108 research outputs found
Mitochondrial dysfunction and dynamics in tumourigenesis
No full textMitochondria are essential in many cancer-related processes, but the link between mitochondrial DNA (mtDNA) dysfunction and cancer remains poorly understood, partly due to a historical focus on the nuclear genome and challenges in mtDNA analysis. Current understanding identifies three main somatic mtDNA alterations in tumours: sequence variations, changes in copy number, and integrations into the nuclear genome (NUMTs). Although these phenomena are increasingly observed across tumour types, small sample sizes and limited clinical data have hindered our understanding of their clinical significance, highlighting the need for more focused research.
In this thesis, we developed an accurate mtDNA variant-calling pipeline to detect both somatic and germline mtDNA variants across heteroplasmies. We applied this, along with established pipelines for mtDNA copy number estimation and NUMT detection, to the largest High-Grade Serous Ovarian Cancer (HGSOC) sequencing cohort to date. Our analysis revealed frequent somatic mtDNA variants and increased copy numbers in HGSOC tumours compared to normal tissue, though no somatic NUMT integrations were detected. Notably, predicted deleterious mtDNA variants were associated with poorer patient prognosis, independent of age, stage and homologous recombination deficiency. This pattern also scaled with heteroplasmy level and was consistent across missense variant effect prediction strategies.
Expanding our analysis to 17,509 tumour samples across various cancer types, we found widespread mtDNA somatic variants, with certain cancers showing higher susceptibility, and significant increases in mtDNA copy number compared to matched normals. Similar survival differences for predicted deleterious mtDNA variants were observed only in a breast tumour cohort. Though survival differences could not be replicated in an independent HGSOC cohort, potentially a result of differing tumour aggressiveness between cohorts.
Finally, we explored the functional impact of predicted deleterious mtDNA variants using matched transcriptomic data from the HGSOC cohort. Our findings suggest that these variants may disrupt oxidative phosphorylation (OXPHOS) and could influence chemotherapy sensitivity. Additionally, we identified mtDNA expression groups weakly associated with patient prognosis, independent of predicted deleterious mtDNA variants.
Overall, this thesis provides strong evidence for the prognostic value of somatic mtDNA variants in HGSOC, underscoring the importance of mitochondrial genetics in cancer research. These findings lay the groundwork for future studies on the mechanisms of predicted deleterious mtDNA variants, with potential implications for improving patient survival and treatment strategies in HGSOC and other cancers
Quantifying single nucleotide variant detection sensitivity in exome sequencing
No full textBACKGROUND: The targeted capture and sequencing of genomic regions has rapidlydemonstrated its utility in genetic studies. Inherent in this technology isconsiderable heterogeneity of target coverage and this is expected tosystematically impact our sensitivity to detect genuine polymorphisms. To fullyinterpret the polymorphisms identified in a genetic study it is often essentialto both detect polymorphisms and to understand where and with what probabilityreal polymorphisms may have been missed.RESULTS: Using down-sampling of 30 deeply sequenced exomes and a set ofgold-standard single nucleotide variant (SNV) genotype calls for each sample, we developed an empirical model relating the read depth at a polymorphic site to theprobability of calling the correct genotype at that site. We find that measuredsensitivity in SNV detection is substantially worse than that predicted from the naive expectation of sampling from a binomial. This calibrated model allows us toproduce single nucleotide resolution SNV sensitivity estimates which can bemerged to give summary sensitivity measures for any arbitrary partition of thetarget sequences (nucleotide, exon, gene, pathway, exome). These metrics aredirectly comparable between platforms and can be combined between samples to give"power estimates" for an entire study. We estimate a local read depth of 13X isrequired to detect the alleles and genotype of a heterozygous SNV 95% of thetime, but only 3X for a homozygous SNV. At a mean on-target read depth of 20X,commonly used for rare disease exome sequencing studies, we predict 5-15% ofheterozygous and 1-4% of homozygous SNVs in the targeted regions will be missed.CONCLUSIONS: Non-reference alleles in the heterozygote state have a high chanceof being missed when commonly applied read coverage thresholds are used despitethe widely held assumption that there is good polymorphism detection at thesecoverage levels. Such alleles are likely to be of functional importance inpopulation based studies of rare diseases, somatic mutations in cancer andexplaining the "missing heritability" of quantitative traits.<br/
