Change-point analysis of paired allele-specific copy number variation data

The recent genome-wide allele-specific copy number variation data enable us to explore two types of genomic information including chromosomal genotype variations as well as DNA copy number variations. For a cancer study, it is common to collect data for paired normal and tumor samples. Then, two types of paired data can be obtained to study a disease subject. However, there is a lack of methods for a simultaneous analysis of these four sequences of data. In this study, we propose a statistical framework based on the change-point analysis approach. The validity and usefulness of our proposed statistical framework are demonstrated through the simulation studies and applications based on an experimental data set

Statistical model-based testing to evaluate the recurrence of genomic aberrations

Motivation: In cancer genomes, chromosomal regions harboring cancer genes are often subjected to genomic aberrations like copy number alteration and loss of heterozygosity. Given this, finding recurrent genomic aberrations is considered an apt approach for screening cancer genes. Although several permutation-based tests have been proposed for this purpose, none of them are designed to find recurrent aberrations from the genomic dataset without paired normal sample controls. Their application to unpaired genomic data may lead to false discoveries, because they retrieve pseudo-aberrations that exist in normal genomes as polymorphisms

An integrated analysis tool for analyzing hybridization intensities and genotypes using new-generation population-optimized human arrays

The cross-sample plot of the multipoint LOH/LCSH analyses of the three samples used in Fig. 5. The plot comprises four panels: (a) The top-left panel is a cross-sample and cross-chromosome plot. The vertical axis is the index of study samples, and the horizontal axis is the physical position (Mb) on each of the 23 chromosomes. The blue and red bars represent SNPs without and with LOH/LSCH, respectively. (b) The top-right panel is a histogram of cross-chromosome aberration frequency. The vertical axis is the index of study samples, and the horizontal axis is the cross-chromosome aberration frequency of the corresponding samples. The pink (skyblue) background represents that the genetic gender of a sample is female (male). The histogram represents the aberration frequency of LOH/LCSH SNPs across the chromosomes of the corresponding samples. (c) The bottom-left panel is a histogram of the cross-sample aberration frequency. The vertical axis is the cross-sample aberration frequency of a SNP, and the horizontal axis is the physical position (Mb) on each of the 23 chromosomes. The purple line represents the aberration proportion of samples carrying the SNPs with LOH/LCSH. (d) The bottom-right panel is the legend of the genetic gender that is used in panel (b), where the pink (skyblue) background represents that the genetic gender of a sample is female (male). (TIFF 1656 kb

Discovery of Variants Underlying Simple and Complex Traits through Genome-Wide Association Study and Whole-Genome Resequencing

If no reference genome exists, then the generation of a de novo genome assembly of an organism is necessary because having a reference genome expedites the discoveries for simple and complex traits. Reference genomes for the domestic dog (Canis lupus familiaris) and chicken (Gallus gallus) have resulted in the development of single nucleotide polymorphism (SNP) arrays for use in genome-wide association studies (GWAS). Next-generation sequencing technologies provide a rapid, increasingly affordable method for the generation of genome resequencing data. The first objective of this work was to utilize existing genomic resources to investigate the genetic basis for traits of the dog and chicken. Episodic falling syndrome (EFS) is a recessive neurological disease of Cavalier King Charles spaniels. Using SNP profiles from only 12 individuals, EFS was mapped to chromosome 7; further experimentation led to the identification of the causative deletion. In a second example, SNP profiles from 197 German shepherd dogs were generated to identify loci underlying numerous diseases afflicting the breed, including recessive pituitary dwarfism, and three complex diseases: degenerative myelopathy, megaesophagus, and pancreatic acinar atrophy. Lastly, SNP profiles for 60 Araucana chickens were used to identify an association with the semi-dominant tailless rump (rumpless) phenotype on chromosome 2, as well as the recessive lethal ear-tufts phenotype on chromosome 15. Positional candidate genes were identified for both traits. The second objective of this work was to identify loci associated with dermatomyositis (DM) and to develop resources to facilitate the identification of the causative mutation. DM is an inflammatory myopathy affecting humans and domestic dogs, primarily the collie and Shetland sheepdog breeds where painful lesions on the face and extremities are characteristics. The second objective was accomplished through 1) assembly of a population of DM-affected and healthy control collies, 2) completion of a GWAS using SNP profiles generated for this population, and 3) establishment of whole-genome resequencing data from 3 DM-affected collies and 2 healthy controls. Results revealed a strong association on chromosome 10. Annotation of the collie genome yielded novel SNPs, structural variants, and selective sweeps, and regions of reduced heterogeneity surrounding a gene(s) under strong positive selection

Exome sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome.

Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder that is characterized by mild thrombocytopenia with large platelets and a paucity of α-granules; these abnormalities cause mostly moderate but in rare cases severe bleeding. We sequenced the exomes of four unrelated individuals and identified NBEAL2 as the causative gene; it has no previously known function but is a member of a gene family that is involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation

High-resolution QTL mapping in Tetranychus urticae reveals acaricide-specific responses and common target-site resistance after selection by different METI-I acaricides

Arthropod herbivores cause dramatic crop losses, and frequent pesticide use has led to widespread resistance in numerous species. One such species, the two-spotted spider mite, Tetranychus urticae, is an extreme generalist herbivore and a major worldwide crop pest with a history of rapidly developing resistance to acaricides. Mitochondrial Electron Transport Inhibitors of complex I (METI-Is) have been used extensively in the last 25 years to control T. urticae around the globe, and widespread resistance to each has been documented. METI-I resistance mechanisms in T. urticae are likely complex, as increased metabolism by cytochrome P450 monooxygenases as well as a target-site mutation have been linked with resistance. To identify loci underlying resistance to the METI-I acaricides fenpyroximate, pyridaben and tebufenpyrad without prior hypotheses, we crossed a highly METI-I-resistant strain of T. urticae to a susceptible one, propagated many replicated populations over multiple generations with and without selection by each compound, and performed bulked segregant analysis genetic mapping. Our results showed that while the known H92R target-site mutation was associated with resistance to each compound, a genomic region that included cytochrome P450-reductase (CPR) was associated with resistance to pyridaben and tebufenpyrad. Within CPR, a single nonsynonymous variant distinguished the resistant strain from the sensitive one. Furthermore, a genomic region linked with tebufenpyrad resistance harbored a non-canonical member of the nuclear hormone receptor 96 (NHR96) gene family. This NHR96 gene does not encode a DNA-binding domain (DBD), an uncommon feature in arthropods, and belongs to an expanded family of 47 NHR96 proteins lacking DBDs in T. urticae. Our findings suggest that although cross-resistance to METI-Is involves known detoxification pathways, structural differences in METI-I acaricides have also resulted in resistance mechanisms that are compound-specific

Analysis of Genetic and Gene-by-Treatment Effectsin the Collaborative Cross Mice

Gene-by-treatment interaction (GxT) describes the phenomenon whereby individuals respond differently to an applied treatment due to differences in genetic background. GxT is considered to be a major source of the variability in drug effects. In human clinical trials, GxT may cause idiosyncratic drug reactions, raising serious safety concerns. There is therefore great interest in identifying genetic variants associated with differential treatment response. It is well-recognized that replicable populations of mice are valuable resources for investigating treatment response. Inbred mice allow researchers to measure treatment response with fixed conditions, including fixed genetic background. Studying GxT can then be achieved through experiments measuring multiple inbred strains, thus making it possible to observe treatment response differences due to genetic background. An ideal mouse model for GxT research is the Collaborative Cross (CC). The CC is a large panel of recombinant inbred mouse strains with high genetic diversity that allows efficient genetic mapping of treatment response. In this work, we performed a set of studies building a framework and developing tools for analyzing genetic and GxT effect in CC. We begin by constructing a variant resource to efficiently query genotype and haplotype variation of CC strains. We then analyzed two drug treatment datasets exploring the GxT mechanism behind idiosyncratic drug-induced liver injury (IDILI). We developed methods to model treatment effects, run QTL mapping and identify candidate variants associated with GxT. We also designed a pipeline to identify GxT associations in expression modules. Several genetic regions and variants associated with IDILI of drug Idelalisib and TAK-875 are identified. Besides GxT, valuable information is also present in the control mice. Control mice from one study were used to identify candidate genetic variation driving mRNA and protein levels of several key drug metabolism enzymes and transporters. Control mice from three studies were used to test between-study replicability of genetic signals. Such analysis will enhance our understanding of replicability and help improve genetic analysis methods. Taken together, methods and analysis in this work represent progress towards a new way of studying genetic and GxT in a diverse and replicable genetic reference population.Doctor of Philosoph

