Matching Items (7)
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Description
Induced pluripotent stem cells (iPSCs) are an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative

Induced pluripotent stem cells (iPSCs) are an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. We describe the establishment of primary dermal fibroblasts cells lines from 28 autopsy donors. These fibroblasts were used to examine the proliferative effects of establishment protocol, tissue amount, biopsy site, and donor age. As proof-of-principle, iPSCs were generated from fibroblasts from a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. To our knowledge, this is the first study describing autopsy donor-derived somatic cells being used for iPSC generation and subsequent neural differentiation. This unique approach also enables us to compare iPSC-derived cell cultures to endogenous tissues from the same donor. We utilized RNA sequencing (RNA-Seq) to evaluate the transcriptional progression of in vitro-differentiated neural cells (over a timecourse of 0, 35, 70, 105 and 140 days), and compared this with donor-identical temporal lobe tissue. We observed in vitro progression towards the reference brain tissue, supported by (i) a significant increasing monotonic correlation between the days of our timecourse and the number of actively transcribed protein-coding genes and long intergenic non-coding RNAs (lincRNAs) (P < 0.05), consistent with the transcriptional complexity of the brain, (ii) an increase in CpG methylation after neural differentiation that resembled the epigenomic signature of the endogenous tissue, and (iii) a significant decreasing monotonic correlation between the days of our timecourse and the percent of in vitro to brain-tissue differences (P < 0.05) for tissue-specific protein-coding genes and all putative lincRNAs. These studies support the utility of autopsy donors' somatic cells for iPSC-based neurological disease models, and provide evidence that in vitro neural differentiation can result in physiologically progression.
ContributorsHjelm, Brooke E (Author) / Craig, David W. (Thesis advisor) / Wilson-Rawls, Norma J. (Thesis advisor) / Huentelman, Matthew J. (Committee member) / Mason, Hugh S. (Committee member) / Kusumi, Kenro (Committee member) / Arizona State University (Publisher)
Created2013
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Description

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), is a rare and understudied cancer with a dismal prognosis. SCCOHT's infrequency has hindered empirical study of its biology and clinical management. However, we and others have recently identified inactivating mutations in the SWI/SNF chromatin remodeling gene SMARCA4 with concomitant loss

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), is a rare and understudied cancer with a dismal prognosis. SCCOHT's infrequency has hindered empirical study of its biology and clinical management. However, we and others have recently identified inactivating mutations in the SWI/SNF chromatin remodeling gene SMARCA4 with concomitant loss of SMARCA4 protein in the majority of SCCOHT tumors. Here we summarize these findings and report SMARCA4 status by targeted sequencing and/or immunohistochemistry (IHC) in an additional 12 SCCOHT tumors, 3 matched germlines, and the cell line SCCOHT-1. We also report the identification of a homozygous inactivating mutation in the gene SMARCB1 in one SCCOHT tumor with wild-type SMARCA4, suggesting that SMARCB1 inactivation may also play a role in the pathogenesis of SCCOHT. To date, SMARCA4 mutations and protein loss have been reported in the majority of 69 SCCOHT cases (including 2 cell lines). These data firmly establish SMARCA4 as a tumor suppressor whose loss promotes the development of SCCOHT, setting the stage for rapid advancement in the biological understanding, diagnosis, and treatment of this rare tumor type.

ContributorsRamos, Pilar (Author) / Kamezis, Anthony N. (Author) / Hendricks, William P. D. (Author) / Wang, Yemin (Author) / Tembe, Waibhav (Author) / Zismann, Victoria L. (Author) / Legendre, Christophe (Author) / Liang, Winnie S. (Author) / Russell, Megan L. (Author) / Craig, David W. (Author) / Farley, John H. (Author) / Monk, Bradley J. (Author) / Anthony, Stephen P. (Author) / Sekulic, Aleksandar (Author) / Cunliffe, Heather E. (Author) / Huntsman, David G. (Author) / Trent, Jeffrey M. (Author) / College of Liberal Arts and Sciences (Contributor)
Created2014-11-03
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Description

Background: Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In rare tumors, where large-scalec linical trials are daunting, this targeted genomic approach offers a new perspective and hope for improved treatments. Cancers of the ampulla of Vater are rare tumors that comprise

Background: Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In rare tumors, where large-scalec linical trials are daunting, this targeted genomic approach offers a new perspective and hope for improved treatments. Cancers of the ampulla of Vater are rare tumors that comprise only about 0.2% of gastrointestinal cancers. Consequently, they are often treated as either distal common bile duct or pancreatic cancers.