Genetic heterogeneity in Cornelia de Lange syndrome (CdLS) and CdLS-like phenotypes with observed and predicted levels of mosaicism
No full textCornelia de Lange syndrome (CdLS) is a multisystem disorder with distinctive facial appearance, intellectual disability and growth failure as prominent features. Most individuals with typical CdLS have de novo heterozygous loss-of-function mutations in NIPBL with mosaic individuals representing a significant proportion. Mutations in other cohesin components, SMC1A, SMC3, HDAC8 and RAD21 cause less typical CdLS
Rapid Evolution of Colistin Resistance in a Bioreactor Model of Infection of Klebsiella pneumoniae
No full textColistin remains an important antibiotic for the therapeutic management of drug-resistant Klebsiella pneumoniae. Despite the numerous reports of colistin resistance in clinical strains, it remains unclear exactly when and how different mutational events arise resulting in reduced colistin susceptibility. Using a bioreactor model of infection, we modelled the emergence of colistin resistance in a susceptible isolate of K. pneumoniae. Genotypic, phenotypic and mathematical analyses of the antibiotic-challenged and un-challenged population indicates that after an initial decline, the population recovers within 24 h due to a small number of “founder cells” which have single point mutations mainly in the regulatory genes encoding crrB and pmrB that when mutated results in up to 100-fold reduction in colistin susceptibility. Our work underlines the rapid development of colistin resistance during treatment or exposure of susceptible K. pneumoniae infections having implications for the use of cationic antimicrobial peptides as a monotherapy.</p
Variant detection sensitivity and biases in whole genome and exome sequencing
No full textBackground: Less than two percent of the human genome is protein coding, yet that small fraction harbours the majority of known disease causing mutations. Despite rapidly falling whole genome sequencing (WGS) costs, much research and increasingly the clinical use of sequence data is likely to remain focused on the protein coding exome. We set out to quantify and understand how WGS compares with the targeted capture and sequencing of the exome (exome-seq), for the specific purpose of identifying single nucleotide polymorphisms (SNPs) in exome targeted regions.Results: We have compared polymorphism detection sensitivity and systematic biases using a set of tissue samples that have been subject to both deep exome and whole genome sequencing. The scoring of detection sensitivity was based on sequence down sampling and reference to a set of gold-standard SNP calls for each sample. Despite evidence of incremental improvements in exome capture technology over time, whole genome sequencing has greater uniformity of sequence read coverage and reduced biases in the detection of non-reference alleles than exome-seq. Exome-seq achieves 95% SNP detection sensitivity at a mean on-target depth of 40 reads, whereas WGS only requires a mean of 14 reads. Known disease causing mutations are not biased towards easy or hard to sequence areas of the genome for either exome-seq or WGS.Conclusions: From an economic perspective, WGS is at parity with exome-seq for variant detection in the targeted coding regions. WGS offers benefits in uniformity of read coverage and more balanced allele ratio calls, both of which can in most cases be offset by deeper exome-seq, with the caveat that some exome-seq targets will never achieve sufficient mapped read depth for variant detection due to technical difficulties or probe failures. As WGS is intrinsically richer data that can provide insight into polymorphisms outside coding regions and reveal genomic rearrangements, it is likely to progressively replace exome-seq for many applications
Abstract 4161: Integrated analysis of whole exome sequencing and hormone receptor expression data in endometrioid ovarian cancer
No full textHeterochromatin delays CRISPR-Cas9 mutagenesis but does not influence repair outcome
No full textAbstractCRISPR-Cas9 genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem. Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of Cas9-induced mutations can be attributed unequivocally to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. Surprisingly, the outcome of mutagenic DNA repair was independent of chromatin state, with similar efficiencies of homology directed repair and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint of CRISPR-Cas9 genome editing, and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.</jats:p
