Publications by authors named "Ilker Tunc"

33 Publications

Higher levels of allograft injury in black patients early after heart transplantation.

J Heart Lung Transplant 2021 Dec 23. Epub 2021 Dec 23.

Genomic Research Alliance for Transplantation (GRAfT), Bethesda, Maryland; Laborarory of Applied Precision Omics (APO), Division of Intramural Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland; Department of Medicine, Stanford University School of Medicine, Palo Alto, California. Electronic address:

Black patients suffer higher rates of antibody-mediated rejection and have worse long-term graft survival after heart transplantation. Donor-derived cell free DNA (ddcfDNA) is released into the blood following allograft injury. This study analyzed %ddcfDNA in 63 heart transplant recipients categorized by Black and non-Black race, during the first 200 days after transplant. Immediately after transplant, %ddcfDNA was higher for Black patients (mean [SE]: 8.3% [1.3%] vs 3.2% [1.2%], p = 0.001). In the first week post-transplant, the rate of decay in %ddcfDNA was similar (0.7% [0.68] vs 0.7% [0.11], p = 0.78), and values declined in both groups to a comparable plateau at 7 days post-transplant (0.46% [0.03] vs 0.45% [0.04], p = 0.78). The proportion of Black patients experiencing AMR was higher than non-Black patients (21% vs 9% [hazard ratio of 2.61 [95% confidence interval: 0.651-10.43], p = 0.18). Black patients were more likely to receive a race mismatched organ than non-Black patients (69% vs 35%, p = 0.01), which may explain the higher levels of early allograft injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.healun.2021.12.006DOI Listing
December 2021

Response by Shah et al to Letter Regarding Article, "Cell-Free DNA to Detect Heart Allograft Acute Rejection".

Circulation 2021 09 7;144(10):e198-e199. Epub 2021 Sep 7.

Genomic Research Alliance for Transplantation (GRAfT), Bethesda, MD (P.S., S.A-E., I.T., S.H., E.F., K.S., M.E.R., S.S.N., H.K., U.F., A.B., A.M., K.B., Y.Y., M.K.J., C.Marboe, G.J.B., H.A.V.).

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.121.055697DOI Listing
September 2021

Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques.

Mol Ther 2022 Jan 24;30(1):209-222. Epub 2021 Jun 24.

Translational Stem Cell Biology Branch, NHLBI, NIH, Building 10-CRC, 5E-3332, 9000 Rockville Pike, Bethesda, MD 20892, USA. Electronic address:

The programmable nuclease technology CRISPR-Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR-Cas9 therapeutically. Here, we utilized a rhesus macaque model to compare the predictive values of CIRCLE-seq, an in vitro off-target prediction method, with in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-seq and ISP for a CD33 guide RNA (gRNA) with thousands of off-target sites predicted by ISP and CIRCLE-seq. We found poor correlation between the sites predicted by the two methods. When almost 500 sites predicted by each method were analyzed by error-corrected sequencing of hematopoietic cells following transplantation, 19 off-target sites revealed insertion or deletion mutations. Of these sites, 8 were predicted by both methods, 8 by CIRCLE-seq only, and 3 by ISP only. The levels of cells with these off-target edits exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In addition, we utilized an unbiased method termed CAST-seq to search for translocations between the on-target site and off-target sites present in animals following transplantation, detecting one specific translocation that persisted in blood cells for at least 1 year following transplantation. In conclusion, neither CIRCLE-seq or ISP predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods, may be required for optimizing safety of clinical development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymthe.2021.06.016DOI Listing
January 2022

Donor-derived cell-free DNA accurately detects acute rejection in lung transplant patients, a multicenter cohort study.

J Heart Lung Transplant 2021 08 24;40(8):822-830. Epub 2021 Apr 24.

Genomic Research Alliance for Transplantation (GRAfT), 10 Center Drive, 7S261, Bethesda, Maryland; Division of Intramural Research, National Heart, Lung and Blood Institute, 10 Center Drive, 7S261, Bethesda, Maryland; Division of Pulmonary and Critical Care Medicine, The Johns Hopkins School of Medicine, 1830 East Monument Street, Baltimore, Maryland. Electronic address:

Background: Acute rejection, which includes antibody-mediated rejection and acute cellular rejection, is a risk factor for lung allograft loss. Lung transplant patients often undergo surveillance transbronchial biopsies to detect and treat acute rejection before irreversible chronic rejection develops. Limitations of this approach include its invasiveness and high interobserver variability. We tested the performance of percent donor-derived cell-free DNA (%ddcfDNA), a non-invasive blood test, to detect acute rejection.

