Recovering rearranged cancer chromosomes from karyotype graphs

BACKGROUND: Many cancer genomes are extensively rearranged with highly aberrant chromosomal karyotypes. Structural and copy number variations in cancer genomes can be determined via abnormal mapping of sequenced reads to the reference genome. Recently it became possible to reconcile both of these types of large-scale variations into a karyotype graph representation of the rearranged cancer genomes. Such a representation, however, does not directly describe the linear and/or circular structure of the underlying rearranged cancer chromosomes, thus limiting possible analysis of cancer genomes somatic evolutionary process as well as functional genomic changes brought by the large-scale genome rearrangements. RESULTS: Here we address the aforementioned limitation by introducing a novel methodological framework for recovering rearranged cancer chromosomes from karyotype graphs. For a cancer karyotype graph we formulate an Eulerian Decomposition Problem (EDP) of finding a collection of linear and/or circular rearranged cancer chromosomes that are determined by the graph. We derive and prove computational complexities for several variations of the EDP. We then demonstrate that Eulerian decomposition of the cancer karyotype graphs is not always unique and present the Consistent Contig Covering Problem (CCCP) of recovering unambiguous cancer contigs from the cancer karyotype graph, and describe a novel algorithm CCR capable of solving CCCP in polynomial time. We apply CCR on a prostate cancer dataset and demonstrate that it is capable of consistently recovering large cancer contigs even when underlying cancer genomes are highly rearranged. CONCLUSIONS: CCR can recover rearranged cancer contigs from karyotype graphs thereby addressing existing limitation in inferring chromosomal structures of rearranged cancer genomes and advancing our understanding of both patient/cancer-specific as well as the overall genetic instability in cancer

The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima.

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.This work was supported by the following grants: NHGRIU54HG003273 to R.A.G; EU Marie Curie ITN #215781 “Evonet” to M.A.; a Wellcome Trust Value in People (VIP) award to C.B. and Wellcome Trust graduate studentship WT089615MA to J.E.G; Marine rhythms of Life” of the University of Vienna, an FWF (http://www.fwf.ac.at/) START award (#AY0041321) and HFSP (http://www.hfsp.org/) research grant (#RGY0082/2010) to KT-­‐R; MFPL Vienna International PostDoctoral Program for Molecular Life Sciences (funded by Austrian Ministry of Science and Research and City of Vienna, Cultural Department -­‐Science and Research to T.K; Direct Grant (4053034) of the Chinese University of Hong Kong to J.H.L.H.; NHGRI HG004164 to G.M.; Danish Research Agency (FNU), Carlsberg Foundation, and Lundbeck Foundation to C.J.P.G.; U.S. National Institutes of Health R01AI55624 to J.H.W.; Royal Society University Research fellowship to F.M.J.; P.D.E. was supported by the BBSRC via the Babraham Institute;This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pbio.100200

Farnesyltransferase inhibitor and rapamycin correct aberrant genome organisation and decrease DNA damage respectively, in Hutchinson–Gilford progeria syndrome fibroblasts

Hutchinson–Gilford progeria syndrome (HGPS) is a rare and fatal premature ageing disease in children. HGPS is one of several progeroid syndromes caused by mutations in the LMNA gene encoding the nuclear structural proteins lamins A and C. In classic HGPS the mutation G608G leads to the formation of a toxic lamin A protein called progerin. During post-translational processing progerin remains farnesylated owing to the mutation interfering with a step whereby the farnesyl moiety is removed by the enzyme ZMPSTE24. Permanent farnesylation of progerin is thought to be responsible for the proteins toxicity. Farnesyl is generated through the mevalonate pathway and three drugs that interfere with this pathway and hence the farnesylation of proteins have been administered to HGPS children in clinical trials. These are a farnesyltransferase inhibitor (FTI), statin and a bisphosphonate. Further experimental studies have revealed that other drugs such as N-acetyl cysteine, rapamycin and IGF-1 may be of use in treating HGPS through other pathways. We have shown previously that FTIs restore chromosome positioning in interphase HGPS nuclei. Mis-localisation of chromosomes could affect the cells ability to regulate proper genome function. Using nine different drug treatments representing drug regimes in the clinic we have shown that combinatorial treatments containing FTIs are most effective in restoring specific chromosome positioning towards the nuclear periphery and in tethering telomeres to the nucleoskeleton. On the other hand, rapamycin was found to be detrimental to telomere tethering, it was, nonetheless, the most effective at inducing DNA damage repair, as revealed by COMET analyses.Sparks (BRU011)SPARKs children’s charity, FP6 EURO-laminopathies monies, and y Brunel Progeria Research Fun

