Epigenetics and genetics of ageing
Epigenetics and genetics of ageing
We have investigated the change in methylation status of three regulatory regions at the human IGF 2 gene. These regions were the biallelically-expressed promoter P1, imprinted promoter P3 and the downstream differentially methylated region (DMR) 2. The two cohorts were “Young” <30 years and “Elderly” >80 years. In order to increase sensitivity and throughput of the methylation detection, a methylation-sensitive/bisulphite sequencing protocol was applied to human peripheral blood lymphocyte derived, bisulphite modified genomic DNA.
Promoter 1 (p=0.13) and the downstream DMR2 (p=0.38) show neither hyper- nor hypo-methylation during ageing in peripheral blood lymphocytes. However, the distal region of the imprinted promoter 3 shows significant hypermethylation, in peripheral blood lymphocyte derived genomic DNA, with increasing age, p=0.004. The P3 result is in concordance with the report of Issa et al 1996 in which age-related hypermethylation of the IGF2 P3 region was demonstrated in colon tissue. As methylation is promoter, gene and tissue specific, the previous demonstration of age-related methylation in colon was insufficient to prove that age-related hypermethylation occurs in other tissues. This validates the use of peripheral blood lymphocyte derived DNA as a proxy for the methylation status of less easily available tissue and removes one of the barriers to high throughput studies of population methylation.
A high throughput assay was designed, based upon the basic strategy of using a methylation sensitive enzyme to quantify methylation, and then aiming to achieve the necessary additional sensitivity by linking this analysis to quantitative PCR. One CpG site in the promoter 3 region (base 1130, sequence accession number X03562) was chosen for the initial target of this quantitation strategy. The bisulphite sequencing described above demonstrated age-related hypermethylation at this CpG site. This CpG site lies within an Hpall restriction site.
University of Southampton
Hoffman, Elizabeth
c965407f-0971-43ea-8c86-f03cc770cc9f
2003
Hoffman, Elizabeth
c965407f-0971-43ea-8c86-f03cc770cc9f
Hoffman, Elizabeth
(2003)
Epigenetics and genetics of ageing.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
We have investigated the change in methylation status of three regulatory regions at the human IGF 2 gene. These regions were the biallelically-expressed promoter P1, imprinted promoter P3 and the downstream differentially methylated region (DMR) 2. The two cohorts were “Young” <30 years and “Elderly” >80 years. In order to increase sensitivity and throughput of the methylation detection, a methylation-sensitive/bisulphite sequencing protocol was applied to human peripheral blood lymphocyte derived, bisulphite modified genomic DNA.
Promoter 1 (p=0.13) and the downstream DMR2 (p=0.38) show neither hyper- nor hypo-methylation during ageing in peripheral blood lymphocytes. However, the distal region of the imprinted promoter 3 shows significant hypermethylation, in peripheral blood lymphocyte derived genomic DNA, with increasing age, p=0.004. The P3 result is in concordance with the report of Issa et al 1996 in which age-related hypermethylation of the IGF2 P3 region was demonstrated in colon tissue. As methylation is promoter, gene and tissue specific, the previous demonstration of age-related methylation in colon was insufficient to prove that age-related hypermethylation occurs in other tissues. This validates the use of peripheral blood lymphocyte derived DNA as a proxy for the methylation status of less easily available tissue and removes one of the barriers to high throughput studies of population methylation.
A high throughput assay was designed, based upon the basic strategy of using a methylation sensitive enzyme to quantify methylation, and then aiming to achieve the necessary additional sensitivity by linking this analysis to quantitative PCR. One CpG site in the promoter 3 region (base 1130, sequence accession number X03562) was chosen for the initial target of this quantitation strategy. The bisulphite sequencing described above demonstrated age-related hypermethylation at this CpG site. This CpG site lies within an Hpall restriction site.
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Published date: 2003
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Local EPrints ID: 465099
URI: http://eprints.soton.ac.uk/id/eprint/465099
PURE UUID: 7101fbdb-8f55-43e0-beef-94bdfa02a61d
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Date deposited: 05 Jul 2022 00:23
Last modified: 16 Mar 2024 19:57
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Author:
Elizabeth Hoffman
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