Methods: This multicenter cohort study monitored 148 lung transplant subjects over a median of 19.6 months. We collected serial plasma samples contemporaneously with TBBx to measure %ddcfDNA. Clinical data was collected to adjudicate for acute rejection. The primary analysis consisted of computing the area-under-the-receiver-operating-characteristic-curve of %ddcfDNA to detect acute rejection. Secondary analysis determined %ddcfDNA rule-out thresholds for acute rejection.

Results: ddcfDNA levels were high after transplant surgery and decayed logarithmically. With acute rejection, ddcfDNA levels rose six-fold higher than controls. ddcfDNA levels also correlated with severity of lung function decline and histological grading of rejection. %ddcfDNA area-under-the-receiver-operating-characteristic-curve for acute rejection, AMR, and ACR were 0.89, 0.93, and 0.83, respectively. ddcfDNA levels of <0.5% and <1.0% showed a negative predictive value of 96% and 90% for acute rejection, respectively. Histopathology detected one-third of episodes with ddcfDNA levels ≥1.0%, even though >90% of these events were coincident to clinical complications missed by histopathology.

Conclusions: This study demonstrates that %ddcfDNA reliably detects acute rejection and other clinical complications potentially missed by histopathology, lending support to its use as a non-invasive marker of allograft injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.healun.2021.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8319066PMC
August 2021

Use of donor-derived-cell-free DNA as a marker of early allograft injury in primary graft dysfunction (PGD) to predict the risk of chronic lung allograft dysfunction (CLAD).

J Heart Lung Transplant 2021 06 20;40(6):488-493. Epub 2021 Feb 20.

Laborarory of Applied Precision Omics (APO), Lung and Blood Institute (NHLBI), National Institutes of Health, Bethesda, Maryland; Genomic Research Alliance for Transplantation (GRAfT), National Heart, Lung and Blood Institute, Bethesda, Maryland; Department of Surgery, Johns Hopkins Hospital, Baltimore, Maryland. Electronic address:

Background: Primary graft dysfunction (PGD) is a risk factor for chronic lung allograft dysfunction (CLAD). However, the association between PGD and degree of allograft injury remains poorly defined. In this study, we leverage a novel biomarker for allograft injury, percentage donor-derived cell-free DNA (%ddcfDNA), to study the association between PGD, degree of allograft injury, and the development of CLAD.

Methods: This prospective cohort study recruited 99 lung transplant recipients and collected plasma samples on days 1, 3, and 7 for %ddcfDNA measurements. Clinical data on day 3 was used to adjudicate for PGD. %ddcfDNA levels were compared between PGD grades. In PGD patients, %ddcfDNA was compared between those who developed CLAD and those who did not.

Results: On posttransplant day 3, %ddcfDNA was higher in PGD than in non-PGD patients (median [IQR]: 12.2% [8.2, 22.0] vs 8.5% [5.6, 13.2] p = 0.01). %ddcfDNA correlated with the severity grade of PGD (r = 0.24, p = 0.02). Within the PGD group, higher levels of %ddcfDNA correlated with increased risk of developing CLAD (log OR(SE) 1.38 (0.53), p = 0.009). PGD patients who developed CLAD showed ∼2-times higher %ddcfDNA levels than patients who did not develop CLAD (median [IQR]: 22.4% [11.8, 27.6] vs 9.9% [6.7, 14.9], p = 0.007).

Conclusion: PGD patients demonstrated increased early posttransplant allograft injury, as measured by %ddcfDNA, in comparison to non-PGD patients, and these high %ddcfDNA levels were associated with subsequent development of CLAD. This study suggests that %ddcfDNA identifies PGD patients at greater risk of CLAD than PGD alone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.healun.2021.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8169618PMC
June 2021

Network Analysis and Transcriptome Profiling Identify Autophagic and Mitochondrial Dysfunctions in SARS-CoV-2 Infection.

Front Genet 2021 16;12:599261. Epub 2021 Mar 16.

Bioinformatics and Computational Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, United States.

Analyzing host cells' transcriptional response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection will help delineate biological processes underlying viral pathogenesis. First, analysis of expression profiles of lung cell lines A549 and Calu3 revealed upregulation of antiviral interferon signaling genes in response to all three SARS-CoV-2, MERS-CoV, or influenza A virus (IAV) infections. However, perturbations in expression of genes involved in inflammatory, mitochondrial, and autophagy processes were specifically observed in SARS-CoV-2-infected cells. Next, a validation study in infected human nasopharyngeal samples also revealed perturbations in autophagy and mitochondrial processes. Specifically, mTOR expression, mitochondrial ribosomal, mitochondrial complex I, lysosome acidification, and mitochondrial fission promoting genes were concurrently downregulated in both infected cell lines and human samples. SARS-CoV-2 infection impeded autophagic flux either by upregulating GSK3B in lung cell lines or by downregulating autophagy genes, SNAP29, and lysosome acidification genes in human samples, contributing to increased viral replication. Therefore, drugs targeting lysosome acidification or autophagic flux could be tested as intervention strategies. Finally, age-stratified SARS-CoV-2-positive human data revealed impaired upregulation of chemokines, interferon-stimulated genes, and tripartite motif genes that are critical for antiviral signaling. Together, this analysis has revealed specific aspects of autophagic and mitochondrial function that are uniquely perturbed in SARS-CoV-2-infected host cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fgene.2021.599261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008150PMC
March 2021

Circulating mitochondrial DNA is a proinflammatory DAMP in sickle cell disease.

