DNA methylation in human

The epigenome is determined by DNA methylation and histone modifications, which together is part of the cell-specific gene expression regulation.

Transcriptional gene expression regulation is mediated by an interplay between DNA methylation and histone modifications making the genome either accessible to the transcriptional machinery for active transcription or silencing of a gene by creating a closed unaccessible chromatin structure.

This section will focus on the influence of the DNA methylation pattern changes associated with a high BMI and altered expression of genes found in patients with type 2 diabetes (T2D).

 

DNA methylation mechanisms

DNA methyltransferases (DNMTs) are responsible for the addition of methyl groups to the carbon 5 at cytosines (5mC) within a CpG dinucleotide site.

DNMT1 maintains the methylation pattern of the genomic regions during replication, while DNMT3A and DNMT3B are responsible for de-novo attachment of methyl groups. CpG dinucleotide sites are not evenly distributed throughout the genome, but are clustered in CpG-rich regions, known as CpG islands (CGI). CpG islands span regulatory regions of the genome as gene promoters, enhancers as well as housekeeping genes.

While the majority of CpG islands in a normal cell are maintained in an unmethylated state, allowing for expression of the associated gene, a number of repressed genes show promoter methylation of CpG islands in somatic cells.

More than half of the protein coding genes contain a CpG island spanning their promoter region, in which the methylation level is instrumental in regulating the transcriptional activity of the gene.

Consequently, changes of the cell’s normal methylation pattern can have severe consequences and contribute to a large variety of diseases.

The ten-eleven-translocation (TET) enzymes are involved in the demethylation of genomic regions, by oxidation of the methyl-groups to hydroxymethyl, which eventually is substituted by unmethylated cytosines via DNA repair.

The DNA methylation patterns across the epigenome are dynamic, the methylation status can be reversed, processes, which are regulated by an interplay between DNMTs and TET proteins.

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Genome wide DNA methylation

An epigenome wide association study is an important tool to interrogate the methylation status of a substantial number of CpG dinucleotides throughout the genome.

The infinitum 450K methylation BeadChip contains approximately 450,000 CpG sites, which only covers about 1.5% of all CpG sites in the genome. Still, this array has been intensively used for years to identify differentially methylated regions (DMRs), pointing at an altered gene expression profile, which together may play an important role in the development or progression of a large variety of diseases.

The Infinium MethylationEpic BeadChip array contains 850,000 CpG sites, and is a step on the road to provide a more comprehensive picture of the global DNA methylation pattern of a pathological changed cell’s epigenome.

Whole genome bisulfite sequencing utilises next-generation sequencing technologies where the DNA methylation levels can be analysed at single-nucleotide resolution, as with the methylation arrays described above.

These technologies together allow for at highly detailed analysis of the methylome and improved understanding of the role of DNA methylation related gene expression alterations implicated in diseases.

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Test material

Peripheral blood samples are often easily accessible as source for different DNA analyses.

When changes in the epigenetic pattern is investigated, it is important to carefully select the test material, to provide a meaningful picture of phenotypical changes of the affected tissue.

For epigenetic analysis of changes specific for obese individuals and patients diagnosed with type 2 diabetes, it would be meaningful to analyse human adipose tissue, human skeletal muscle, human pancreatic islets, and human liver tissue. These types of samples may be difficult to collect in a sufficiently high number to perform a valid differential DNA methylation profiling.

Therefore , a large number of individuals can be analysed by epigenome wide DNA methylation analysis, which instead may be performed using blood DNA as the accessible source, but the specific altered DNA methylation pattern should be carefully validated using the proper tissue in the light of the asked questions.

 

Type 2 Diabetes (T2D)

More than 400 million people suffer from T2D world wide, and the incidence is increasing.

The risk of developing cardiovascular diseases for individuals with T2D is high, and responsible for the death of around 5 million people each year.

Type 2 diabetes mellitus is a chronic disease characterised by a persistent high level of blood glucose and an impaired insulin secretion combined with insulin resistance.

Insulin is secreted from the human pancreatic β cells as response to food intake, and it regulates blood glucose levels by enhancing glucose uptake and glycolysis in skeletal muscle and adipose tissue.

As response to long term exposure to high glucose and lipid levels the pancreatic β cells loose function resulting in a persistent high level of glucose in the blood.

T2D is a multifactorial disease, and Genome-Wide Association Studies (GWAS) have been used to identify more than 200 genetic loci specific for T2D patients, which together with environmental factors as obesity and sedentary lifestyle contributed to the aetiology of the disease.

These genetic variants may only explain less than 20% of the factors responsible for the risk of developing diabetes, which is why the focus for the past decade has been turned to map epigenetic changes in T2D patients (see Low M. et al; 2019) The epigenome responds to certain environmental influences, and histone modifications as well as DNA methylation changes are associated with high BMI and obesity, which are main risk factors for T2D.

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Significance of methylation alterations in T2D

In the discovery of epigenetic events, affecting genes important for the onset of T2D, the test tissues have to be carefully selected.

