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Title: Age-dependent microsatellite somatic mosaicism in humans
Author: Alghamdi, Saeed
ISNI:       0000 0004 5347 2001
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2014
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Several inherited diseases such as Huntington disease and myotonic dystrophy type 1 are associated with the expansion of repeats. A high level of age-dependent instability has been observed in the expanded alleles present in the germline and soma. The present study covers the investigation of other non-disease-associated expanded microsatellites in order to explore their variability, which in turn could be used to predict age in the general population. First, using Tandem Repeats Finder, 23 pure loci with alleles >50 repeats in at least one of two reference genomes were identified. Several loci among those show high levels of variation in the general population with a considerable proportion of large expanded alleles. SP-PCR analysis revealed that these loci showed a relatively low level of somatic instability and mostly only small length changes. The SP-PCR approach is laborious and time consuming; these disadvantages could potentially be overcome by the use of new technologies based on high throughput analysis. In this study a target enrichment sequencing approach using custom bait and Illumina Paired-End Sequencing using Illumina GA IIx platform to capture and sequence sequences in question was used. 25,539 pure (100% match) mono, di, and trinucleotides with unique flanking sequences and copy number ≥5 were investigated. Similarly, telomeric regions, DNA repair genes, the DMPK region and SNPs were captured using custom designed baits. The experiment generated millions of reads for each sample, ranging from 5.5 to 9.5 million reads. Subsequently, CLC Genomics Workbench and Bowtie 2 were used to map reads to the HG19 reference genome and to count the number of reads aligned to each DNA sequence. The effect of age at sampling on the generated reads was also investigated. The data showed a significant correlation between unique sequence reads and age at sampling. Microsatellites, including mono, di and tri nucleotides, were successfully captured using custom baits based on unique flanking sequences. The microsatellite total reads were found to be correlated with age at sampling. Genotyping was found to be successful for di, tri and tetra nucleotide loci however PCR slippage was commonly found in the dinucleotide repeats. By contrast due to the extensive PCR slippage observed in mononucleotides, reliable genotyping was difficult to achieve. Unlike microsatellites, no significant correlation was observed between DNA sequences of repair genes or the autosome gene read counts with age at sampling. Thereby, this feature could be exploited to normalise the read counts such as of telomeres. Thousands of human SNPs were successfully captured using baits having a single mismatch. Baits were designed to ensure an equal capture for both alleles that could exist at a single SNP. The overall coverage of SNPs was noticeably good and used to generate reliable genotype. Age-dependent telomere shortening was estimated. The relative telomere length showed a significant correlation with age at sampling. The data show four major variants of telomeric repeats, TTAGGG, TTGGGG, TGAGGG and TAAGGG with a shortening rate of 60 bp/year. In summary, the primary aim of this project was not achieved due to the low level somatic instability of microsatellite and low-resolution power of traditional SP-PCR. However, the success with the NGS observed in this study, indicate a reasonable potential of using microsatellites to estimate ageing in humans.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH426 Genetics