Methods: We analyzed DNA from a resected cancer of the ampulla of Vater and whole blood DNAfrom a 63 year-old man who underwent a pancreaticoduodenectomy by whole genomesequencing, achieving 37× and 40× coverage, respectively. We determined somatic mutations and structural alterations.

Results: We identified relevant aberrations, including deleterious mutations of KRAS and SMAD4 as well as a homozygous focal deletion of the PTEN tumor suppressor gene. These findings suggest that these tumors have a distinct oncogenesis from either common bile duct cancer or pancreatic cancer. Furthermore, this combination of genomic aberrations suggests a therapeutic context for dual mTOR/PI3K inhibition.

Conclusions: Whole genome sequencing can elucidate an oncogenic context and expose potential therapeutic vulnerabilities in rare cancers.

ContributorsDemeure, Michael J. (Author) / Craig, David W. (Author) / Sinari, Shripad (Author) / Moses, Tracy M. (Author) / Christoforides, Alexis (Author) / Dinh, Jennifer (Author) / Izatt, Tyler (Author) / Aldrich, Jessica (Author) / Decker, Ardis (Author) / Baker, Angela (Author) / Cherni, Irene (Author) / Watanabe, April (Author) / Koep, Lawrence (Author) / Lake, Douglas (Author) / Hostetter, Galen (Author) / Trent, Jeffrey M. (Author) / Von Hoff, Daniel D. (Author) / Carpten, John D. (Author) / College of Liberal Arts and Sciences (Contributor)
Created2012-07-04
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Description

Pancreatic adenocarcinoma (PAC) is among the most lethal malignancies. While research has implicated multiple genes in disease pathogenesis, identification of therapeutic leads has been difficult and the majority of currently available therapies provide only marginal benefit. To address this issue, our goal was to genomically characterize individual PAC patients to

Pancreatic adenocarcinoma (PAC) is among the most lethal malignancies. While research has implicated multiple genes in disease pathogenesis, identification of therapeutic leads has been difficult and the majority of currently available therapies provide only marginal benefit. To address this issue, our goal was to genomically characterize individual PAC patients to understand the range of aberrations that are occurring in each tumor. Because our understanding of PAC tumorigenesis is limited, evaluation of separate cases may reveal aberrations, that are less common but may provide relevant information on the disease, or that may represent viable therapeutic targets for the patient. We used next generation sequencing to assess global somatic events across 3 PAC patients to characterize each patient and to identify potential targets. This study is the first to report whole genome sequencing (WGS) findings in paired tumor/normal samples collected from 3 separate PAC patients. We generated on average 132 billion mappable bases across all patients using WGS, and identified 142 somatic coding events including point mutations, insertion/deletions, and chromosomal copy number variants. We did not identify any significant somatic translocation events. We also performed RNA sequencing on 2 of these patients' tumors for which tumor RNA was available to evaluate expression changes that may be associated with somatic events, and generated over 100 million mapped reads for each patient. We further performed pathway analysis of all sequencing data to identify processes that may be the most heavily impacted from somatic and expression alterations. As expected, the KRAS signaling pathway was the most heavily impacted pathway (P<0.05), along with tumor-stroma interactions and tumor suppressive pathways. While sequencing of more patients is needed, the high resolution genomic and transcriptomic information we have acquired here provides valuable information on the molecular composition of PAC and helps to establish a foundation for improved therapeutic selection.