Blood 2021 06;137(22):3116-3126

Sickle Cell Branch.

The pathophysiology of sickle cell disease (SCD) is driven by chronic inflammation fueled by damage associated molecular patterns (DAMPs). We show that elevated cell-free DNA (cfDNA) in patients with SCD is not just a prognostic biomarker, it also contributes to the pathological inflammation. Within the elevated cfDNA, patients with SCD had a significantly higher ratio of cell-free mitochondrial DNA (cf-mtDNA)/cell-free nuclear DNA compared with healthy controls. Additionally, mitochondrial DNA in patient samples showed significantly disproportionately increased hypomethylation compared with healthy controls, and it was increased further in crises compared with steady-state. Using flow cytometry, structured illumination microscopy, and electron microscopy, we showed that circulating SCD red blood cells abnormally retained their mitochondria and, thus, are likely to be the source of the elevated cf-mtDNA in patients with SCD. Patient plasma containing high levels of cf-mtDNA triggered the formation of neutrophil extracellular traps (NETs) that was substantially reduced by inhibition of TANK-binding kinase 1, implicating activation of the cGAS-STING pathway. cf-mtDNA is an erythrocytic DAMP, highlighting an underappreciated role for mitochondria in sickle pathology. These trials were registered at www.clinicaltrials.gov as #NCT00081523, #NCT03049475, and #NCT00047996.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2020009063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8176765PMC
June 2021

Cell-free DNA maps COVID-19 tissue injury and risk of death and can cause tissue injury.

JCI Insight 2021 04 8;6(7). Epub 2021 Apr 8.

Genomic Research Alliance for Transplantation (GRAfT) and Laboratory of Applied Precision Omics, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, Maryland, USA.

INTRODUCTIONThe clinical course of coronavirus 2019 (COVID-19) is heterogeneous, ranging from mild to severe multiorgan failure and death. In this study, we analyzed cell-free DNA (cfDNA) as a biomarker of injury to define the sources of tissue injury that contribute to such different trajectories.METHODSWe conducted a multicenter prospective cohort study to enroll patients with COVID-19 and collect plasma samples. Plasma cfDNA was subject to bisulfite sequencing. A library of tissue-specific DNA methylation signatures was used to analyze sequence reads to quantitate cfDNA from different tissue types. We then determined the correlation of tissue-specific cfDNA measures to COVID-19 outcomes. Similar analyses were performed for healthy controls and a comparator group of patients with respiratory syncytial virus and influenza.RESULTSWe found markedly elevated levels and divergent tissue sources of cfDNA in COVID-19 patients compared with patients who had influenza and/or respiratory syncytial virus and with healthy controls. The major sources of cfDNA in COVID-19 were hematopoietic cells, vascular endothelium, hepatocytes, adipocytes, kidney, heart, and lung. cfDNA levels positively correlated with COVID-19 disease severity, C-reactive protein, and D-dimer. cfDNA profile at admission identified patients who subsequently required intensive care or died during hospitalization. Furthermore, the increased cfDNA in COVID-19 patients generated excessive mitochondrial ROS (mtROS) in renal tubular cells in a concentration-dependent manner. This mtROS production was inhibited by a TLR9-specific antagonist.CONCLUSIONcfDNA maps tissue injury that predicts COVID-19 outcomes and may mechanistically propagate COVID-19-induced tissue injury.FUNDINGIntramural Targeted Anti-COVID-19 grant, NIH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.147610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8119224PMC
April 2021

Cell-Free DNA to Detect Heart Allograft Acute Rejection.

Circulation 2021 03 13;143(12):1184-1197. Epub 2021 Jan 13.

Genomic Research Alliance for Transplantation, Bethesda, MD (S.A.-E., P.S., I.T., S.H., E.F., K.S., M.E.R., S.S.N., H.K., U.F., A.B., A.M., K.B., Y.Y., M.K.J., C.M., G.J.B., H.A.V.).

