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Allele-specific, age-dependent and BMI-associated DNA methylation of human MCHR1.

Stepanow S, Reichwald K, Huse K, Gausmann U, Nebel A, Rosenstiel P, Wabitsch M, Fischer-Posovszky P, Platzer M - PLoS ONE (2011)

Bottom Line: The AC haplotype shows a significantly higher methylation level than the GT haplotype.Interestingly, the GT allele shows a decrease in methylation status with increasing BMI, whereas the methylation of the AC allele is not associated with this phenotype.We show that DNA methylation at MCHR1 is allele-specific, age-dependent, BMI-associated and affects transcription.

View Article: PubMed Central - PubMed

Affiliation: Genome Analysis, Leibniz Institute for Age Research, Jena, Germany. stepanow@fli-leibniz.de

ABSTRACT

Background: Melanin-concentrating hormone receptor 1 (MCHR1) plays a significant role in regulation of energy balance, food intake, physical activity and body weight in humans and rodents. Several association studies for human obesity showed contrary results concerning the SNPs rs133072 (G/A) and rs133073 (T/C), which localize to the first exon of MCHR1. The variations constitute two main haplotypes (GT, AC). Both SNPs affect CpG dinucleotides, whereby each haplotype contains a potential methylation site at one of the two SNP positions. In addition, 15 CpGs in close vicinity of these SNPs constitute a weak CpG island. Here, we studied whether DNA methylation in this sequence context may contribute to population- and age-specific effects of MCHR1 alleles in obesity.

Principal findings: We analyzed DNA methylation of a 315 bp region of MCHR1 encompassing rs133072 and rs133073 and the CpG island in blood samples of 49 individuals by bisulfite sequencing. The AC haplotype shows a significantly higher methylation level than the GT haplotype. This allele-specific methylation is age-dependent. In young individuals (20-30 years) the difference in DNA methylation between haplotypes is significant; whereas in individuals older than 60 years it is not detectable. Interestingly, the GT allele shows a decrease in methylation status with increasing BMI, whereas the methylation of the AC allele is not associated with this phenotype. Heterozygous lymphoblastoid cell lines show the same pattern of allele-specific DNA methylation. The cell line, which exhibits the highest difference in methylation levels between both haplotypes, also shows allele-specific transcription of MCHR1, which can be abolished by treatment with the DNA methylase inhibitor 5-aza-2'-deoxycytidine.

Conclusions: We show that DNA methylation at MCHR1 is allele-specific, age-dependent, BMI-associated and affects transcription. Conceivably, this epigenetic regulation contributes to the age- and/or population specific effects reported for MCHR1 in several human obesity studies.

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Related in: MedlinePlus

Allele-specific DNA methylation at MCHR1.The box plot shows average methylation levels of the GT and the AC allele observed in 49 individuals, comprised of 18 homozygous for GT, 13 homozygous for AC and 18 heterozygotes. The median methylation level for the GT allele is 20.9% and for the AC allele 29.9%. This difference is significant (***: P<0.001, Mann-Whitney-test). The methylation analysis was done by PCR on bisulfite treated DNA. PCR products were cloned and sequenced. We analyzed on average 41 clones per individual. The methylation level for each allele per individual was calculated by dividing the number of methylated sites in all clones harboring the respective allele by the number of possible methylation sites.
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pone-0017711-g002: Allele-specific DNA methylation at MCHR1.The box plot shows average methylation levels of the GT and the AC allele observed in 49 individuals, comprised of 18 homozygous for GT, 13 homozygous for AC and 18 heterozygotes. The median methylation level for the GT allele is 20.9% and for the AC allele 29.9%. This difference is significant (***: P<0.001, Mann-Whitney-test). The methylation analysis was done by PCR on bisulfite treated DNA. PCR products were cloned and sequenced. We analyzed on average 41 clones per individual. The methylation level for each allele per individual was calculated by dividing the number of methylated sites in all clones harboring the respective allele by the number of possible methylation sites.

Mentions: We next analyzed MCHR1 methylation in blood of 49 individuals, including 18 individuals homozygous for GT, 13 individuals homozygous for AC and 18 heterozygotes after bisulfite treatment by cloning and sequencing. We analyzed on average 41 clones per individual. The average clone number for the GT allele is 29 (number of allele carrier: nind = 36) and for the AC allele 31 (nind = 31). The methylation intensity of the GT allele was significantly lower than that of the AC allele (median: 20.9% vs. 29.9%; P<0.001, Mann-Whitney-test; Figure 2). We also checked for methylation level differences according to gender and could not detect differences for both alleles (data not shown). As expected for a CpG island, the analyzed clones show a high abundance of unmethylated sequences (methyl-CpG/all CpG<20%), but heterogeneously (20–80%) and highly methylated clones (>80%) are also observed. Moreover, the AC allele shows a higher abundance in heterogeneously and highly methylated and fewer less methylated clones than the GT allele, which is significant (P<0.001, Chi-square-test) (Figure 3).


