The source and fate of mitochondrial DNA mutations using high-sensitivity next-generation sequencing technologies

Pathogenic mutations in mitochondrial DNA (mtDNA) are known to cause numerous inherited diseases. However, the lack of methods to transgenically manipulate the mtDNA limits the possibilities to learn about mtDNA sequence and function. The mtDNA mutator mouse is used as a saturation mutagenesis model to generate high variant load into mtDNA. With this model, it has been previously shown that, for instance OriL is essential for mtDNA replication or that strong purifying selection of potentially deleterious mtDNA mutations takes place in the germ line. Traditionally, mtDNA mutations have been detected by Sanger sequencing or post-PCR cloning and sequencing, which are unable to represent the entire mtDNA genome, and are laborious, expensive, or of low sensitivity. More recently, high-throughput sequencing methods have been utilized as cheaper approaches to detect mtDNA variants over the entire genome. However, the high error-rate of these technologies is considered as a limiting factor regarding variant detection sensitivity. On the other hand, high-sensitivity high-throughput sequencing methods, such as Duplex Sequencing, are often laborious requiring extensive optimization and high sequencing depth, ultimately raising the costs to a prohibitive level. In this thesis, various mitochondria enrichment and amplification methods are explored in order to enrich mtDNA free from nuclear DNA contamination. Standard Illumina HiSeq sequencing is utilized and data analysis steps are carefully optimized to be suitable for mtDNA genome, which has characteristics very different from the nuclear genome. Finally, the optimized mtDNA enrichment and sequencing protocol, mtDNA-seq, is validated utilizing a titration of spike-in samples harboring known mtDNA variants. With mtDNA-seq it is possible to detect mtDNA variants reliably even below allele frequency of 0.05 %, which is approximately ten times lower variant detection threshold than what has been generally applied in other studies. The optimized mtDNA-seq is applied to address open mitochondrial biology research questions. The variant profile of the entire mtDNA genome is generated, and several complete mutational coldspots are discovered at the control region of the mtDNA. These novel coldspots are hypothesized to be potential regulation sites for mtDNA replication and replication-associated transcription by as-yet-unknown molecular mechanisms. To clarify the developmental stage and mechanism of purifying selection, hemizygote mtDNA mutator mouse is utilized to isolate mtDNA variants into female lineages. As it is possible to detect extremely rare mtDNA variants by mtDNA‑seq, these new results expand the previous study showing strong purifying selection by N2 generation of mice. The results suggest that by chance any mtDNA variant may be transmitted to the offspring, however, the most deleterious mutations do not seem to clonally expand even in N1 generation mice. To understand the mtRNA processing, amplicon sequencing approach is utilized in a preliminary study. The aim in this study is to detect allelic mismatches between mtDNA and mtRNA variants, which potentially indicate mtRNA processing defects

Analysis of the pattern and trend of human genomic variations in the form of single nucleotide polymorphisms (SNPs) and small insertions and deletions (INDELs)

Single nucleotide polymorphisms (SNPs) and small insertions/deletions (INDELs) are the most common genetic variations in the human genome. They have been shown to associate with phenotype variation including genetic disease. Based on data in a recent version of the NCBI dbSNP database (Build 150), there are 305,651,992 SNPs and 19,177,943 INDELs, and together as all small sequence variants, they represent approximately 11% of the human reference genome sequences. In this study, we aimed first to examine the characteristics of SNPs and INDELs based on their location and variation type. We then identified the ancestral alleles for these variants and examined the patterns of variation from the ancestral state. Our results show that the occurrence of small variants averages at 104 SNPs/kb and 6.5 INDELs/kb for a total of ~11% of the genome. Chromosome 16 and 21 represent the least and most conserved autosomes, respectively, while the sex chromosomes are shown to have a much lower density of SNPs and INDELs being more than 30% lower in the X chromosome and more than 85% lower in the Y chromosome. By gene context, SNPs are biased towards genic regions and INDELs are biased towards intergenic regions, and further, INDELs are biased towards protein-coding genes and intron regions within the genic regions and SNPs are biased towards non-coding genes in the genic regions. Within the coding regions, SNPs and INDELs are biased towards missense and frameshift variations, respectively. Some of the biases were due to biased sources of the variation data targeting at genic regions, while the bias towards intron regions is due to selection pressure. Further, genes with the highest level of variation showed enrichment in functions related to environmental sensing and immune responses, while those with least variation associate with critical processes such as mRNA splicing and processing. Through a comparative genomics approach, we determined the ancestral state for most of these variants and our results indicate that ~0.79% of the genome has been subject to SNP and INDEL variation since the last common human ancestor. Our study represents the first comprehensive data analysis of human variation in SNPs and INDELs and the determination of their ancestral state, providing useful resources for human genetics study and new insights into human evolution

A new mouse model of radiation-induced liver disease reveals mitochondrial dysfunction as an underlying fibrotic stimulus.

