Transposon Extermination Reveals Their Adaptive Fitness Contribution

Transposable Elements are molecular parasites that persist in their host genome by generating new copies to outpace natural selection. Here we measure the parameters governing the copy number dynamics of the fission yeast Tf2 retrotransposons, using experimental and natural populations and a strain where all Tf2 copies are removed. Natural population genomes display active and persistent Tf2 colonies, but in the absence of selection mitotic recombination deletes Tf2 elements at rates that far exceed transposition. We show that Tf2 elements provide a fitness contribution to their host by dynamically rewiring the transcriptional response to metabolic stress. Therefore, Tf2 elements exhibit a mutualistic rather than parasitic behavior toward their host.

Clonal evolution in serially passaged Cryptococcus neoformans x deneoformans hybrids reveals a heterogenous landscape of mitotic recombination

Cryptococcus neoformans x deneoformans hybrids (also known as serotype AD hybrids) are basidiomycete yeasts that are common in a clinical setting. Like many hybrids, the AD hybrids are largely locked at the F1 stage and are mostly unable to undergo normal meiotic reproduction. However, these F1 hybrids, which display a high (∼10%) sequence divergence are known to genetically diversify through mitotic recombination and aneuploidy, and this diversification may be adaptive. In this study, we evolved a single AD hybrid genotype to six diverse environments by serial passaging and then used genome resequencing of evolved clones to determine evolutionary mechanisms of adaptation. The evolved clones generally increased fitness after passaging, accompanied by an average of 3.3 point mutations and 2.9 loss of heterozygosity (LOH) events per clone. LOH occurred through nondisjunction of chromosomes, crossing over consistent with break-induced replication, and gene conversion, in that order of prevalence. The breakpoints of these recombination events were significantly associated with regions of the genome with lower sequence divergence between the parents and clustered in subtelomeric regions, notably in regions that had undergone introgression between the two parental species. Parallel evolution was observed, particularly through repeated homozygosity via nondisjunction, yet there was little evidence of environment-specific parallel change for either LOH, aneuploidy, or mutations. These data show that AD hybrids have both a remarkable genomic plasticity and yet are challenged in the ability to recombine through sequence divergence and chromosomal rearrangements, a scenario likely limiting the precision of adaptive evolution to novel environments.

Mitotic interhomolog recombination drives genomic diversity in diatoms

Diatoms, an evolutionarily successful group of microalgae, display high levels of intraspecific variability in natural populations. However, the process generating such diversity is unknown. Here we estimated the variability within a natural diatom population and subsequently mapped the genomic changes arising within cultures clonally propagated from single diatom cells. We demonstrate that genome rearrangements and mitotic recombination between homologous chromosomes underlie clonal variability, resulting in haplotype diversity accompanied by the appearance of novel protein variants and loss of heterozygosity resulting in the fixation of alleles. The frequency of interhomolog mitotic recombination exceeds 4 out of 100 cell divisions and increases under environmental stress. We propose that this plastic response in the interhomolog mitotic recombination rate increases the evolutionary potential of diatoms, contributing to their ecological success.One Sentence SummaryRecombination between homologous chromosomes in diatom vegetative cells leads to extensive genomic diversity in clonal populations.

NF1 Tumor Suppressor Gene Inactivation in Juvenile Myelomonocytic Leukemia: New Genetic Evidence and Consequences for Diagnostic Work-up

