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Epigenetics and genetics of ageing

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
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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