'HOXA9' as a risk factor in Acute Myeloid Leukaemia

Acute myeloid leukaemia (AML), also known as acute myelogenous leukaemia is a cancer of the myeloid line of blood cells, characterized by the rapid proliferation of abnormal myeloid cells accumulating in the bone marrow that interfere with normal haematopoiesis. Although AML is a relatively rare disease, which accounts for almost 1% of cancer deaths in the United Kingdom and 1.2% of cancer deaths in the United States, it is the most common acute leukaemia; primarily affecting adults, and its incidence is known to increase with age. To date, there is still very little understood about the risk factors for the development of AML beyond the onset of old age. However, some key pre-existing haematological disorders such as myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN) are now known to have a relatively high incidence of developing AML (15-52% and 7% respectively). In recent years, there have been great strides made towards understanding some of the major genetic changes that occur in AML. Normal haematopoiesis is strictly regulated by the HOX family of transcription factors which have been shown to play a key role in the development of cancer when elevated in the normal peripheral blood mononuclear cells (PBMCs) of the elderly. Together with its known role as an oncogene in AML, and a predictor of survival in this disease, it is suggested that HOXA9 expression in PBMCs could be a predictive marker of non-therapy related AML. This study suggests that elevated HOXA9 expression is indeed a predictive marker for AML development and attempts to shine a light on the progression of AML from pre-leukemic diseases such as MDS and MPN as well as offering a possible solution which may be used as a therapeutic drug either alone or in combination with current commonly used therapeutics

Beyond DNA sequencing : integrative approaches to resolving selected higher and lower taxonomic problems in Afrotropical Chiroptera.

Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.Of the approximate 300 currently recognised bat species known from the Afrotropics, very few have been studied in sufficient detail to a) provide accurate species and distributional limits for extant taxa, b) identify possible cryptic species, and c) ascertain the closest sister lineage of numerous taxonomic groups. For those species where DNA-based phylogenies are available, the use of additional taxonomic markers and methods has provided further insights into the evolutionary history of certain extant chiropteran groups. This work comprises a series of systematic studies of African and Malagasy Chiroptera aimed at investigating sequence-based evolutionary hypotheses of higher and lower level taxa using comparative molecular cytogenetic and morphometric techniques. Efforts were directed at resolving taxonomic inconsistencies of chiropteran taxa from the African subregion and/or Madagascar, for which there is a general paucity of comprehensive and/or resolved phylogenies. Taxa belonging to the families Pteropodidae, Hipposideridae, Myzopodidae, and Molossidae were chosen for study because molecular-based have failed to provide consensus regarding evolutionary relationships amongst the above-mentioned taxonomic groups, or are in stark contrast to phylogenies based on morphological data. In addition, molecular cytogenetics and geometric morphometric approaches were used because they have had been applied in few evolutionary studies of Afrotropical bats. With the exception of a few karyotypic descriptions, scant data are available that details the chromosomal diversity and karyotypic evolution of bats from Madagascar in relation to their conspecifics or congenerics on other continents. To understand better the mechanisms that may have structured the karyotypes of extant Malagasy Chiroptera and the utility of chromosomal characters in retracing their evolutionary history, eight species from seven families were analysed using G- and C-banding and chromosome painting. Robertsonian (Rb) fusions and fissions were the dominant mode of genome restructuring amongst taxa and, for the most part, proved useful characters for investigations of phylogenomic relationships amongst families and genera. Chromosomal data generated from painting studies employing Myotis myotis (MMY) chromosomal probes, produced phylogenetically important characters that supported two conflicting hypotheses regarding the evolutionary affinities of the Myzopodidae, a family of bats endemic to Madagascar. The Rb fusion MMY 9+11 detected in Myzopodidae, also common to Phyllostomidae, could suggest a close association of Myzopoda aurita with the superfamily Noctilionoidea. However, the Rb fusion MMY 3+4 that is also present in vesper bats, suggests closer evolutionary ties between M. aurita and the Vespertilionoidea. A sex-autosome translocation, a cytogenetic character previously confined to phyllostomid and vespertilionid bats, was also detected in M. aurita casting further uncertainties on the evolutionary origins of this deep-branching species. This study highlighted the need for more refined cytogenetic investigations based on human-derived chromosomal paints and the application of highresolution bacterial artificial chromosomal (BACs) probes to map intrachromosomal breakpoints and/or subchromosomal rearrangements in the genome of Myzopoda. Heterochromatic polymorphisms and inversions appear to be important mechanisms of karyotypic evolution amongst pteropodid genera. Painting data revealed that at least five structural arrangements might be linked to the evolutionary divergence of pteropodine and rousettine fruit bats. A cryptic pericentric inversion was detected in the genome of Pteropus rufus corresponding to the homologue of MMY 4+19 (equivalent to HSA3+21); an ancestral syntenic character proposed for eutherian mammals. Proposed synapomorphies of the rousettine clade, as defined by molecular DNA studies, include the derived state of the MMY 4+19 homologue and the non-centric fusion of MMY 16/17+24 homologue. Integration of painting data on Hipposideros commersoni with published comparative maps of other hipposiderids enabled a brief revision of the postulated ancestral hipposiderid chromosomal complement. These data disputed previously proposed chromosomal synapomorphies of Hipposideridae and supported the basal position of H. commersoni within the genus. The inclusion of other hipposiderid genera, in particular Malagasy Paratriaenops and southern African Cloeotis, in chromosome painting studies may allow for further inferences regarding the evolutionary history of this diverse family. Morphometric approaches were employed to resolve uncertainties concerning species-level relationships within Afrotropical Otomops. Multivariate analyses delineated three well-supported morphological groups that corresponded to recently described genetic lineages and revealed several species-specific morphological traits for taxonomic diagnoses. Otomops from Djibouti, Ethiopia, Kenya, and Yemen constitute an undescribed morphologically and genetically cohesive group that requires a formal taxonomic description. Understanding the ecological and possible physiological adaptive value of morphological variation can provide valuable insights into the evolutionary history of this Afrotropical species complex. This work has provided further insights into the systematics of certain Afrotropical Chiroptera through the use of molecular cytogenetic and geometric morphometric techniques. Specifically, it has facilitated the interpretation of ancestral, independent and convergent chromosomal characters in the evolution of Afrotropical taxa belonging to the families Pteropodidae, Hipposideridae, and Myzopodidae, and has also elucidated lineage-specific morphological attributes in members of the genus Otomops thereby advancing our understanding of chiropteran diversity within the region

Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common type of leukemia. The Cancer Genome Atlas Research Network has demonstrated the increasing genomic complexity of acute myeloid leukemia (AML). In addition, the network has facilitated our understanding of the molecular events leading to this deadly form of malignancy for which the prognosis has not improved over past decades. AML is a highly heterogeneous disease, and cytogenetics and molecular analysis of the various chromosome aberrations including deletions, duplications, aneuploidy, balanced reciprocal translocations and fusion of transcription factor genes and tyrosine kinases has led to better understanding and identification of subgroups of AML with different prognoses. Furthermore, molecular classification based on mRNA expression profiling has facilitated identification of novel subclasses and defined high-, poor-risk AML based on specific molecular signatures. However, despite increased understanding of AML genetics, the outcome for AML patients whose number is likely to rise as the population ages, has not changed significantly. Until it does, further investigation of the genomic complexity of the disease and advances in drug development are needed. In this review, leading AML clinicians and research investigators provide an up-to-date understanding of the molecular biology of the disease addressing advances in diagnosis, classification, prognostication and therapeutic strategies that may have significant promise and impact on overall patient survival

Nuclear architecture explored by live-cell fluorescence microscopy using laser and ion microbeam irradiation.