Regionally enriched rare deleterious exonic variants in the UK and Ireland
No full textIt is unclear how patterns of regional genetic differentiation in the UK and Ireland might impact the protein-coding fraction of the genome. We exploit UK Biobank (UKB) and Viking Genes whole exome sequencing data to study regional genetic differentiation across the UK and Ireland in protein coding genes, encompassing 44,696 unrelated individuals from 20 regions of origin. We demonstrate substantial exonic differentiation among Shetlanders, Orcadians, individuals with full or partial Ashkenazi Jewish ancestry and in several mainland regions (particularly north and south Wales, southeast Scotland and Ireland). With stringent filtering criteria, we find 67 regionally enriched (≥5-fold) variants likely to have adverse biomedical consequences in homozygous individuals. Here, we show that regional genetic variation across the UK and Ireland should be considered in the design of genetic studies and may inform effective genetic screening and counselling
Robust Genetic Analysis of the X-Linked Anophthalmic (Ie) Mouse
No full textAnophthalmia (missing eye) describes a failure of early embryonic ocular development. Mutations in a relatively small set of genes account for 75% of bilateral anophthalmia cases, yet 25% of families currently are left without a molecular diagnosis. Here, we report our experimental work that aimed to uncover the developmental and genetic basis of the anophthalmia characterising the X-linked Ie (eye-ear reduction) X-ray-induced allele in mouse that was first identified in 1947. Histological analysis of the embryonic phenotype showed failure of normal eye development after the optic vesicle stage with particularly severe malformation of the ventral retina. Linkage analysis mapped this mutation to a ~6 Mb region on the X chromosome. Short- and long-read whole-genome sequencing (WGS) of affected and unaffected male littermates confirmed the Ie linkage but identified no plausible causative variants or structural rearrangements. These analyses did reduce the critical candidate interval and revealed evidence of multiple variants within the ancestral DNA, although none were found that altered coding sequences or that were unique to Ie. To investigate early embryonic events at a genetic level, we then generated mouse ES cells derived from male Ie embryos and wild type littermates. RNA-seq and accessible chromatin sequencing (ATAC-seq) data generated from cultured optic vesicle organoids did not reveal any large differences in gene expression or accessibility of putative cis-regulatory elements between Ie and wild type. However, an unbiased TF-footprinting analysis of accessible chromatin regions did provide evidence of a genome-wide reduction in binding of transcription factors associated with ventral eye development in Ie, and evidence of an increase in binding of the Zic-family of transcription factors, including Zic3, which is located within the Ie-refined critical interval. We conclude that the refined Ie critical region at chrX: 56,145,000–58,385,000 contains multiple genetic variants that may be linked to altered cis regulation but does not contain a convincing causative mutation. Changes in the binding of key transcription factors to chromatin causing altered gene expression during development, possibly through a subtle mis-regulation of Zic3, presents a plausible cause for the anophthalmia phenotype observed in Ie, but further work is required to determine the precise causative allele and its genetic mechanism
Loss of ALDH18A1 function is associated with a cellular lipid droplet phenotype suggesting a link between autosomal recessive cutis laxa type 3A and Warburg Micro syndrome
No full textAutosomal recessive cutis laxa type 3A is caused by mutations in ALDH18A1, a gene encoding the mitochondrial enzyme ∆1-pyrroline-5-carboxylate synthase (P5CS). It is a rare disorder with only six pathogenic mutations and 10 affected individuals from five families previously described in the literature. Here we report the identification of novel compound heterozygous missense mutations in two affected siblings from a Lebanese family by whole-exome sequencing. The mutations alter a conserved C-terminal domain of the encoded protein and reduce protein stability as determined through Western blot analysis of patient fibroblasts. Patient fibroblasts exhibit a lipid droplet phenotype similar to that recently reported in Warburg Micro syndrome, a disorder with similar features but hitherto unrelated cellular etiology
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