ContributorsLiang, Winnie S. (Author) / Craig, David W. (Author) / Carpten, John (Author) / Borad, Mitesh J. (Author) / Demeure, Michael J. (Author) / Weiss, Glen J. (Author) / Izatt, Tyler (Author) / Sinari, Shripad (Author) / Christoforides, Alexis (Author) / Aldrich, Jessica (Author) / Kurdoglu, Ahmet (Author) / Barrett, Michael (Author) / Phillips, Lori (Author) / Benson, Hollie (Author) / Tembe, Waibhav (Author) / Braggio, Esteban (Author) / Kiefer, Jeffrey A. (Author) / Legendre, Christophe (Author) / Posner, Richard (Author) / Hostetter, Galen H. (Author) / Baker, Angela (Author) / Egan, Jan B. (Author) / Han, Haiyong (Author) / Lake, Douglas (Author) / Stites, Edward C. (Author) / Ramanathan, Ramesh K. (Author) / Fonseca, Rafael (Author) / Stewart, A. Keith (Author) / Von Hoff, Daniel (Author) / College of Liberal Arts and Sciences (Contributor)
Created2012-10-10
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Description

Background: The field of cancer genomics has rapidly adopted next-generation sequencing (NGS) in order to study and characterize malignant tumors with unprecedented resolution. In particular for cancer, one is often trying to identify somatic mutations--changes specific to a tumor and not within an individual's germline. However, false positive and false

Background: The field of cancer genomics has rapidly adopted next-generation sequencing (NGS) in order to study and characterize malignant tumors with unprecedented resolution. In particular for cancer, one is often trying to identify somatic mutations--changes specific to a tumor and not within an individual's germline. However, false positive and false negative detections often result from lack of sufficient variant evidence, contamination of the biopsy by stromal tissue, sequencing errors, and the erroneous classification of germline variation as tumor-specific.

Results: We have developed a generalized Bayesian analysis framework for matched tumor/normal samples with the purpose of identifying tumor-specific alterations such as single nucleotide mutations, small insertions/deletions, and structural variation. We describe our methodology, and discuss its application to other types of paired-tissue analysis such as the detection of loss of heterozygosity as well as allelic imbalance. We also demonstrate the high level of sensitivity and specificity in discovering simulated somatic mutations, for various combinations of a) genomic coverage and b) emulated heterogeneity.

Conclusion: We present a Java-based implementation of our methods named Seurat, which is made available for free academic use. We have demonstrated and reported on the discovery of different types of somatic change by applying Seurat to an experimentally-derived cancer dataset using our methods; and have discussed considerations and practices regarding the accurate detection of somatic events in cancer genomes. Seurat is available at https://sites.google.com/site/seuratsomatic.

ContributorsChristoforides, Alexis (Author) / Carpten, John D. (Author) / Weiss, Glen J. (Author) / Demeure, Michael J. (Author) / Von Hoff, Daniel D. (Author) / Craig, David W. (Author) / College of Health Solutions (Contributor)
Created2013-05-04
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Description
Rapid advancements in genomic technologies have increased our understanding of rare human disease. Generation of multiple types of biological data including genetic variation from genome or exome, expression from transcriptome, methylation patterns from epigenome, protein complexity from proteome and metabolite information from metabolome is feasible. "Omics" tools provide comprehensive view

