. We have efficiently knocked-in both a truncated CD19 (hΔCD19) marker gene a non-immunogenic and clinical relevant marker, or green fluorescent protein (GFP) at the homologous AAVS1 site in rhesus iPSCs (RhiPSCs) using the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR-Cas9) system. PCR and Southern blot analyses demonstrated highly efficient knock-in into the AAVS1 locus, with over one third of clones screened containing only targeted but not random integrations. (Table 1Table 1). Edited RhiPSC-GFP/hΔCD19 clones retained a normal karyotype and pluripotency - as shown by teratoma formation. Directed differentiation of these clones to neutrophils, hepatocytes or cardiomyocytes was not hindered by the knock-in of marker genes into the AAVS1 sites. Notably, transgene expression was stable in undifferentiated RhiPSCs and differentiated cell types derived from the RhiPSC (Figure 1Figure 1), in contrast to prior experience with viral vector delivery. We have established a computational platform to assess off-target effects of guide RNAs in the rhesus genome. Genetically marked RhiPSCs afford a unique opportunity to develop clinically relevant models for iPSC-based cell therapies.Table 1Summary of CRISPR-mediated gene editing in rhesus iPSCsOriginal iPSC cloneReporter geneClones with TI/Clones screened1Clones without RI/Clones with TI2ZG15-M11-10hΔCD194/42/8GFP14/145/9ZG32-3-4hΔCD19ND1/4GFPND2/4ZH26-HS41hΔ CD19ND1/4Total18/18(100%)11/29 (37.9%) View Table in HTML RI: random integration, TI; targeted integration, ND: not determined1based on by PCR analysis2based on Southern blot analysisView Large Image | Download PowerPoint Slide