Background: After heart transplantation, endomyocardial biopsy (EMBx) is used to monitor for acute rejection (AR). Unfortunately, EMBx is invasive, and its conventional histological interpretation has limitations. This is a validation study to assess the performance of a sensitive blood biomarker-percent donor-derived cell-free DNA (%ddcfDNA)-for detection of AR in cardiac transplant recipients.

Methods: This multicenter, prospective cohort study recruited heart transplant subjects and collected plasma samples contemporaneously with EMBx for %ddcfDNA measurement by shotgun sequencing. Histopathology data were collected to define AR, its 2 phenotypes (acute cellular rejection [ACR] and antibody-mediated rejection [AMR]), and controls without rejection. The primary analysis was to compare %ddcfDNA levels (median and interquartile range [IQR]) for AR, AMR, and ACR with controls and to determine %ddcfDNA test characteristics using receiver-operator characteristics analysis.

Results: The study included 171 subjects with median posttransplant follow-up of 17.7 months (IQR, 12.1-23.6), with 1392 EMBx, and 1834 %ddcfDNA measures available for analysis. Median %ddcfDNA levels decayed after surgery to 0.13% (IQR, 0.03%-0.21%) by 28 days. Also, %ddcfDNA increased again with AR compared with control values (0.38% [IQR, 0.31-0.83%], versus 0.03% [IQR, 0.01-0.14%]; <0.001). The rise was detected 0.5 and 3.2 months before histopathologic diagnosis of ACR and AMR. The area under the receiver operator characteristic curve for AR was 0.92. A 0.25%ddcfDNA threshold had a negative predictive value for AR of 99% and would have safely eliminated 81% of EMBx. In addition, %ddcfDNA showed distinctive characteristics comparing AMR with ACR, including 5-fold higher levels (AMR ≥2, 1.68% [IQR, 0.49-2.79%] versus ACR grade ≥2R, 0.34% [IQR, 0.28-0.72%]), higher area under the receiver operator characteristic curve (0.95 versus 0.85), higher guanosine-cytosine content, and higher percentage of short ddcfDNA fragments.

Conclusions: We found that %ddcfDNA detected AR with a high area under the receiver operator characteristic curve and negative predictive value. Monitoring with ddcfDNA demonstrated excellent performance characteristics for both ACR and AMR and led to earlier detection than the EMBx-based monitoring. This study supports the use of %ddcfDNA to monitor for AR in patients with heart transplant and paves the way for a clinical utility study. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02423070.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.120.049098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8221834PMC
March 2021

Identification of Genes Contributing to a Long Circadian Period in .

J Biol Rhythms 2021 06 4;36(3):239-253. Epub 2020 Dec 4.

Laboratory of Systems Genetics, Systems Biology Center, National Heart, Lung, and Blood Institute, Bethesda, Maryland.

The endogenous circadian period of animals and humans is typically very close to 24 h. Individuals with much longer circadian periods have been observed, however, and in the case of humans, these deviations have health implications. Previously, we observed a line of with a very long average period of 31.3 h for locomotor activity behavior. Preliminary mapping indicated that the long period did not map to known canonical clock genes but instead mapped to multiple chromosomes. Using RNA-Seq, we surveyed the whole transcriptome of fly heads from this line across time and compared it with a wild-type control. A three-way generalized linear model revealed that approximately two-thirds of the genes were expressed differentially among the two genotypes, while only one quarter of the genes varied across time. Using these results, we applied algorithms to search for genes that oscillated over 24 h, identifying genes not previously known to cycle. We identified 166 differentially expressed genes that overlapped with a previous Genome-wide Association Study (GWAS) of circadian behavior, strongly implicating them in the long-period phenotype. We tested mutations in 45 of these genes for their effect on the circadian period. Mutations in (), , and had significant effects on the circadian period, with seven of these mutations increasing the circadian period of locomotor activity behavior. Genetic rescue of mutant restored the circadian period to wild-type levels, suggesting it has a critical role in determining period length in constant darkness.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/0748730420975946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8390771PMC
June 2021

lncRNAKB, a knowledgebase of tissue-specific functional annotation and trait association of long noncoding RNA.

Sci Data 2020 10 5;7(1):326. Epub 2020 Oct 5.

Bioinformatics and Computational Biology Core, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA.

