Identifying DNase I hypersensitive sites as driver distal regulatory elements in breast cancer.

Efforts to identify driver mutations in cancer have largely focused on genes, whereas non-coding sequences remain relatively unexplored. Here we develop a statistical method based on characteristics known to influence local mutation rate and a series of enrichment filters in order to identify distal regulatory elements harboring putative driver mutations in breast cancer. We identify ten DNase I hypersensitive sites that are significantly mutated in breast cancers and associated with the aberrant expression of neighboring genes. A pan-cancer analysis shows that three of these elements are significantly mutated across multiple cancer types and have mutation densities similar to protein-coding driver genes. Functional characterization of the most highly mutated DNase I hypersensitive sites in breast cancer (using in silico and experimental approaches) confirms that they are regulatory elements and affect the expression of cancer genes. Our study suggests that mutations of regulatory elements in tumors likely play an important role in cancer development.Cancer driver mutations can occur within noncoding genomic sequences. Here, the authors develop a statistical approach to identify candidate noncoding driver mutations in DNase I hypersensitive sites in breast cancer and experimentally demonstrate they are regulatory elements of known cancer genes.

Aberrant DNA Methylation in Human iPSCs Associates with MYC-Binding Motifs in a Clone-Specific Manner Independent of Genetics.

Induced pluripotent stem cells (iPSCs) show variable methylation patterns between lines, some of which reflect aberrant differences relative to embryonic stem cells (ESCs). To examine whether this aberrant methylation results from genetic variation or non-genetic mechanisms, we generated human iPSCs from monozygotic twins to investigate how genetic background, clone, and passage number contribute. We found that aberrantly methylated CpGs are enriched in regulatory regions associated with MYC protein motifs and affect gene expression. We classified differentially methylated CpGs as being associated with genetic and/or non-genetic factors (clone and passage), and we found that aberrant methylation preferentially occurs at CpGs associated with clone-specific effects. We further found that clone-specific effects play a strong role in recurrent aberrant methylation at specific CpG sites across different studies. Our results argue that a non-genetic biological mechanism underlies aberrant methylation in iPSCs and that it is likely based on a probabilistic process involving MYC that takes place during or shortly after reprogramming.

Large-Scale Profiling Reveals the Influence of Genetic Variation on Gene Expression in Human Induced Pluripotent Stem Cells.

In this study, we used whole-genome sequencing and gene expression profiling of 215 human induced pluripotent stem cell (iPSC) lines from different donors to identify genetic variants associated with RNA expression for 5,746 genes. We were able to predict causal variants for these expression quantitative trait loci (eQTLs) that disrupt transcription factor binding and validated a subset of them experimentally. We also identified copy-number variant (CNV) eQTLs, including some that appear to affect gene expression by altering the copy number of intergenic regulatory regions. In addition, we were able to identify effects on gene expression of rare genic CNVs and regulatory single-nucleotide variants and found that reactivation of gene expression on the X chromosome depends on gene chromosomal position. Our work highlights the value of iPSCs for genetic association analyses and provides a unique resource for investigating the genetic regulation of gene expression in pluripotent cells.

High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost.

iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types.

Large-scale collections of induced pluripotent stem cells (iPSCs) could serve as powerful model systems for examining how genetic variation affects biology and disease. Here we describe the iPSCORE resource: a collection of systematically derived and characterized iPSC lines from 222 ethnically diverse individuals that allows for both familial and association-based genetic studies. iPSCORE lines are pluripotent with high genomic integrity (no or low numbers of somatic copy-number variants) as determined using high-throughput RNA-sequencing and genotyping arrays, respectively. Using iPSCs from a family of individuals, we show that iPSC-derived cardiomyocytes demonstrate gene expression patterns that cluster by genetic background, and can be used to examine variants associated with physiological and disease phenotypes. The iPSCORE collection contains representative individuals for risk and non-risk alleles for 95% of SNPs associated with human phenotypes through genome-wide association studies. Our study demonstrates the utility of iPSCORE for examining how genetic variants influence molecular and physiological traits in iPSCs and derived cell lines.

Regression/eradication of gliomas in mice by a systemically-deliverable ATF5 dominant-negative peptide.

Malignant gliomas have poor prognosis and urgently require new therapies. Activating Transcription Factor 5 (ATF5) is highly expressed in gliomas, and interference with its expression/function precipitates targeted glioma cell apoptosis in vitro and in vivo. We designed a novel deliverable truncated-dominant-negative (d/n) form of ATF5 fused to a cell-penetrating domain (Pen-d/n-ATF5-RP) that can be intraperitoneally/subcutaneously administered to mice harboring malignant gliomas generated; (1) by PDGF-B/sh-p53 retroviral transformation of endogenous neural progenitor cells; and (2) by human U87-MG xenografts. In vitro Pen-d/n-ATF5-RP entered into glioma cells and triggered massive apoptosis. In vivo, subcutaneously-administered Pen-d/n-ATF5-RP passed the blood brain barrier, entered normal brain and tumor cells, and then caused rapid selective tumor cell death. MRI verified elimination of retrovirus-induced gliomas within 8-21 days. Histopathology revealed growth-suppression of intracerebral human U87-MG cells xenografts. For endogenous PDGF-B gliomas, there was no recurrence or mortality at 6-12 months versus 66% mortality in controls at 6 months. Necropsy and liver-kidney blood enzyme analysis revealed no adverse effects on brain or other tissues. Our findings thus identify Pen-d/n-ATF5-RP as a potential therapy for malignant gliomas.