Neurofibromatosis type 1 (NF-1) predisposes to juvenile myelomonocytic leukemia (JMML) via loss of function of the NF1 tumor suppressor gene and consecutive deregulation of Ras signal transduction. Affected individuals usually carry one defective NF1 allele in the germline; somatic inactivation of the second NF1 allele in hematopoietic cells is associated with transformation to leukemia. We previously demonstrated that a major mechanism for biallelic loss of NF1 function in patients with JMML/NF-1 is mitotic recombination leading to uniparental disomy (UPD) of the 17q chromosome arm (Flotho, 2007; Steinemann, 2010). Using contemporary resequencing and microarray technology, we have now revisited the genetics of NF1 inactivation in JMML. Specifically, we addressed two questions: 1) Are genetic findings in leukemic cells of JMML/NF-1 patients consistent with the clinical diagnosis and the two-hit concept? 2) Does the quintuple-negative (QN) group of JMML (patients without clinical evidence of NF-1 and negative for mutations in PTPN11, KRAS, NRAS, or CBL) contain unrecognized cases likely driven by NF1? We investigated 156 children with JMML registered in studies EWOG-MDS 98 or 2006 and tested for mutations in PTPN11, KRAS, NRAS, and CBL. Twenty-five children (16%) were clinically diagnosed as NF-1 based on >=6 café-au-lait spots (CALS) or family history plus CALS. Family history was positive in 9 JMML/NF-1 patients; >=6, 4, and 1 CALS were described in 23, 1, and 1 patients, respectively. The median age at diagnosis of JMML in the NF-1 group was 35.9 months (range, 4.2 to 71.4). Sixteen children (10%) were grouped as JMML-QN. Granulocyte DNA from bone marrow or peripheral blood collected at time of diagnosis was used for next-generation sequencing of the entire NF1 coding sequence (custom Ampliseq enrichment and Miseq, Illumina). Pathogenicity of NF1 variants was assessed according to ACMG criteria. Affymetrix Cytoscan HD array analysis was applied to detect segmental deletions or copy number-neutral loss of heterozygosity (LOH). Among 25 JMML/NF-1 cases, 8 exhibited an NF1 loss-of-function mutation at near-100% variant allelic frequency (VAF) in combination with UPD involving almost the entire 17q arm, suggesting single mitotic recombination as the leukemic driver. One case had an NF1 mutation at near-100% VAF and segmental 17q UPD, suggesting the unusual occurrence of double mitotic recombination. Nine cases carried two independent pathogenic NF1 mutations at near-50% VAF each; here, germline and somatic events could not be distinguished due to unavailability of non-hematopoietic or parental DNA. Four cases exhibited an NF1 microdeletion in combination with a pathogenic NF1 mutation at near-100% VAF; non-hematopoietic tissue available in 2 of these 4 cases failed to display the mutation, indicating the microdeletion as the constitutional event. A deleterious mutation at 71% VAF but no LOH was revealed in one sample. Only monoallelic evidence of NF1 deficiency was found in 2 cases. In the JMML-QN group, 9/16 cases had previously unrecognized NF1 alterations. Compound-heterozygous pathogenic NF1 mutations were found in 2 and homozygous pathogenic NF1 mutations combined with focal LOH in 3 patients, strongly suggesting NF1 as the JMML driver. In the absence of clinical NF-1 features, the findings in these 5 children may be explained by postzygotic NF1 mosaicism, onset of JMML before clinical manifestation of NF-1, or double somatic NF1 hits in the hematopoietic lineage. Four cases carried monoallelic pathogenic or likely pathogenic NF1 mutations with VAF at ~50% or less, providing inconclusive evidence of driver function as it is also possible that these are secondary hits in a myeloproliferative neoplasm (MPN) driven by an unidentified event. There was no genetic evidence of NF1 involvement in the remaining 7 JMML-QN cases. Several important conclusions can be drawn from our study: The clinical diagnosis is reliable in children with JMML/NF-1; we propose that it can be made on the basis of CALS and JMML alone as the patients are generally too young to display the conventional spectrum of NF-1 features. Children without features of NF-1 should only be assigned to JMML-QN after genetic work-up of NF1 because this will unmask involvement of NF1 in a significant number of cases. In suspected JMML-QN without identifiable NF1 lesion, other forms of MPN should be considered. Disclosures Locatelli: Jazz Pharmaceeutical: Speakers Bureau; Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Niemeyer:Celgene: Consultancy; Novartis: Consultancy.

Genome-wide mapping of spontaneous genetic alterations in diploid yeast cells

Genomic alterations including single-base mutations, deletions and duplications, translocations, mitotic recombination events, and chromosome aneuploidy generate genetic diversity. We examined the rates of all of these genetic changes in a diploid strain ofSaccharomyces cerevisiaeby whole-genome sequencing of many independent isolates (n= 93) subcloned about 100 times in unstressed growth conditions. The most common alterations were point mutations and small (<100 bp) insertion/deletions (n= 1,337) and mitotic recombination events (n= 1,215). The diploid cells of most eukaryotes are heterozygous for many single-nucleotide polymorphisms (SNPs). During mitotic cell divisions, recombination can produce derivatives of these cells that have become homozygous for the polymorphisms, termed loss-of-heterozygosity (LOH) events. LOH events can change the phenotype of the cells and contribute to tumor formation in humans. We observed two types of LOH events: interstitial events (conversions) resulting in a short LOH tract (usually less than 15 kb) and terminal events (mostly cross-overs) in which the LOH tract extends to the end of the chromosome. These two types of LOH events had different distributions, suggesting that they may have initiated by different mechanisms. Based on our results, we present a method of calculating the probability of an LOH event for individual SNPs located throughout the genome. We also identified several hotspots for chromosomal rearrangements (large deletions and duplications). Our results provide insights into the relative importance of different types of genetic alterations produced during vegetative growth.