Long non-coding RNA Knowledgebase (lncRNAKB) is an integrated resource for exploring lncRNA biology in the context of tissue-specificity and disease association. A systematic integration of annotations from six independent databases resulted in 77,199 human lncRNA (224,286 transcripts). The user-friendly knowledgebase covers a comprehensive breadth and depth of lncRNA annotation. lncRNAKB is a compendium of expression patterns, derived from analysis of RNA-seq data in thousands of samples across 31 solid human normal tissues (GTEx). Thousands of co-expression modules identified via network analysis and pathway enrichment to delineate lncRNA function are also accessible. Millions of expression quantitative trait loci (cis-eQTL) computed using whole genome sequence genotype data (GTEx) can be downloaded at lncRNAKB that also includes tissue-specificity, phylogenetic conservation and coding potential scores. Tissue-specific lncRNA-trait associations encompassing 323 GWAS (UK Biobank) are also provided. LncRNAKB is accessible at http://www.lncrnakb.org/ , and the data are freely available through Open Science Framework ( https://doi.org/10.17605/OSF.IO/RU4D2 ).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41597-020-00659-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536183PMC
October 2020

Activation and inhibition of nonsense-mediated mRNA decay control the abundance of alternative polyadenylation products.

Nucleic Acids Res 2020 07;48(13):7468-7482

Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Alternative polyadenylation (APA) produces transcript 3' untranslated regions (3'UTRs) with distinct sequences, lengths, stabilities and functions. We show here that APA products include a class of cryptic nonsense-mediated mRNA decay (NMD) substrates with extended 3'UTRs that gene- or transcript-level analyses of NMD often fail to detect. Transcriptome-wide, the core NMD factor UPF1 preferentially recognizes long 3'UTR products of APA, leading to their systematic downregulation. Counteracting this mechanism, the multifunctional RNA-binding protein PTBP1 regulates the balance of short and long 3'UTR isoforms by inhibiting NMD, in addition to its previously described modulation of co-transcriptional polyadenylation (polyA) site choice. Further, we find that many transcripts with altered APA isoform abundance across multiple tumor types are controlled by NMD. Together, our findings reveal a widespread role for NMD in shaping the outcomes of APA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkaa491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367170PMC
July 2020

Network Analysis and Transcriptome Profiling Identify Autophagic and Mitochondrial Dysfunctions in SARS-CoV-2 Infection.

bioRxiv 2020 Jul 20. Epub 2020 Jul 20.

Bioinformatics and Computational Biology Laboratory, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Analyzing host transcriptional changes in response to SARS-CoV-2 infection will help delineate biological processes underlying viral pathogenesis. Comparison of expression profiles of lung cell lines A549 (infected with either SARS-CoV-2 (with ACE2 expression)) or Influenza A virus (IAV)) and Calu3 (infected with SARS-CoV-2 or MERS-CoV) revealed upregulation of the antiviral interferon signaling in all three viral infections. However, perturbations in inflammatory, mitochondrial, and autophagy processes were specifically observed in SARS-CoV-2 infected cells. Validation of findings from cell line data revealed perturbations in autophagy and mitochondrial processes in the infected human nasopharyngeal samples. Specifically, downregulation of mTOR expression, mitochondrial ribosomal, mitochondrial complex I, and lysosome acidification genes were concurrently observed in both infected cell lines and human datasets. Furthermore, SARS-CoV-2 infection impedes autophagic flux by upregulating GSK3B in lung cell lines, or by downregulating autophagy genes, SNAP29 and lysosome acidification genes in human samples, contributing to increased viral replication. Therefore, drugs targeting lysosome acidification or autophagic flux could be tested as intervention strategies. Additionally, downregulation of MTFP1 (in cell lines) or SOCS6 (in human samples) results in hyperfused mitochondria and impede proper interferon response. Coexpression networks analysis identifies correlated clusters of genes annotated to inflammation and mitochondrial processes that are misregulated in SARS-CoV-2 infected cells. Finally, comparison of age stratified human gene expression data revealed impaired upregulation of chemokines, interferon stimulated and tripartite motif genes that are critical for antiviral signaling. Together, this analysis has revealed specific aspects of autophagic and mitochondrial function that are uniquely perturbed in SARS-CoV-2 infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.05.13.092536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241104PMC
July 2020

Misregulation of ELK1, AP1, and E12 Transcription Factor Networks Is Associated with Melanoma Progression.

Cancers (Basel) 2020 Feb 17;12(2). Epub 2020 Feb 17.

Bioinformatics and Computational Biology Laboratory, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Melanoma is among the most malignant cutaneous cancers and when metastasized results in dramatically high mortality. Despite advances in high-throughput gene expression profiling in cancer transcriptomic studies, our understanding of mechanisms driving melanoma progression is still limited. We present here an in-depth bioinformatic analysis of the melanoma RNAseq, chromatin immunoprecipitation (ChIP)seq, and single-cell (sc)RNA seq data to understand cancer progression. Specifically, we have performed a consensus network analysis of RNA-seq data from clinically re-grouped melanoma samples to identify gene co-expression networks that are conserved in early (stage 1) and late (stage 4/invasive) stage melanoma. Overlaying the fold-change information on co-expression networks revealed several coordinately up or down-regulated subnetworks that may play a critical role in melanoma progression. Furthermore, by incorporating histone lysine-27 acetylation information and highly expressed genes identified from the single-cell RNA data from melanoma patient samples, we present a comprehensive list of pathways, putative protein-protein interactions (PPIs) and transcription factor (TF) networks that are driving cancer progression. From this analysis, we have identified Elk1, AP1 and E12 TF networks that coordinately change expression in late melanoma when compared to early melanoma, implicating these TFs in melanoma progression. Additionally, the sumoylation-associated interactome is upregulated in invasive melanoma. Together, this bioinformatic analysis potentially implicates a combination of TF networks and PPIs in melanoma progression, which if confirmed in the experimental systems, could be used as targets for drug intervention in melanoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12020458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072154PMC
February 2020