Genome-wide mutational landscape of mucinous carcinomatosis peritonei of appendiceal origin.

BACKGROUND: Mucinous neoplasms of the appendix (MNA) are rare tumors which may progress from benign to malignant disease with an aggressive biological behavior. MNA is often diagnosed after metastasis to the peritoneal surfaces resulting in mucinous carcinomatosis peritonei (MCP). Genetic alterations in MNA are poorly characterized due to its low incidence, the hypo-cellularity of MCPs, and a lack of relevant pre-clinical models. As such, application of targeted therapies to this disease is limited to those developed for colorectal cancer and not based on molecular rationale. METHODS: We sequenced the whole exomes of 10 MCPs of appendiceal origin to identify genome-wide somatic mutations and copy number aberrations and validated significant findings in 19 additional cases. RESULTS: Our study demonstrates that MNA has a different molecular makeup than colorectal cancer. Most tumors have co-existing oncogenic mutations in KRAS (26/29) and GNAS (20/29) and are characterized by downstream PKA activation. High-grade tumors are GNAS wild-type (5/6), suggesting they do not progress from low-grade tumors. MNAs do share some genetic alterations with colorectal cancer including gain of 1q (5/10), Wnt, and TGFß pathway alterations. In contrast, mutations in TP53 (1/10) and APC (0/10), common in colorectal cancer, are rare in MNA. Concurrent activation of the KRAS and GNAS mediated signaling pathways appears to be shared with pancreatic intraductal papillary mucinous neoplasm. CONCLUSIONS: MNA genome-wide mutational analysis reveals genetic alterations distinct from colorectal cancer, in support of its unique pathophysiology and suggests new targeted therapeutic opportunities.

Genetic ancestry of participants in the National Children's Study.

BACKGROUND: The National Children's Study (NCS) is a prospective epidemiological study in the USA tasked with identifying a nationally representative sample of 100,000 children, and following them from their gestation until they are 21 years of age. The objective of the study is to measure environmental and genetic influences on growth, development, and health. Determination of the ancestry of these NCS participants is important for assessing the diversity of study participants and for examining the effect of ancestry on various health outcomes. RESULTS: We estimated the genetic ancestry of a convenience sample of 641 parents enrolled at the 7 original NCS Vanguard sites, by analyzing 30,000 markers on exome arrays, using the 1000 Genomes Project superpopulations as reference populations, and compared this with the measures of self-reported ethnicity and race. For 99% of the individuals, self-reported ethnicity and race agreed with the predicted superpopulation. NCS individuals self-reporting as Asian had genetic ancestry of either South Asian or East Asian groups, while those reporting as either Hispanic White or Hispanic Other had similar genetic ancestry. Of the 33 individuals who self-reported as Multiracial or Non-Hispanic Other, 33% matched the South Asian or East Asian groups, while these groups represented only 4.4% of the other reported categories. CONCLUSIONS: Our data suggest that self-reported ethnicity and race have some limitations in accurately capturing Hispanic and South Asian populations. Overall, however, our data indicate that despite the complexity of the US population, individuals know their ancestral origins, and that self-reported ethnicity and race is a reliable indicator of genetic ancestry.

Organotypic distribution of stem cell markers in formalin-fixed brain harboring glioblastoma multiforme.

The role of stem cells in the origin, growth patterns, and infiltration of glioblastoma multiforme is a subject of intense investigation. One possibility is that glioblastoma may arise from transformed stem cells in the ventricular zone. To explore this hypothesis, we examined the distribution of two stem cell markers, activating transcription factor 5 (ATF5) and CD133, in an autopsy brain specimen from an individual with glioblastoma multiforme. A 41-year-old male with a right posterior temporal glioblastoma had undergone surgery, radiation, and chemotherapy. The brain was harvested within several hours after death. After formalin fixation, sectioning, and mapping of tumor location in the gross specimen, histologic specimens were prepared from tumor-bearing and grossly normal hemispheres. Fluorescence immunohistochemistry and colorimetric staining were performed for ATF5 and CD133. Both markers co-localized to the ependymal and subependymal zones on the side of the tumor, but not in the normal hemisphere or more rostrally in the affected hemisphere. ATF5 staining was especially robust within the diseased hemisphere in histologically normal ependyma. To our knowledge, this is the first in situ demonstration of stem cell markers in whole human brain. These preliminary results support the hypothesis that some glioblastomas may arise from the neurogenic zone of the lateral ventricle. The robust staining for ATF5 and CD133 in histologically normal ventricular zone suggests that an increase in periventricular stem cell activity occurred in this patient on the side of the tumor, either as a localized response to brain injury or as an integral component of oncogenesis and tumor recurrence.