Genome Evolution and Innovation across the Four Major Lineages of Cryptococcus gattii

We acknowledge the Broad Institute Sequencing Platform and Imperial College London for generating the DNA sequence described here (and R265 Illumina sequences described previously [4]). We thank Sinéad Chapman for coordinating sequencing at the Broad Institute and Margaret Priest for assistance in submitting assemblies to NCBI. This project was supported by the National Human Genome Research Institute, grant no. U54HG003067. R.A.F. is supported by the Wellcome Trust. R.C.M. is supported by the Lister Institute for Preventive Medicine, the Medical Research Council UK, and the European Research Council.Peer reviewedPublisher PD

Microevolution of Serial Clinical Isolates of Cryptococcus neoformans var. grubii and C. gattii

We thank the Broad Institute Sequencing Platform for generating the Illumina sequences. We thank Chen-Hsin Yu for helping on the data processing of the phenotypic tests. We acknowledge the South African National Institute for Communicable Diseases’ GERMS-SA surveillance network through which these isolates were originally collected. This project has been funded in whole or in part by the following U.S. Health and Human Services grants from the National Institute of Allergy and Infectious Diseases: U19 AI110818 (Broad Institute), R01 AI93257 (J.R.P.), R01 AI73896 (J.R.P.), and R01 AI025783 (T.G.M.). R.A.F. was supported by the Wellcome Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The content is solely our responsibility and does not necessarily represent the official views of the funders. The use of product names in this manuscript does not imply their endorsement by the U.S. Department of Health and Human Services. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the CDC.Peer reviewedPublisher PD

Genome Diversity, Recombination, and Virulence across the Major Lineages of Paracoccidioides

We thank Angela Restrepo, Rosana Puccia, Zoilo Pires de Camargo, and Maria Sueli Felipe for kindly providing the isolates for this study. This project has been funded in whole or in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract no. HHSN272200900018C. This work was partly supported by Colciencias via the grants “A Gene Atlas for Human Pathogenic Fungi” (122256934875) and “A Comprehensive Genomic and Transcriptomic Analysis of Dimorphic Human Pathogen Fungi and Its Relation with Virulence” (221365842971) and by the Universidad de Antioquia via a “Sostenibilidad 2015/2016” grant. Colciencias National Doctorate Program funding supported J.F.M.; Enlaza Mundos partly supported his fellowship. The Wellcome Trust supported R.A.F.Peer reviewedPublisher PD

Tracing Genetic Exchange and Biogeography of Cryptococcus neoformans var. grubii at the Global Population Level

Cryptococcus neoformans var. grubii is the causative agent of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals, typically human immunodeficiency virus/AIDS patients from developing countries. Despite the worldwide emergence of this ubiquitous infection, little is known about the global molecular epidemiology of this fungal pathogen. Here we sequence the genomes of 188 diverse isolates and characterize the major subdivisions, their relative diversity, and the level of genetic exchange between them. While most isolates of C. neoformans var. grubii belong to one of three major lineages (VNI, VNII, and VNB), some haploid isolates show hybrid ancestry including some that appear to have recently interbred, based on the detection of large blocks of each ancestry across each chromosome. Many isolates display evidence of aneuploidy, which was detected for all chromosomes. In diploid isolates of C. neoformans var. grubii (serotype AA) and of hybrids with C. neoformans var. neoformans (serotype AD) such aneuploidies have resulted in loss of heterozygosity, where a chromosomal region is represented by the genotype of only one parental isolate. Phylogenetic and population genomic analyses of isolates from Brazil reveal that the previously “African” VNB lineage occurs naturally in the South American environment. This suggests migration of the VNB lineage between Africa and South America prior to its diversification, supported by finding ancestral recombination events between isolates from different lineages and regions. The results provide evidence of substantial population structure, with all lineages showing multi-continental distributions; demonstrating the highly dispersive nature of this pathogen

Evolution of pathogenicity and sexual reproduction in eight Candida genomes

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.publishe

Comparative Genomic Analysis of Human Fungal Pathogens Causing Paracoccidioidomycosis

Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.National Institute of Allergy and Infectious Diseases (U.S.)National Institutes of Health. Department of Health and Human Services (contract HHSN266200400001C)National Institutes of Health. Department of Health and Human Services(contract HHSN2722009000018C)Brazil. National Council for Scientific and Technological Developmen

Characterizing the spectrum of somatic alterations in canine and human cancers

Cancers arise as a result of deleterious somatic alterations accumulating in the genome during the process of cell division. These alterations arise either via exposure to mutagens or due to errors occurring during DNA replication. In this thesis, a systematic exploration, from discovery to analyses of somatic alterations in three diverse cancers that affect dogs and humans, was undertaken. In Studies I and II, whole-exome sequencing of dogs affected by the cancers of osteosarcoma and hemangiosarcoma were done to delineate coding mutations that can contribute to their carcinogenesis. Besides, as these cancers mirror the corresponding human disease in clinical manifestation and histological features, a secondary objective was to confirm the molecular drivers found in the canines were also influencing factors in the human cancer(s). In the osteosarcoma investigations with three breeds, we found that tumors show a high frequency of somatic copy-number alterations, affecting key cancer genes. TP53 was the most frequently altered gene, akin to human osteosarcoma. The second most mutated gene, histone methyltransferase SETD2, has known epigenetic roles in multiple cancers but not in osteosarcoma. Our study highlights the strong genetic similarities between human and dog osteosarcoma, suggesting that canine disease may serve as an excellent model for developing treatment strategies in both species. In the hemangiosarcoma study in golden retrievers, putative driver alterations were identified in the tumor suppressor TP53 and in genes involved in the cell cycle regulating PI3K pathway, including PIK3CA and PIK3R1. Furthermore, we find several somatic alterations between the dog hemangiosarcoma and human angiosarcoma overlap, indicating we can use the canine model to apprise the infrequently occurring human disease. In Study III, we implemented whole-genome sequencing methodologies to define both coding and non-coding alterations in the glioblastoma cancer genome. We find the coding somatic alterations recapitulate what has been previously seen for the cancer, including driver alterations in the genes of EGFR, PTEN, and TP53. Significantly though, using the concept of evolutionary constraint, we find an enrichment of non-coding mutations in regulatory regions, around GBM-implicated genes. The mutated regions include splice sites, promoters, and transcription factor binding sites, suggesting the importance of regulatory mutations for the pathogenesis of glioblastoma. Overall, the insights garnered from the above exome- and genome-wide surveys provide novel insights into unraveling some of the complexities associated with somatic genomic alterations in cancer genomes. It also convincingly underscores the benefits of using sequencing technologies to comprehend complex biological diseases

Characterizing the spectrum of somatic alterations in canine and human cancers

Cancers arise as a result of deleterious somatic alterations accumulating in the genome during the process of cell division. These alterations arise either via exposure to mutagens or due to errors occurring during DNA replication. In this thesis, a systematic exploration, from discovery to analyses of somatic alterations in three diverse cancers that affect dogs and humans, was undertaken. In Studies I and II, whole-exome sequencing of dogs affected by the cancers of osteosarcoma and hemangiosarcoma were done to delineate coding mutations that can contribute to their carcinogenesis. Besides, as these cancers mirror the corresponding human disease in clinical manifestation and histological features, a secondary objective was to confirm the molecular drivers found in the canines were also influencing factors in the human cancer(s). In the osteosarcoma investigations with three breeds, we found that tumors show a high frequency of somatic copy-number alterations, affecting key cancer genes. TP53 was the most frequently altered gene, akin to human osteosarcoma. The second most mutated gene, histone methyltransferase SETD2, has known epigenetic roles in multiple cancers but not in osteosarcoma. Our study highlights the strong genetic similarities between human and dog osteosarcoma, suggesting that canine disease may serve as an excellent model for developing treatment strategies in both species. In the hemangiosarcoma study in golden retrievers, putative driver alterations were identified in the tumor suppressor TP53 and in genes involved in the cell cycle regulating PI3K pathway, including PIK3CA and PIK3R1. Furthermore, we find several somatic alterations between the dog hemangiosarcoma and human angiosarcoma overlap, indicating we can use the canine model to apprise the infrequently occurring human disease. In Study III, we implemented whole-genome sequencing methodologies to define both coding and non-coding alterations in the glioblastoma cancer genome. We find the coding somatic alterations recapitulate what has been previously seen for the cancer, including driver alterations in the genes of EGFR, PTEN, and TP53. Significantly though, using the concept of evolutionary constraint, we find an enrichment of non-coding mutations in regulatory regions, around GBM-implicated genes. The mutated regions include splice sites, promoters, and transcription factor binding sites, suggesting the importance of regulatory mutations for the pathogenesis of glioblastoma. Overall, the insights garnered from the above exome- and genome-wide surveys provide novel insights into unraveling some of the complexities associated with somatic genomic alterations in cancer genomes. It also convincingly underscores the benefits of using sequencing technologies to comprehend complex biological diseases