Affected Sib-Pair Analyses Identify Signaling Networks Associated With Social Behavioral Deficits in Autism.

Front Genet 2019 27;10:1186. Epub 2019 Nov 27.

McKusick-Nathans Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States.

Autism spectrum disorders (ASDs) are characterized by deficits in three core behavioral domains: reciprocal social interactions, communication, and restricted interests and/or repetitive behaviors. Several hundreds of risk genes for autism have been identified, however, it remains a challenge to associate these genes with specific core behavioral deficits. In multiplex autism families, affected sibs often show significant differences in severity of individual core phenotypes. We hypothesize that a higher mutation burden contributes to a larger difference in the severity of specific core phenotypes between affected sibs. We tested this hypothesis on social behavioral deficits in autism. We sequenced synaptome genes (n = 1,886) in affected male sib-pairs (n = 274) in families from the Autism Genetics Research Exchange (AGRE) and identified rare (MAF ≤ 1%) and predicted functional variants. We selected affected sib-pairs with a large (≥10; n = 92 pairs) or a small (≤4; n = 108 pairs) difference in total cumulative Autism Diagnostic Interview-Revised (ADI-R) social scores (SOCT_CS). We compared burdens of unshared variants present only in sibs with severe social deficits and found a higher burden in SOCT_CS≥10 compared to SOCT_CS ≤ 4 (SOCT_CS≥10: 705.1 ± 16.2; SOCT_CS ≤ 4, 668.3 ± 9.0; = 0.025). Unshared SOCT_CS≥10 genes only in sibs with severe social deficits are significantly enriched in the SFARI gene set. Network analyses of these genes using InWeb_IM, molecular signatures database (MSigDB), and GeNetMeta identified enrichment for phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) (Enrichment Score [eScore] value = 3.36E-07; n = 8 genes) and Nerve growth factor (NGF) (eScore value = 8.94E-07; n = 9 genes) networks. These studies support a key role for these signaling networks in social behavioral deficits and present a novel approach to associate risk genes and signaling networks with core behavioral domains in autism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fgene.2019.01186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892440PMC
November 2019

Circulating Lymphangioleiomyomatosis Tumor Cells With Loss of Heterozygosity in the TSC2 Gene Show Increased Aldehyde Dehydrogenase Activity.

Chest 2019 08 26;156(2):298-307. Epub 2019 Apr 26.

Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda. Electronic address:

Background: Lymphangioleiomyomatosis (LAM) is a destructive metastasizing neoplasm of the lung characterized by proliferation of LAM cells in specialized lung nodules. LAM cells are characterized by expression of the prometastatic and cancer-initiating hyaluronan receptor CD44v6, and loss of heterozygosity (LOH) of TSC1 and TSC2. The circulating neoplastic LAM cells are thought to be involved in metastasis. Because LAM cells display properties of neoplastic, metastatic, and stem cell-like cancer cells, we hypothesized that elevated aldehyde dehydrogenase (ALDH) activity, characteristic of cancer and stem cells, is a property of LAM cells.

Methods: We performed an in silico search of ALDH genes in microdissected LAM lung nodules. To identify circulating LAM cells, we osmotically removed red blood cells from whole blood to obtain peripheral blood mononuclear cells, which were then sorted by fluorescence-activated cell sorting based on their level of ALDH activity.

Results: Microdissected LAM lung nodules possess a distinctive ALDH gene profile. The cell subpopulation with high ALDH activity, isolated from circulating cells, possessed TSC2 LOH in 8 of 14 patients with LAM. Approximately 60% of the circulating cells with high ALDH activity expressed CD44v6. Cells with TSC2 LOH from patients with LAM and LAM/TSC exhibited different properties in different body locations, but all cell types showed high ALDH activity.

Conclusions: This new procedure allows for isolation of circulating LAM cells from cultured cells, blood, and chylous effusions and shows that circulating LAM cells are heterogeneous with neoplastic, metastatic, and cancer-stem cell-like properties.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chest.2019.03.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859254PMC
August 2019

Neutrophil Subsets, Platelets, and Vascular Disease in Psoriasis.