Background & Aims High-dose irradiation is an essential tool to help control the growth of hepatic tumors, but it can cause radiation-induced liver disease (RILD). This life-threatening complication manifests itself months following radiation therapy and is characterized by fibrosis of the pericentral sinusoids. In this study, we aimed to establish a mouse model of RILD to investigate the underlying mechanism of radiation-induced liver fibrosis. Methods Using a small animal image-guided radiation therapy platform, an irradiation scheme delivering 50 Gy as a single dose to a focal point in mouse livers was designed. Tissues were analyzed 1 and 6 days, and 6 and 20 weeks post-irradiation. Irradiated livers were assessed by histology, immunohistochemistry, imaging mass cytometry and RNA sequencing. Mitochondrial function was assessed using high-resolution respirometry. Results At 6 and 20 weeks post-irradiation, pericentral fibrosis was visible in highly irradiated areas together with immune cell infiltration and extravasation of red blood cells. RNA sequencing analysis showed gene signatures associated with acute DNA damage, p53 activation, senescence and its associated secretory phenotype and fibrosis. Moreover, gene profiles of mitochondrial damage and an increase in mitochondrial DNA heteroplasmy were detected. Respirometry measurements of hepatocytes in vitro confirmed irradiation-induced mitochondrial dysfunction. Finally, the highly irradiated fibrotic areas showed markers of reactive oxygen species such as decreased glutathione and increased lipid peroxides and a senescence-like phenotype. Conclusions Based on our mouse model of RILD, we propose that irradiation-induced mitochondrial DNA instability contributes to the development of fibrosis via the generation of excessive reactive oxygen species, p53 pathway activation and a senescence-like phenotype. Lay summary Irradiation is an efficient cancer therapy, however, its applicability to the liver is limited by life-threatening radiation-induced hepatic fibrosis. We have developed a new mouse model of radiation-induced liver fibrosis, that recapitulates the human disease. Our model highlights the role of mitochondrial DNA instability in the development of irradiation-induced liver fibrosis. This new model and subsequent findings will help increase our understanding of the hepatic reaction to irradiation and to find strategies that protect the liver, enabling the expanded use of radiotherapy to treat hepatic tumors

The Off-Targets of Clustered Regularly Interspaced Short Palindromic Repeats Gene Editing

Funding: The work of VMB was funded by iNOVA4Health – UIDB/Multi/04462/2020 and UIDP/Multi/04462/2020, a program financially supported by Fundação para a Ciência e Tecnologia (FCT)/Ministério da Educação e Ciência through national funds, and the FCT grant PTDC/BEX-BCM/5900/2014.The repurposing of the CRISPR/Cas bacterial defense system against bacteriophages as simple and flexible molecular tools has revolutionized the field of gene editing. These tools are now widely used in basic research and clinical trials involving human somatic cells. However, a global moratorium on all clinical uses of human germline editing has been proposed because the technology still lacks the required efficacy and safety. Here we focus on the approaches developed since 2013 to decrease the frequency of unwanted mutations (the off-targets) during CRISPR-based gene editing.publishersversionpublishe

Epigenetic Modification of mitochondrial genes in Alzheimer's disease (AD)

Alzheimer’s disease is a chronic, neurodegenerative disease characterised by amyloid plaque accumulation, neurofibrillary tangles and eventual neuronal cell loss. The complex aetiology exhibited in late-onset Alzheimer’s disease presents a considerable challenge in the field of genetics, with identified variants from genome-wide association studies collectively only explaining about a third of disease incidence. As such, new avenues are being explored to elucidate underlying mechanisms associated with disease onset and progression. In 2014, the first epigenome-wide association studies in Alzheimer’s disease were published, identifying several, novel differentially methylated loci in the nuclear genome in cortical brain samples, highlighting that epigenetic mechanisms may play a role in disease aetiology. Further, a growing body of evidence has implicated mitochondrial dysfunction as an early feature of disease pathogenesis. Despite this, few studies have investigated the role of mitochondrial DNA epigenetics in Alzheimer’s disease. Indeed, the relatively nascent field of mitochondrial epigenetics has largely been restricted to candidate-based gene approaches to identify differential methylation associated with disease. The main aim of this thesis was therefore to design an experimental and bioinformatic pipeline for the analysis of mitochondrial DNA methylation in post- mortem human brain tissue; first in healthy non-demented control donors, and subsequently in individuals with Alzheimer’s disease. Our work therefore represents the first epigenome wide studies of mitochondrial DNA methylation at single nucleotide resolution, providing a framework not only for mitochondrial DNA methylation in Alzheimer’s disease, but also in a number of complex diseases characterised by mitochondrial dysfunction

Mitophagy and the dynamics of mitochondrial DNA inheritance in early development and reproduction