Whole-genome sequencing of glioblastoma reveals enrichment of non-coding constraint mutations in known and novel genes

Background Glioblastoma (GBM) has one of the worst 5-year survival rates of all cancers. While genomic studies of the disease have been performed, alterations in the non-coding regulatory regions of GBM have largely remained unexplored. We apply whole-genome sequencing (WGS) to identify non-coding mutations, with regulatory potential in GBM, under the hypothesis that regions of evolutionary constraint are likely to be functional, and somatic mutations are likely more damaging than in unconstrained regions. Results We validate our GBM cohort, finding similar copy number aberrations and mutated genes based on coding mutations as previous studies. Performing analysis on non-coding constraint mutations and their position relative to nearby genes, we find a significant enrichment of non-coding constraint mutations in the neighborhood of 78 genes that have previously been implicated in GBM. Among them, SEMA3C and DYNC1I1 show the highest frequencies of alterations, with multiple mutations overlapping transcription factor binding sites. We find that a non-coding constraint mutation in the SEMA3C promoter reduces the DNA binding capacity of the region. We also identify 1776 other genes enriched for non-coding constraint mutations with likely regulatory potential, providing additional candidate GBM genes. The mutations in the top four genes, DLX5, DLX6, FOXA1, and ISL1, are distributed over promoters, UTRs, and multiple transcription factor binding sites. Conclusions These results suggest that non-coding constraint mutations could play an essential role in GBM, underscoring the need to connect non-coding genomic variation to biological function and disease pathology.De tre första författarna delar förstaförfattarskapetTitle in thesis list of papers: Whole Genome Sequencing of Glioblastoma Reveals Enrichment of Non-Coding Constraint Mutations in Known and Novel Genes</p

PIK3CA is recurrently mutated in canine mammary tumors, similarly to in human mammary neoplasia

Abstract Biological features of neoplastic disease affecting mammary gland tissue are shared between canines and humans. Research performed in either species has translational value and early phase clinical trials performed in canines with spontaneous disease could be informative for human trials. The purpose of this study was to investigate the somatic genetic aberrations occurring in canine mammary neoplasia by exome capture and next generation sequencing. Based on 55 tumor-normal pairs we identified the PIK3CA gene as the most commonly mutated gene in canine mammary tumors, with 25% of samples carrying mutations in this gene. A recurrent missense mutation was identified, p.H1047R, which is homologous to the human PIK3CA hotspot mutation found in different types of breast neoplasia. Mutations homologous to other known human mutation hotspots such as the PIK3CA p.E545K and the KRAS p.G12V/D were also identified. We identified copy number aberrations affecting important tumor suppressor and oncogenic pathways including deletions affecting the PTEN tumor suppressor gene. We suggest that activation of the KRAS or PIK3CA oncogenes or loss of the PTEN suppressor gene may be important for mammary tumor development in dogs. This data endorses the conservation of cancer across species and the validity of studying cancer in non-human species