Single-cell Genomics of Post-mitotic Human Tissues in Aging and Disease

Abstract

Somatic mutations occur in human tissues due to errors in DNA replication and DNA damage repair during aging as well as under disease conditions. Previous studies have focused on somatic mutations in clonal cell populations, such as tumors, and found that specific somatic mutations are critical in driving cancer progression. However, the prevalence and impact of somatic mutations in post-mitotic tissues remain largely unknown. Recent advancements in single-cell whole-genome sequencing (scWGS) technologies have enabled us to detect these private mutations. This thesis aims to characterize somatic mutations in single human neurons using scWGS to better understand the role of somatic mutations in neurodegeneration. Whole-genome duplex consensus sequencing has emerged to provide an enhanced accuracy of mutation calling at the single-molecule level as two DNA strands are sequenced independently to form consensus calls. Duplex sequencing can be applied to both bulk tissues and single cells. Existing tools focus on detecting single-nucleotide variants (SNVs) and often neglect other types of mutations such as small insertions and deletions (Indels). The first aim of this thesis was to develop a computational pipeline for somatic Indel calling in duplex sequencing data to allow more comprehensive somatic mutation characterization. Benchmarking analysis on cancer cell lines demonstrated the accuracy and robustness of the pipeline. The second and third aims of this thesis focused on the burden and pattern of somatic mutations in neurodegeneration. Chronic traumatic encephalopathy (CTE), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD) are neurodegenerative diseases with distinct neuropathologies. The work presented in the second aim profiled somatic SNVs and Indels in single neurons from CTE individuals, revealing significantly elevated somatic mutation burdens in CTE compared to neurotypical controls. Comparisons between CTE and AD pointed to potentially common mutagenic processes underlying the two diseases. The third aim turned to ALS and FTD, two closely related neurodegenerative conditions with nuclear TDP-43 depletion as the hallmark, and unveiled a recurrent and widespread pattern of somatic Indels shared by all four neurodegenerative diseases. This thesis investigates how somatic mutations accumulate in human neurons across multiple major neurodegenerative conditions and highlights the application of the latest sequencing technologies in making biological discoveries. Furthermore, this thesis can serve as the basis for an array of future studies. The new pipeline can be applied to other tissues, and the recurrent somatic Indel pattern may provide valuable insight into identifying potential therapeutic targets for a broad range of neurodegenerative diseases.