Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis.

BACKGROUND: Cryptococcal meningitis (CM) causes an estimated 180,000 deaths annually, predominantly in sub-Saharan Africa, where most patients receive fluconazole (FLC) monotherapy. While relapse after FLC monotherapy with resistant strains is frequently observed, the mechanisms and impact of emergence of FLC resistance in human CM are poorly understood. Heteroresistance (HetR) - a resistant subpopulation within a susceptible strain - is a recently described phenomenon in Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg), the significance of which has not previously been studied in humans. METHODS: A cohort of 20 patients with HIV-associated CM in Tanzania was prospectively observed during therapy with either FLC monotherapy or in combination with flucytosine (5FC). Total and resistant subpopulations of Cryptococcus spp. were quantified directly from patient cerebrospinal fluid (CSF). Stored isolates underwent whole genome sequencing and phenotypic characterization. RESULTS: Heteroresistance was detectable in Cryptococcus spp. in the CSF of all patients at baseline (i.e., prior to initiation of therapy). During FLC monotherapy, the proportion of resistant colonies in the CSF increased during the first 2 weeks of treatment. In contrast, no resistant subpopulation was detectable in CSF by day 14 in those receiving a combination of FLC and 5FC. Genomic analysis revealed high rates of aneuploidy in heteroresistant colonies as well as in relapse isolates, with chromosome 1 (Chr1) disomy predominating. This is apparently due to the presence on Chr1 of ERG11, which is the FLC drug target, and AFR1, which encodes a drug efflux pump. In vitro efflux levels positively correlated with the level of heteroresistance. CONCLUSION: Our findings demonstrate for what we believe is the first time the presence and emergence of aneuploidy-driven FLC heteroresistance in human CM, association of efflux levels with heteroresistance, and the successful suppression of heteroresistance with 5FC/FLC combination therapy. FUNDING: This work was supported by the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377/Z/11/Z and the Daniel Turnberg Travel Fellowship

T-loop phosphorylation of Arabidopsis CDKA;1 is required for its function and can be partially substituted by an aspartate residue

As in other eukaryotes, progression through the cell cycle in plants is governed by cyclin-dependent kinases. Phosphorylation of a canonical Thr residue in the T-loop of the kinases is required for high enzyme activity in animals and yeast. We show that the Arabidopsis thaliana Cdc21/Cdc28 homolog CDKA; 1 is also phosphorylated in the T-loop and that phosphorylation at the conserved Thr-161 residue is essential for its function. A phospho-mimicry T161D substitution restored the primary defect of cdka; 1 mutants, and although the T161D substitution displayed a dramatically reduced kinase activity with a compromised ability to bind substrates, homozygous mutant plants were recovered. The rescue by the T161D substitution, however, was not complete, and the resulting plants displayed various developmental abnormalities. For instance, even though flowers were formed, these plants were completely sterile as a result of a failure of the meiotic program, indicating that different requirements for CDKA; 1 function are needed during plant development

Uniparental Genetic Heritage of Belarusians: Encounter of Rare Middle Eastern Matrilineages with a Central European Mitochondrial DNA Pool

Ethnic Belarusians make up more than 80% of the nine and half million people inhabiting the Republic of Belarus. Belarusians together with Ukrainians and Russians represent the East Slavic linguistic group, largest both in numbers and territory, inhabiting East Europe alongside Baltic-, Finno-Permic- and Turkic-speaking people. Till date, only a limited number of low resolution genetic studies have been performed on this population. Therefore, with the phylogeographic analysis of 565 Y-chromosomes and 267 mitochondrial DNAs from six well covered geographic sub-regions of Belarus we strove to complement the existing genetic profile of eastern Europeans. Our results reveal that around 80% of the paternal Belarusian gene pool is composed of R1a, I2a and N1c Y-chromosome haplogroups – a profile which is very similar to the two other eastern European populations – Ukrainians and Russians. The maternal Belarusian gene pool encompasses a full range of West Eurasian haplogroups and agrees well with the genetic structure of central-east European populations. Our data attest that latitudinal gradients characterize the variation of the uniparentally transmitted gene pools of modern Belarusians. In particular, the Y-chromosome reflects movements of people in central-east Europe, starting probably as early as the beginning of the Holocene. Furthermore, the matrilineal legacy of Belarusians retains two rare mitochondrial DNA haplogroups, N1a3 and N3, whose phylogeographies were explored in detail after de novo sequencing of 20 and 13 complete mitogenomes, respectively, from all over Eurasia. Our phylogeographic analyses reveal that two mitochondrial DNA lineages, N3 and N1a3, both of Middle Eastern origin, might mark distinct events of matrilineal gene flow to Europe: during the mid-Holocene period and around the Pleistocene-Holocene transition, respectively