Allele-specific, age-dependent and BMI-associated DNA methylation of human MCHR1.

Stepanow S, Reichwald K, Huse K, Gausmann U, Nebel A, Rosenstiel P, Wabitsch M, Fischer-Posovszky P, Platzer M - PLoS ONE (2011)

Allele-specific DNA methylation at MCHR1.The box plot shows average methylation levels of the GT and the AC allele observed in 49 individuals, comprised of 18 homozygous for GT, 13 homozygous for AC and 18 heterozygotes. The median methylation level for the GT allele is 20.9% and for the AC allele 29.9%. This difference is significant (***: P<0.001, Mann-Whitney-test). The methylation analysis was done by PCR on bisulfite treated DNA. PCR products were cloned and sequenced. We analyzed on average 41 clones per individual. The methylation level for each allele per individual was calculated by dividing the number of methylated sites in all clones harboring the respective allele by the number of possible methylation sites.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102661&req=5

pone-0017711-g002: Allele-specific DNA methylation at MCHR1.The box plot shows average methylation levels of the GT and the AC allele observed in 49 individuals, comprised of 18 homozygous for GT, 13 homozygous for AC and 18 heterozygotes. The median methylation level for the GT allele is 20.9% and for the AC allele 29.9%. This difference is significant (***: P<0.001, Mann-Whitney-test). The methylation analysis was done by PCR on bisulfite treated DNA. PCR products were cloned and sequenced. We analyzed on average 41 clones per individual. The methylation level for each allele per individual was calculated by dividing the number of methylated sites in all clones harboring the respective allele by the number of possible methylation sites.
Mentions: We next analyzed MCHR1 methylation in blood of 49 individuals, including 18 individuals homozygous for GT, 13 individuals homozygous for AC and 18 heterozygotes after bisulfite treatment by cloning and sequencing. We analyzed on average 41 clones per individual. The average clone number for the GT allele is 29 (number of allele carrier: nind = 36) and for the AC allele 31 (nind = 31). The methylation intensity of the GT allele was significantly lower than that of the AC allele (median: 20.9% vs. 29.9%; P<0.001, Mann-Whitney-test; Figure 2). We also checked for methylation level differences according to gender and could not detect differences for both alleles (data not shown). As expected for a CpG island, the analyzed clones show a high abundance of unmethylated sequences (methyl-CpG/all CpG<20%), but heterogeneously (20–80%) and highly methylated clones (>80%) are also observed. Moreover, the AC allele shows a higher abundance in heterogeneously and highly methylated and fewer less methylated clones than the GT allele, which is significant (P<0.001, Chi-square-test) (Figure 3).

Bottom Line: The AC haplotype shows a significantly higher methylation level than the GT haplotype.Interestingly, the GT allele shows a decrease in methylation status with increasing BMI, whereas the methylation of the AC allele is not associated with this phenotype.We show that DNA methylation at MCHR1 is allele-specific, age-dependent, BMI-associated and affects transcription.

View Article: PubMed Central - PubMed

Affiliation: Genome Analysis, Leibniz Institute for Age Research, Jena, Germany. stepanow@fli-leibniz.de

ABSTRACT

Background: Melanin-concentrating hormone receptor 1 (MCHR1) plays a significant role in regulation of energy balance, food intake, physical activity and body weight in humans and rodents. Several association studies for human obesity showed contrary results concerning the SNPs rs133072 (G/A) and rs133073 (T/C), which localize to the first exon of MCHR1. The variations constitute two main haplotypes (GT, AC). Both SNPs affect CpG dinucleotides, whereby each haplotype contains a potential methylation site at one of the two SNP positions. In addition, 15 CpGs in close vicinity of these SNPs constitute a weak CpG island. Here, we studied whether DNA methylation in this sequence context may contribute to population- and age-specific effects of MCHR1 alleles in obesity.

Principal findings: We analyzed DNA methylation of a 315 bp region of MCHR1 encompassing rs133072 and rs133073 and the CpG island in blood samples of 49 individuals by bisulfite sequencing. The AC haplotype shows a significantly higher methylation level than the GT haplotype. This allele-specific methylation is age-dependent. In young individuals (20-30 years) the difference in DNA methylation between haplotypes is significant; whereas in individuals older than 60 years it is not detectable. Interestingly, the GT allele shows a decrease in methylation status with increasing BMI, whereas the methylation of the AC allele is not associated with this phenotype. Heterozygous lymphoblastoid cell lines show the same pattern of allele-specific DNA methylation. The cell line, which exhibits the highest difference in methylation levels between both haplotypes, also shows allele-specific transcription of MCHR1, which can be abolished by treatment with the DNA methylase inhibitor 5-aza-2'-deoxycytidine.

Conclusions: We show that DNA methylation at MCHR1 is allele-specific, age-dependent, BMI-associated and affects transcription. Conceivably, this epigenetic regulation contributes to the age- and/or population specific effects reported for MCHR1 in several human obesity studies.

Show MeSH
Related in: MedlinePlus