Rapid advancements in genomic technologies have increased our understanding of rare human disease. Generation of multiple types of biological data including genetic variation from genome or exome, expression from transcriptome, methylation patterns from epigenome, protein complexity from proteome and metabolite information from metabolome is feasible. "Omics" tools provide comprehensive view into biological mechanisms that impact disease trait and risk. In spite of available data types and ability to collect them simultaneously from patients, researchers still rely on their independent analysis. Combining information from multiple biological data can reduce missing information, increase confidence in single data findings, and provide a more complete view of genotype-phenotype correlations. Although rare disease genetics has been greatly improved by exome sequencing, a substantial portion of clinical patients remain undiagnosed. Multiple frameworks for integrative analysis of genomic and transcriptomic data are presented with focus on identifying functional genetic variations in patients with undiagnosed, rare childhood conditions. Direct quantitation of X inactivation ratio was developed from genomic and transcriptomic data using allele specific expression and segregation analysis to determine magnitude and inheritance mode of X inactivation. This approach was applied in two families revealing non-random X inactivation in female patients. Expression based analysis of X inactivation showed high correlation with standard clinical assay. These findings improved understanding of molecular mechanisms underlying X-linked disorders. In addition multivariate outlier analysis of gene and exon level data from RNA-seq using Mahalanobis distance, and its integration of distance scores with genomic data found genotype-phenotype correlations in variant prioritization process in 25 families. Mahalanobis distance scores revealed variants with large transcriptional impact in patients. In this dataset, frameshift variants were more likely result in outlier expression signatures than other types of functional variants. Integration of outlier estimates with genetic variants corroborated previously identified, presumed causal variants and highlighted new candidate in previously un-diagnosed case. Integrative genomic approaches in easily attainable tissue will facilitate the search for biomarkers that impact disease trait, uncover pharmacogenomics targets, provide novel insight into molecular underpinnings of un-characterized conditions, and help improve analytical approaches that use large datasets.
ContributorsSzelinger, Szabolcs (Author) / Craig, David W. (Thesis advisor) / Kusumi, Kenro (Thesis advisor) / Narayan, Vinodh (Committee member) / Rosenberg, Michael S. (Committee member) / Huentelman, Matthew J (Committee member) / Arizona State University (Publisher)
Created2015
Description
In 2014, the Centers for Medicare and Medicaid Services (CMS), which oversees the federal Clinical Laboratories Improvement Amendments (CLIA) program, issued guidance that the CLIA requirements apply when researchers seek to return individual-level research findings to study participants or their physician (Centers for Medicare & Medicaid Services, 2014). The present

In 2014, the Centers for Medicare and Medicaid Services (CMS), which oversees the federal Clinical Laboratories Improvement Amendments (CLIA) program, issued guidance that the CLIA requirements apply when researchers seek to return individual-level research findings to study participants or their physician (Centers for Medicare & Medicaid Services, 2014). The present study explores the stance of U.S. Institutional Review Boards (IRBs) toward the applicability of and compliance with the CLIA regulations when studies plan to return individual research results (RIRR). I performed a document content analysis of 73 IRB policies and supporting documents from 30 United States (U.S.) institutions funded for biomedical research by the National Institutes of Health in 2017. Documents analyzed included policies, procedures, guidance, protocol and consent templates, and miscellaneous documents (such as IRB presentations) found to address the RIRR to study participants. I used qualitative content and document analysis to identify themes across institutions related to the CLIA regulations and the RIRR. Basic descriptive statistics were used to represent the data quantitatively. The study found that 96.67% (n=29) of institutions had documents that addressed the RIRR to participants. The majority of the institutions had at least one document that referenced the CLIA regulations when discussing the practice of disclosing participant-specific results [76.67% (n=23)]. The majority of institutions [56.67% (n=17)] indicated that they require compliance with the CLIA regulations for returning individual study findings to participants, while 13.33% (n=4) recommended compliance. The intent of two (6.67%) institutions was vague or unclear, while seven (26.67%) institutions were silent on the topic altogether. Of the 23 institutions that referenced “CLIA” in their documents, 52.17% only mentioned CLIA in a one or two-sentence blurb, providing very little guidance to investigators. The study results provide evidence that the majority of U.S. biomedical institutions require or recommend compliance with CLIA stipulations when investigators intend to return individual research results to study participants. However, the data indicates there is heterogeneity and variation in the quality of the guidance provided.
ContributorsBuchholtz, Stephanie (Author) / Robert, Jason S. (Thesis advisor) / Ellison, Karin D. (Committee member) / Carpten, John D. (Committee member) / Craig, David W. (Committee member) / Marchant, Gary E. (Committee member) / Arizona State University (Publisher)
Created2021