Rapid PCR assay for detecting common genetic variants arising in human pluripotent stem cell cultures

Human pluripotent stem cells (hPSCs) are prone to acquiring genetic changes upon prolonged culture. Particularly common are copy number changes, including gains of chromosomes 1q, 12p, 17q, and 20q, and/or loss of chromosomes 10p and 18q. The variant cells harboring common genetic changes display altered behaviors compared to their diploid counterparts, thus potentially impacting upon the validity of experimental results and safety of hPSC-derived cellular therapies. Hence, a critical quality attribute in hPSC maintenance should include frequent monitoring for genetic changes arising in cultures. This in turn places large demands on the genotyping assays for detection of genetic changes. Traditional methods for screening cells entail specialized cytogenetic analyses, but their high costs and a lengthy turnaround time make them impractical for high-throughput analyses and routine laboratory use. Here, we detail a protocol for a rapid, accessible, and affordable PCR-based method for detection of frequently occurring copy number changes in hPSCs

Genome-wide analysis reveals extensive functional interaction between DNA replication initiation and transcription in the genome of trypanosoma brucei

Identification of replication initiation sites, termed origins, is a crucial step in understanding genome transmission in any organism. Transcription of the Trypanosoma brucei genome is highly unusual, with each chromosome comprising a few discrete transcription units. To understand how DNA replication occurs in the context of such organization, we have performed genome-wide mapping of the binding sites of the replication initiator ORC1/CDC6 and have identified replication origins, revealing that both localize to the boundaries of the transcription units. A remarkably small number of active origins is seen, whose spacing is greater than in any other eukaryote. We show that replication and transcription in T. brucei have a profound functional overlap, as reducing ORC1/CDC6 levels leads to genome-wide increases in mRNA levels arising from the boundaries of the transcription units. In addition, ORC1/CDC6 loss causes derepression of silent Variant Surface Glycoprotein genes, which are critical for host immune evasion

Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.

The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes1-7. Stem cells from normal livers have a low mutational burden and limited diversity of signatures8, which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1-5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others-arising from exogenous exposures-were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.This work was supported by a Wellcome Trust and Cancer Research UK (CRUK) Grand Challenge Award (C98/A24032). P.J.C. is a Wellcome Trust Senior Clinical Fellow (WT088340MA); S.F.B. was supported by the Swiss National Science Foundation (P2SKP3-171753 and P400PB-180790); M.A.S. is supported by a Rubicon fellowship from NWO (019.153LW.038); the Cambridge Human Research Tissue Bank is supported by the NIHR Cambridge Biomedical Research Centre; and M.H. is supported by a CRUK Clinician Scientist Fellowship (C52489/A19924)

Systematic Analysis of Molecular and Cellular Dysfunction in Accelerated Aging Phenotypes