JACC Basic Transl Sci 2019 Feb 25;4(1):1-14. Epub 2019 Feb 25.

Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland.

Psoriasis is an inflammatory skin disease associated with increased cardiovascular risk and serves as a reliable model to study inflammatory atherogenesis. Because neutrophils are implicated in atherosclerosis development, this study reports that the interaction among low-density granulocytes, a subset of neutrophils, and platelets is associated with a noncalcified coronary plaque burden assessed by coronary computed tomography angiography. Because early atherosclerotic noncalcified burden can lead to fatal myocardial infarction, the low-density granulocyte-platelet interaction may play a crucial target for clinical intervention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jacbts.2018.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390681PMC
February 2019

Donor-derived cell-free DNA predicts allograft failure and mortality after lung transplantation.

EBioMedicine 2019 Feb 26;40:541-553. Epub 2019 Jan 26.

Genomic Research Alliance for Transplantation (GRAfT), 10 Center Drive, 7S261, Bethesda, MD 20982, United States; Division of Intramural Research, National Heart, Lung and Blood Institute, 10 Center Drive, 7S261, Bethesda, MD 20982, United States. Electronic address:

Background: Allograft failure is common in lung-transplant recipients and leads to poor outcomes including early death. No reliable clinical tools exist to identify patients at high risk for allograft failure. This study tested the use of donor-derived cell-free DNA (%ddcfDNA) as a sensitive marker of early graft injury to predict impending allograft failure.

Methods: This multicenter, prospective cohort study enrolled 106 subjects who underwent lung transplantation and monitored them after transplantation for the development of allograft failure (defined as severe chronic lung allograft dysfunction [CLAD], retransplantation, and/or death from respiratory failure). Plasma samples were collected serially in the first three months following transplantation and assayed for %ddcfDNA by shotgun sequencing. We computed the average levels of ddcfDNA over three months for each patient (avddDNA) and determined its relationship to allograft failure using Cox-regression analysis.

Findings: avddDNA was highly variable among subjects: median values were 3·6%, 1·6% and 0·7% for the upper, middle, and low tertiles, respectively (range 0·1%-9·9%). Compared to subjects in the low and middle tertiles, those with avddDNA in the upper tertile had a 6·6-fold higher risk of developing allograft failure (95% confidence interval 1·6-19·9, p = 0·007), lower peak FEV1 values, and more frequent %ddcfDNA elevations that were not clinically detectable.

Interpretation: Lung transplant patients with early unresolving allograft injury measured via %ddcfDNA are at risk of subsequent allograft injury, which is often clinically silent, and progresses to allograft failure. FUND: National Institutes of Health.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2018.12.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412014PMC
February 2019

The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage.

Elife 2018 12 6;7. Epub 2018 Dec 6.

Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Maryland, United States.

The transcription factors TFE3 and TFEB cooperate to regulate autophagy induction and lysosome biogenesis in response to starvation. Here we demonstrate that DNA damage activates TFE3 and TFEB in a p53 and mTORC1 dependent manner. RNA-Seq analysis of TFEB/TFE3 double-knockout cells exposed to etoposide reveals a profound dysregulation of the DNA damage response, including upstream regulators and downstream p53 targets. TFE3 and TFEB contribute to sustain p53-dependent response by stabilizing p53 protein levels. In TFEB/TFE3 DKOs, p53 half-life is significantly decreased due to elevated Mdm2 levels. Transcriptional profiles of genes involved in lysosome membrane permeabilization and cell death pathways are dysregulated in TFEB/TFE3-depleted cells. Consequently, prolonged DNA damage results in impaired LMP and apoptosis induction. Finally, expression of multiple genes implicated in cell cycle control is altered in TFEB/TFE3 DKOs, revealing a previously unrecognized role of TFEB and TFE3 in the regulation of cell cycle checkpoints in response to stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.40856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292694PMC
December 2018

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

Nat Commun 2018 10 10;9(1):4186. Epub 2018 Oct 10.

School of Immunology and Microbial Sciences, King's College London, London, UK.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-06706-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180132PMC
October 2018

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

Haematologica 2019 02 31;104(2):297-304. Epub 2018 Aug 31.

Laboratory of Myeloid Malignancies, Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2018.203133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355494PMC
February 2019

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

J Heart Lung Transplant 2018 08 26;37(8):967-975. Epub 2018 Apr 26.

Genomic Research Alliance for Transplantation (GRAfT), Division of Intramural Research, National Institutes of Health, Bethesda, Maryland; Division of Intramural Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.healun.2018.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707066PMC
August 2018

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

J Heart Lung Transplant 2018 07 31;37(7):925-932. Epub 2018 Jan 31.