Pax5 Haploinsufficiency Cooperates with BCR-ABL1 to Induce Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the commonest pediatric malignancy and comprises several distinct subtypes each with its own unique pathogenesis, clinical behavior, and response to therapy. Chromosomal aberrations are a hallmark of ALL but alone fail to induce leukemia. Pediatric ALLs can be divided into several categories based on the expression of several genetically conserved chromosomal translocations including the t(9,22)[BCR-ABL1], t(1,19)[TCF3-PBX1], t(12,21)[ETV6-RUNX1], MLLrearranged leukemia’s, hyperdiploid and hypodiploid karyotypes, and T-lineage leukemia. Each translocation confers a characteristic transforming phenotype within the cell in which it originates but is alone insufficient to induce overt leukemia. In order to identify oncogenic lesions that cooperate with the aforementioned initiator lesions, we have performed genome-wide analysis of leukemia cells from 242 pediatric ALL patients using high resolution, single-nucleotide polymorphism microarrays. Our analysis revealed deletion, amplification, point mutation, and structural rearrangements in genes encoding principal regulators of B lymphocyte development and differentiation in 40% of B-progenitor ALL cases. The PAX5 gene was the most frequent target of somatic mutation, being altered in 31.7% of cases. The identified PAX5 mutations resulted in reduced levels of PAX5 protein or the generation of hypomorphic alleles. Deletions were also detected in TCF3 (also known as E2A), EBF1, LEF1, IKZF1 (IKAROS), and IKZF3(AIOLOS). These findings suggest that direct disruption of pathways controlling B-cell development and differentiation contributes to B-progenitor ALL pathogenesis. Moreover, these data demonstrate the power of high-resolution, genome-wide approaches to identify new molecular lesions in cancer. The Philadelphia chromosome, a chromosomal abnormality that encodes BCR-ABL1, is the defining lesion of chronic myelogenous leukemia (CML) and a subset of ALL. To specifically define oncogenic lesions that cooperate with BCR-ABL1 to induce ALL, we subsequently performed genome-wide analysis of diagnostic leukemia samples from 304 individuals with ALL, including 43 BCR-ABL1 B-progenitor ALLs and 23 CMLcases. IKZF1 (encoding the transcription factor Ikaros) was deleted in 83.7% of BCR-ABL1 ALL, but not chronic-phase CML. Deletion of IKZF1 was also identified as an acquired lesion at the time of transformation of CML to ALL (lymphoid blast crisis). The IKZF1 deletions resulted in haploinsufficiency, expression of a dominant-negative Ikaros isoform, or the complete loss of Ikaros expression. Sequencing of IKZF1deletion breakpoints suggested that aberrant RAG-mediated recombination is responsible for the deletions. These findings suggest that genetic lesions resulting in the loss of Ikaros function are an important event in the development of BCR-ABL1 ALL. In order to assess the contribution of the loss of B-cell developmental regulatory genes with BCR-ABL1 we performed bone marrow transplant assays. Pax5haploinsufficiency was shown to cooperate with BCR-ABL1 during leukemogenesis. Furthermore, as seen in human ALL, both Pax5 and p19Arf haploinsufficiency further cooperate during leukemogenesis. Pathological analysis of the leukemias revealed a B-lymphoid phenotype suggesting that this model results in the development of ALL. Secondary transplant studies confirmed that this was ALL and not a lymphoproliferative disorder. Immunophenotypic analysis confirmed the B-ALL phenotype and further revealed striking differences between Pax5+/+, Pax5+/-, and Pax5-/- leukemias. The leukemias that have lost either one or both Pax5 alleles revealed a more immature immunophenotype that was most pronounced in those with bi-allelic Pax5 loss. The wild-type leukemias were consistent with a Hardy fraction C immunophenotype while the Pax5 null leukemias were akin to Hardy fraction A with no expression of any definitive B-cell surface antigens except B220 and CD43. The Pax5+/+ and Pax5+/- leukemias were monoclonal while the Arf+/- and the compound heterozygous (Pax5+/- Arf+/-) leukemias were oligoclonal suggesting that the loss of p19Arf confers greater leukemogenic properties to a cell than does Pax5 loss. This is substantiated by the data that Arf heterozygous animals are tumor prone alone while Pax5 heterozygous animals live full normal lives. Genomic analysis of 50 murine leukemias (15 WT, 25 Pax5+/- and 10 Arf+/-) revealed that some of the same genomic abnormalities found in human ALL also develop in our murine ALL model. This finding suggests that our model is accurately recapitulating the development of human ALL. Furthermore, we have found through extensive gene expression studies that our mouse BCR-ABL1 leukemias share a gene expression profile similar to human BCR-ABL1 leukemias further supporting our murine model of ALL. The gene expression studies that we have performed have also given us insight into the role that Pax5 haploinsufficiency may be playing during leukemogenesis. Recently D.J Wong et al performed an exhaustive study of embryonic and adult stem cell gene expression profiling. He found that there are modules of genes that are common to either embryonic or adult stem cells. He went further to delineate a group of genes that are commonly expressed in both murine and human embryonic stem cells and call this the core ESC-like module. We performed gene set enrichment analysis (GSEA) using this core ESC-like module and found that this geneset is significantly enriched in our murine leukemias. In addition, we found that this core ESC-like module was significantly enriched in normal Hardy fraction B cells, but not in A, or C-F. We also found, using a principal component analysis method, that Pax5+/+ leukemias are most similar to Hardy fraction C while leukemias that have lost either one or both Pax5 alleles are most similar to Hardy fraction B suggesting that the loss of Pax5 blocks B-cell development and results in cells that are more similar to a stage that shares similar expression of embryonic stem cell genes. Taken together this data suggests that by losing Pax5 the leukemia becomes more stem cell like and may gain advantages that other B-cells do not because they continue down the road of differentiation