Zur Gruppe segmentaler progeroider Erkrankungen gehören genetische Erkrankungen, denen beschleunigte Alterungsprozesse in mehreren Geweben zugrunde liegen. Klinische Merkmale dieser Patienten sind altersassoziierte Pathologien in jungen Jahren wie graue Haare, Lipodystrophie, Osteoporose, grauer Star, Schwerhörigkeit, Arteriosklerose, Diabetes mellitus und Tumore. In den vergangenen Jahren wurden viele krankheitsverursachende Gendefekte für segmentale progeroide Erkrankungen identifiziert. Die betroffenen Proteine sind an Genomstabilität und der Funktion der Mitochondrien beteiligt. Die Identifizierung der zugrundeliegenden genetischen Defekte deckte molekulare und zelluläre Mechanismen auf, die unser Verständnis von Alterungspozessen und altersassoziierten Erkankungen im Allgemeinen erweiterte. Das Ziel meiner Arbeit war, unser Wissen über diese zellulären und molekularen Mechanismen der Alterungsprozesse zu erweitern, indem ich eine einzigartige Sammlung von Fibroblasten und DNA-Proben von Patienten mit verschiedenen progeroiden Erkrankungen untersucht habe. Dazu habe ich eine Methode zur Sequenzierung der mitochondrialen DNA mit sehr hoher Abdeckung etabliert, um niederfrequente mtDNA-Varianten in Patientenproben zu quantifizieren. Durch die Behandlung von Kontrollfibroblasten mit genotoxischen Substanzen konnte ich zeigen, dass dieser Ansatz die Detektion von niederfrequenten Varianten im mitochondrialen Genom möglich macht. Die Analyse einer DNA-Probe eines von Cutis Laxa Typ IC betroffen Patienten, der eine homozygote Mutation im LTBP4 Gen trägt, deckte eine signifikant erhöhte Anzahl an mtDNA-Varianten auf. LTBP4 kodiert ein sekretiertes Protein, das den TGF-beta Signalweg reguliert und das bisher noch nicht mit mitochondrialer Dysfunktion assoziiert war. Dieses Ergebnis weist auf eine Verbindung von LTBP4 und der Integrität des mitochondrialen Genoms hin. VII Weiterhin habe ich eine Real-Time PCR-basierte Methode zur Analyse der Telomerlänge etabliert und damit die Telomerlänge in DNA-Proben von Patienten gemessen, die an segmentalen progeroiden Erkrankungen leiden. So habe ich die erwartete sigmoidale Verteilung der Telomerlänge mit dem Alter und eine hohe Varianz der Telomerlänge in Kontrollproben zeigen können. Die Verkürzung der Telomerlänge in einer Probe eines Bloom-Syndrom-Patienten und einer Probe eines Patienten mit Cutis Laxa Typ 1C kommt der Signifikanz nahe. Um die Verringerung der Telomerlänge detaillierter charakterisieren zu können, habe ich bei der Optimierung einer Telomer-qFISH-Methode kollaboriert und diese Methode dann eingesetzt, um die Telomerlänge in drei Fibroblasten von Bloom-Syndrom-Patienten zu messen. Erstaunlicherweise war in einer Patientenprobe die Signalintensität, die der Telomerlänge entspricht, und die Anzahl der Telomersignale erhöht. In dieser Patientenprobe konnte eine Chromosomenaberration und ein verzögerter Zellzyklus mit einer erhöhten Anzahl an Zellen in der G2/M-Phase nachgewiesen werden. Dies deutet darauf hin, dass der doppelte Chromosomensatz die Messung beeinträchtigte. Mit einem Southernblot konnte ich bestätigen, dass die Telomerlänge in diesen Patientenproben nicht verändert war. Des Weiteren habe ich die Anreicherung von DNA-Schäden und die DNA-Reparatur in Patientenfibroblasten mittels der Quantifizierung der γH2AX Foci nach Bestrahlung etabliert und analysiert. So konnte ich eine erhöhte Anzal von γH2AX Foci in zwei unbehandelten Fibroblastenproben mit Mutationen in den Genen GORAB und SLC25A24 identifizieren, was auf eine höhrere Anfälligkeit für DNA-Schäden oder eine Schwäche der DNA-Reparatur hinweist. Des Weiteren hat die Bestrahlung signifikant mehr γH2AX Foci in zwei Patientenfibroblasten hervorgerufen, die Mutationen in den Genen PYCR1 und GORAB tragen, was ebenfalls auf eine Beeinträchtigung der DNA-Reparatur in diesen Zellen hindeutet. Zusammengefasst habe ich in meiner Arbeit neue Methoden zur Quantifizierung von altersassoziierten zellulären Prozessen etabliert und diese Methoden genutzt, um Fibroblasten und DNA-Proben von Patienten mit segmentalen progeroiden Erkrankungen zu analysieren. So konnte ich neue Erkenntnisse zu den beteiligten Pathomechanismen gewinnen, indem ich eine Verbindung von LTBP4 mit der Integrität des mitochondrialen Genoms und einen potentiellen Einfluss von BLM und PYCR1 auf die Telomerlänge identifiziert habe.Accelerated aging in multiple tissues is the connecting characteristic of the group of genetic disorders called segmental progeroid syndromes. At young ages, affected patients show many clinical features of aging-associated pathologies such as hair graying, lipodystrophy, osteoporosis, cataracts, hearing loss, arteriosclerosis, diabetes mellitus, and malignancies. Several disease-causing genes for segmental progeroid syndromes have been identified within the last years, and the affected proteins are often involved in genome maintenance or mitochondrial function. Identifying these underlying genetic alterations revealed molecular and cellular mechanisms involved in the pathology of these diseases and furthered our understanding of aging processes and aging-associated diseases in general. The aim of my thesis was to expand our knowledge of the cellular and molecular mechanisms of aging using a unique collection of fibroblast and DNA samples of patients suffering from a large variety of different progeroid syndromes. I established an ultra-high coverage mtDNA sequencing approach to detect and quantify low-frequency mtDNA variants in patient samples. By treatment of control fibroblasts with genotoxic agents, I could show that this approach allows the detection of low-frequency variants in the mitochondrial genome. Analysis of a DNA sample of a patient suffering from Cutis laxa type IC and carrying a homozygous mutation in LTBP4 revealed a significantly increased number of mtDNA variants. LTBP4 encodes a secreted protein that regulates TGF-beta signaling and has previously not been associated with mitochondrial dysfunction. Therefore, these results indicate for the first time a link between LTBP4 and the integrity of the mitochondrial genome. Additionally, I established a real-time PCR-based method in order to analyze telomere length in DNA samples of patients suffering from progeroid syndromes. I found the anticipated sigmoidal distribution of telomere length by age as well as a high variance of telomere length in the control samples. The decrease of telomere length in two patient samples, a Bloom syndrome patient and a patient suffering from Cutis laxa type IIB, approached significance. V To characterize telomere attrition in more detail, I collaborated on the optimization of a telomere qFISH method, which I then used to measure telomere length in three fibroblast samples of Bloom syndrome patients. Strikingly, the telomere signal intensity corresponding to telomere length as well as the number of telomere signals were increased in one patient sample. A chromosomal aberration and a delayed cell cycle progression with an increased amount of cells in the G2/M phase were then detected in these fibroblasts, indicating that to the double set of chromosomes interfered with the measurement. A Southern blot confirmed that the telomere length in these patient samples was not different from the controls. Further, I analyzed the accumulation of DNA damage and induction of the DNA damage response in patient fibroblasts using the quantification of γH2AX foci upon treatment with genotoxic reagents. I found an elevated level of γH2AX foci in two untreated fibroblast samples carrying mutations in GORAB and SLC25A24 indicative of either higher susceptibility of these cells to DNA damage or deficiencies in DNA damage repair processes. Further irradiation treatment caused a significantly elevated level of γH2AX in two fibroblast samples carrying mutations in the PYCR1 and GORAB genes suggestive of DNA damage repair impairment in these cells. In summary, the results of my Ph.D. thesis help to establish new methods for the analysis and quantification of aging-associated cellular processes. Using these methods on cells and DNA samples of patients with segmental progeroid syndromes, I could provide new insights into the involved pathomechanisms by identifying a link between LTBP4 and mitochondrial DNA as well as a possible influence of BLM and PYCR1 on telomere length.2021-09-3