Many epigenome studies are performed using peripheral blood cells, but as the effect of epigenetic changes are tissue specific, it is important to analyse pancreatic islets as well as adipose and skeletal muscle tissues, to identify the gene-specific epigenetic events, playing an important role in pathways leading to T2D.

Combining epigenome – and genome wide expression analysis of pancreatic islets from T2D patients and non-diabetic controls have shown a number of genes, in which epigenetic changes have proved to affect gene expression.

For genes as CDKN1A, PDE7B, and SEPT9 hypomethylation was followed by an increased gene expression. CDKN1A is a regulator of the cell-cycle progression from G₀ to G_1, and together with PDE7B and SEPT9 over expression of these genes resulted in a decreased glucose-stimulated insulin secretion. Other genes from DMRs as: ADCY5, FTO, HHEX, IRS1, KCNQ1, PPARG, and TCF7L2 are associated with both obesity and risk of T2D (see Ling C. and Rönn T.; 2019).

By using whole genome bisulfite sequencing to interrogate more than 2.5 x 10⁷ CpG dinucleotides in pancreatic islets from T2D patients, especially, altered DNA methylation status of the PDX1 gene was interesting, being a key transcription factor in pancreatic β cells, where it regulates insulin expression.

 

Significance of methylation alterations in obesity

More than 1.5 billion people suffer from overweight or obesity, and the incidence is increasing.

The estimated number of overweight or obese children are exceeding 40 million (see Gallardo-Escribano C. et al; 2020). Childhood obesity is associated with a high risk of being overweight as adult.

The origin of obesity is multifactorial and heterogenous, it is induced by dietary habits, lack of physical activity, determined by neuroendocrine status as well as genetic and epigenetic alterations.

High Body Mass Index (BMI) or obesity increases the individual’s risk of type 2 diabetes, cardiovascular disease, metabolic and inflammatory diseases.

Obesity is estimated to be the underlying cause of T2D and projected to affect 552 million people in 2030.

Epigenome wide analysis using blood leucocyte DNA comparing the profile from lean and obese individuals has showed a higher variability in methylation of CpG sites in the obese cases compared to the lean controls, and that the DNA methylation changes might predict obesity with approx. 70% confidence.

Over the past decades analysis of DNA methylation has identified a large number of specific genes within DMRs from adipose tissue, skeletal muscle, and blood, which are associated with high BMI and obesity.

As examples can be mentioned: the ABCG1 encoded protein involved in macrophage cholesterol and phospholipid transport, the SREBF1 gene encoding a transcription factor involved in lipid metabolism, the MSIS gene encoding the Musashi RNA binding protein2 involved in eating behaviour, and SOCS3, which is found hypomethylated in obese individuals and thereby has an upregulated gene expression. SOCS3 is found to induce insulin and leptin resistance, which affects blood glucose control.

Interestingly, gastric by-pass surgery followed by weight loss is associated with alterations in DNA methylation of a large number of genes implicated in obesity, reflecting the plasticity of the epigenome.

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How MethylDetect can assist you to analyse methylation-specific targets

In MethylDetect, we provide you with a solution to assess a target specific DNA methylation profile of your test samples.

In our catalogue, we offer you more than 850 specific EpiMelt assays.

The EpiMelt test kits are based on the Methylation-Sensitive High-Resolution Melting (MS-HRM) technology, and can be used with standard laboratory equipment for qPCR and melting assessment.

Each EpiMelt test kit comes with a unique control system, securing the high sensitivity of your analysis provided by the kit.

 

Custom-Tailored EpiMelt kits

We design and produce EpiMelt test kits tailored to target specific areas of the genome, in case your gene or genomic area of interest is not found in our portfolio.

Following methylation-specific array screening analyses, you may have identified targets, which are not yet described in the literature.

In collaboration with you, we can design and produce EpiMelt test kits targeting these specific genomic areas, and tailor the kit to fulfil your needs, for example, whether you  may want analyse for hypo – or hypermethylation.

Likewise, we design your new EpiMelt test kit for highly sensitive analysis of the selected test material available for your analyses.

We take into account if it is FFPE, specific liquid biopsies or maybe high quality DNA. Customer tailored EpiMelt assays are always performed in close collaboration with you.

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Further reading

Ling C and Rönn T. Epigenetics in Human Obesity and Type 2 Diabetes. Cell Metabolism 29, May 7, 2019

Ouni M and Schürmann. Epigenetic contribution to obesity. Mammalian Genome (2020) 31:134–145

Loh M et al. Epigenetic disturbances in obesity and diabetes: Epidemiological and functional insights. Molecular Metabolism 27 (2019)

Ahmed SAH et al. The role of DNA methylation in the pathogenesis of type 2 diabetes mellitus. Clinical Epigenetics 12 (2020)

Gallardo-Escribano C. et al. Epigenetic approach in obesity: DNA methylation in a prepubertal population which underwent a lifestyle modification. Clinical Epigenetics 12 (2020)

Cuevas-Sierra A et al. Diet, Gut Microbiota, and Obesity: Links with Host Genetics and Epigenetics and Potential Applications. Adv Nutr 2019;10:S17–S30