Ph. D. Thesis.Inherited heteroplasmic mitochondrial DNA (mtDNA) mutations are a cause of severe disease of adults and children. Thus, methods have been developed to reduce inheritance of pathogenic mtDNA variants; preimplantation genetic diagnosis (PGD) and pronuclear transfer (PNT). However, it is unclear at which stage of development a biopsy better predicts embryo heteroplasmy in PGD. Meanwhile, a small amount of pathogenic variant-harbouring mtDNA is carried over during PNT. Recent work indicates that embryos undergo mitophagy during late preimplantion development. This coincides with a reported increase in intercellular variation of mtDNA heteroplasmy, raising the question of whether mitophagy contributes to segregation of mtDNA. In addition, manipulation of mitophagy may be applied to reduce carry-over of mtDNA variants during PNT. To investigate the mechanisms of mitophagy, using a large single cell RNA sequencing data from human embryos, I analysed the expression of key mitophagy genes. BNIP3 family genes were identified as probable mediators of mitophagy. Overexpression of a phosphomimetic BNIP3L protein upregulated mitophagy, which may be useful in preventing survival of carried over mtDNA in PNT. Furthermore, correlations emerged between mitophagy genes and genes driving the establishment of cell lineages in the blastocyst, suggesting lineage-specific regulation of mitophagy. Using the tRNAalanine (tRNAala) mouse model carrying a pathogenic mtDNA variant, I verified an increase in intercellular variation of heteroplasmy in the blastocyst. Analysis of the segregation of mtDNA suggested that the 8-cell embryo is the optimal stage for PGD biopsy. Upregulation of mitophagy had no detectable influence on the segregation of mtDNA, suggesting that mitophagy may not be a major source of intercellular variation in heteroplasmy. I also revealed an accumulation of mtDNA variants in the oocytes of aged tRNAala mice. Combined with the presence of a pathogenic mutation, mtDNA variants did not manifest a selection mechanism. This suggests acquired mtDNA variants only modestly impair the health of aged oocytes and age-related loss of fertility is primarily due to other causes. These findings elucidate mechanisms which may modulate the inheritance of mtDNA in early development. Furthermore, they provide insight into the impact of mitophagy in the preimplantation embryo, and identify an 8-cell embryo biopsy as preferable to that of a blastocyst in predicting embryo heteroplasmy during PGD. Identification of a method to upregulate mitophagy will help guide the process of preventing the inheritance of mtDNA mutations using PN

STATISTICAL ANALYSES TO DETECT AND REFINE GENETIC ASSOCIATIONS WITH NEURODEGENERATIVE DISEASES

Dementia is a clinical state caused by neurodegeneration and characterized by a loss of function in cognitive domains and behavior. Alzheimer’s disease (AD) is the most common form of dementia. Although the amyloid β (Aβ) protein and hyperphosphorylated tau aggregates in the brain are considered to be the key pathological hallmarks of AD, the exact cause of AD is yet to be identified. In addition, clinical diagnoses of AD can be error prone. Many previous studies have compared the clinical diagnosis of AD against the gold standard of autopsy confirmation and shown substantial AD misdiagnosis Hippocampal sclerosis of aging (HS-Aging) is one type of dementia that is often clinically misdiagnosed as AD. AD and HS-Aging are controlled by different genetic architectures. Familial AD, which often occurs early in life, is linked to mainly mutations in three genes: APP, PSEN1, and PSEN2. Late-onset AD (LOAD) is strongly associated with the ε4 allele of apolipoprotein E (APOE) gene. In addition to the APOE gene, genome-wide association studies (GWAS) have identified several single nucleotide polymorphisms (SNPs) in or close to some genes associated with LOAD. On the other hand, GRN, TMEM106B, ABCC9, and KCNMB2 have been reported to harbor risk alleles associated with HS-Aging pathology. Although GWAS have succeeded in revealing numerous susceptibility variants for dementias, it is an ongoing challenge to identify functional loci and to understand how they contribute to dementia pathogenesis. Until recently, rare variants were not investigated comprehensively. GWAS rely on genotype imputation which is not reliable for rare variants. Therefore, imputed rare variants are typically removed from GWAS analysis. Recent advances in sequencing technologies enable accurate genotyping of rare variants, thus potentially improving our understanding the role of rare variants on disease. There are significant computational and statistical challenges for these sequencing studies. Traditional single variant-based association tests are underpowered to detect rare variant associations. Instead, more powerful and computationally efficient approaches for aggregating the effects of rare variants have become a standard approach for association testing. The sequence-kernel association test (SKAT) is one of the most powerful rare variant analysis methods. A recently-proposed scan-statistic-based test is another approach to detect the location of rare variant clusters influencing disease. In the first study, we examined the gene-based associations of the four putative risk genes, GRN, TMEM106B, ABCC9, and KCNMB2 with HS-aging pathology. We analyzed haplotype associations of a targeted ABCC9 region with HS-Aging pathology and with ABCC9 gene expression. In the second study, we elucidated the role of the non-coding SNPs identified in the International Genomics of Alzheimer’s Project (IGAP) consortium GWAS within a systems genetics framework to understand the flow of biological information underlying AD. In the last study, we identified genetic regions which contain rare variants associated with AD using a scan-statistic-based approach