Somatic Copy Number Alteration detection and Copy Number signature analysis in High-Grade Serous Ovarian Cancer

Somatic copy number alterations (sCNAs) are a type of genomic variation that affects the dosage of DNA sequences promoting tumorigenesis such as in High grade serous ovarian cancer. Their complexity prevents the unravelling of the mechanisms generating them and the molecular stratification of the patients. Here we propose the implementation of a highly sensitive pipeline for their detection based on structural variant calling and a signature analysis for the extraction of recurrent patterns. The precise calling allowed the recovery of small segments missed from the previous pipeline and in turn the clear distinction of patients with Homologous Recombination Deficiency (HRD), which resulted extremely segmented. Although the signature analysis, based on COSMIC copy number signatures, did not provide results consistent with the clinical data, the de novo signature extraction provided 15 new signatures able to proficiently explain the dataset. Two of them were positively associated with HRD, possibly representing a test for the identification of the HRD phenotype. Further insights on these signatures may provide the discovery of their etiology and give the possibility to shed light on association with single nucleotide variations.Somatic copy number alterations (sCNAs) are a type of genomic variation that affects the dosage of DNA sequences promoting tumorigenesis such as in High grade serous ovarian cancer. Their complexity prevents the unravelling of the mechanisms generating them and the molecular stratification of the patients. Here we propose the implementation of a highly sensitive pipeline for their detection based on structural variant calling and a signature analysis for the extraction of recurrent patterns. The precise calling allowed the recovery of small segments missed from the previous pipeline and in turn the clear distinction of patients with Homologous Recombination Deficiency (HRD), which resulted extremely segmented. Although the signature analysis, based on COSMIC copy number signatures, did not provide results consistent with the clinical data, the de novo signature extraction provided 15 new signatures able to proficiently explain the dataset. Two of them were positively associated with HRD, possibly representing a test for the identification of the HRD phenotype. Further insights on these signatures may provide the discovery of their etiology and give the possibility to shed light on association with single nucleotide variations