Systematic Analysis of Molecular and Cellular Dysfunction in Accelerated Aging Phenotypes
by Gesa Werner
Date of Examination:2021-09-09
Date of issue:2021-09-24
Advisor:Prof. Dr. Bernd Wollnik
Referee:Prof. Dr. Bernd Wollnik
Referee:Prof. Dr. Peter Rehling
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Abstract
English
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.
Keywords: Segmental Progeroid Syndromes; mtDNA Sequencing; Telomere Length Measurement