Department of Medicine, Stanford University School of Medicine, Palo Alto, California, USA. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.healun.2018.01.1305DOI Listing
July 2018

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

Genet Epidemiol 2018 03 30;42(2):214-229. Epub 2017 Dec 30.

John P. Hussman Institute for Human Genetics, Miller School of Medicine, University of Miami, Miami, Florida, United States of America.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/gepi.22103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811405PMC
March 2018

CRISPR/Cas9-Based Safe-Harbor Gene Editing in Rhesus iPSCs.

Curr Protoc Stem Cell Biol 2017 11 15;43:5A.11.1-5A.11.14. Epub 2017 Nov 15.

Hematology Branch, National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health, Bethesda, Maryland.

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. © 2017 by John Wiley & Sons, Inc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cpsc.37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709051PMC
November 2017

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.

J Heart Lung Transplant 2017 Sep 20;36(9):1004-1012. Epub 2017 May 20.

Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland; Laboratory of Transplantation Genomics, National Heart, Lung, and Blood Institute, Bethesda, Maryland. Electronic address:

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, R > 0.99, p < 10), as well as across manual and automated platforms (slope = 0.80, R = 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.healun.2017.05.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988434PMC
September 2017

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers.

Mol Ther 2017 01 4;25(1):44-53. Epub 2017 Jan 4.

Hematology Branch, National Heart, Lung and Blood Institute (NHLBI), NIH, Bethesda, MD 20892, USA.

Nonhuman primate (NHP) induced pluripotent stem cells (iPSCs) offer the opportunity to investigate the safety, feasibility, and efficacy of proposed iPSC-derived cellular delivery in clinically relevant in vivo models. However, there is need for stable, robust, and safe labeling methods for NHP iPSCs and their differentiated lineages to study survival, proliferation, tissue integration, and biodistribution following transplantation. Here we investigate the utility of the adeno-associated virus integration site 1 (AAVS1) as a safe harbor for the addition of transgenes in our rhesus macaque iPSC (RhiPSC) model. A clinically relevant marker gene, human truncated CD19 (hΔCD19), or GFP was inserted into the AAVS1 site in RhiPSCs using the CRISPR/Cas9 system. Genetically modified RhiPSCs maintained normal karyotype and pluripotency, and these clones were able to further differentiate into all three germ layers in vitro and in vivo. In contrast to transgene delivery using randomly integrating viral vectors, AAVS1 targeting allowed stable transgene expression following differentiation. Off-target mutations were observed in some edited clones, highlighting the importance of careful characterization of these cells prior to downstream applications. Genetically marked RhiPSCs will be useful to further advance clinically relevant models for iPSC-based cell therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymthe.2016.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363312PMC
January 2017

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

Mol Autism 2016 6;7(1):39. Epub 2016 Sep 6.

Department of Psychology, University of Miami, P.O. Box 248185-0751, Coral Gables, FL USA.

Background: Recent 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.

Methods: The 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.

Results: In 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.

Conclusions: Quantitative 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13229-016-0100-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5013609PMC
October 2017

Kinetics of phytosterol metabolism in neonates receiving parenteral nutrition.

Pediatr Res 2015 Aug 21;78(2):181-9. Epub 2015 Apr 21.

1] Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin [2] Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin.

Background: Phytosterols in soybean oil (SO) lipids likely contribute to parenteral nutrition-associated liver disease (PNALD) in infants. No characterization of phytosterol metabolism has been done in infants receiving SO lipids.

Methods: In a prospective cohort study, 45 neonates (36 SO lipid vs. 9 control) underwent serial blood sample measurements of sitosterol, campesterol, and stigmasterol. Mathematical modeling was used to determine pharmacokinetic parameters of phytosterol metabolism and phytosterol exposure.

Results: Compared to controls, SO lipid-exposed infants had significantly higher levels of sitosterol and campesterol (P < 0.01). During SO lipid infusion, sitosterol and campesterol reached half of steady-state plasma levels within 1.5 and 0.8 d, respectively. Steady-state level was highest for sitosterol (1.68 mg/dl), followed by campesterol (0.98 mg/dl), and lowest for stigmasterol (0.01 mg/dl). Infants born < 28 wk gestational age had higher sitosterol steady-state levels (P = 0.03) and higher area under the curve for sitosterol (P = 0.03) during the first 5 d of SO lipid (AUC5) than infants born ≥ 28 wk gestational age.

Conclusion: Phytosterols in SO lipid accumulate rapidly in neonates. Very preterm infants receiving SO lipid have higher sitosterol exposure, and may have poorly developed mechanisms of eliminating phytosterols that may contribute to their vulnerability to PNALD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/pr.2015.78DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546827PMC
August 2015
-->