Nuclear architecture is a biological field of research that studies the spatio-temporal organization of the components within cell nuclei. Since nuclei are the organelles that harbor the genome and epigenome, they are the place where most of the genetic processes like replication, transcription, splicing, gene-regulation, DNA repair, re- combination etc. are carried out. In the presented doctoral thesis modern 4D live-cell microscopy in combination with laser or ion microbeam irradiation (to label or damage chromatin, respectively) was used to study nuclear architecture in living cells over extended periods of time at the single cell level. The results presented in this thesis can be partitioned into three main parts: (a) chromatin dynamics in cycling cells, (b) adaptation of the ion micro beam facil- ity SNAKE to the needs of live-cell observation (including first experiments) and (c) exploring spatio-temporal dynamics of DNA repair proteins after laser micro ir- radiation. (A) Chromatin dynamics in cycling cells Distribution of interphase chromosomes within cell nuclei has been found to be non- random with respect to gene density and chromosome size. Changes in nuclear orga- nization have been reported in several disorders and diseases. To which extent relative chromosome positioning is conserved through mitosis in cycling cells and whether certain chromatin domains are able change their relative position dramatically in the interphase nucleus has been the subject of various mechanistic models and contro- versial discussions. In 1909 German biologist theodor Boveri was the first one to comment on this topic in his publication: “Die Blastomerenkerne von Ascaris mega- locephala und die Theorie der Chromosomenindividualität” (included as an appendix to this thesis). In order to test Boveri’s hypotheses, 4D live-cell observations were carried out on a modern spinning disc confocal microscope using a human cell line that possesses photoactivatable chromatin. In experiments that used photoactivation and photobleaching of chromatin, it could be demonstrated that – as stated by Boveri – chromatin proximity relationships are in general not conserved through mitosis but destroyed during early prometaphase by the mechanics of mitosis. Other experiments showed that nuclear rotations in a conveyer-belt-like manner are able to bring initially distant chromatin domains into close proximity in a matter of a few minutes. (B) Adaptation of the SNAKE micro beam facility to the needs of live- cell microscopy (including first experiments) Since ordinary irradiation sources lack the ability to perform targeted micro irradia- tion at the micrometer scale and laser micro irradiation produces an artificial mix of various DNA damages, the ion microbeam SNAKE represents an interesting tool to explore the dynamics of repair proteins in a spatio-temporal context. In the course of a collaboration project the ion microbeam was adapted to the needs of long-term live-cell microscopy. These adaptations and first live-cell experiments performed at the refurbished ion micro beam are described in this part of the results. (C) Exploring spatio-temporal dynamics of DNA repair proteins after laser micro irradiation. Mutation of genetic information can cause serious harm to a cell or even a whole or- ganism. DNA repair serves to protect and clean the genome from undirected poten- tially hazardous changes. Compared to the wealth of information which is available about DNA repair at the molecular level only little attention has been payed to it in context of nuclear architecture. In the last part of the results cells stably expressing GFP tagged versions of the repair proteins MDC1, Rad52 and 53BP1 were damaged by laser micro irradiation and imaged over extended periods of time. It could be de- monstrated that at the used damage induction conditions most of the cells show only minor changes with respect to localization of damage signals, kinetochores and nu- cleoli pattern over time. Furthermore, disappearance of spontaneous 53BP1-GFP foci in favor of protein recruitment to damaged chromatin and mutual exclusion between kinetochore signals and Rad52-GFP damage foci could be observed. In a few U2OS Rad52-GFP nuclei DNA damage foci disappeared simultaneously after a dramatic phase in which the total number of foci drastically increased – even adjacent to the laser damaged chromatin