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Identification of novel high-frequency DNA methylation changes in breast cancer.

Ordway JM, Budiman MA, Korshunova Y, Maloney RK, Bedell JA, Citek RW, Bacher B, Peterson S, Rohlfing T, Hall J, Brown R, Lakey N, Doerge RW, Martienssen RA, Leon J, McPherson JD, Jeddeloh JA - PLoS ONE (2007)

Bottom Line: Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis.The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively.Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date.

View Article: PubMed Central - PubMed

Affiliation: Orion Genomics, St. Louis, Missouri, United States of America. jordway@oriongenomics.com

ABSTRACT
Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.

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Correlation between DNA hypermethylation and gene expression.Transcription of GHSR1a was analyzed by RT-PCR. RT-PCR was performed using gene-specific primer pairs designed to flank intronic sequence so that the contribution of contaminating genomic DNA could be excluded. Analysis of GAPDH expression was performed as an internal control. Serial dilutions of first-strand cDNA preparations from tumor samples and a normal breast tissue sample were used as templates for PCR. The DNA methylation measurement (qPCR) for each locus in each tumor sample is indicated (- sparse, + intermediate and ++ dense methylation).
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pone-0001314-g007: Correlation between DNA hypermethylation and gene expression.Transcription of GHSR1a was analyzed by RT-PCR. RT-PCR was performed using gene-specific primer pairs designed to flank intronic sequence so that the contribution of contaminating genomic DNA could be excluded. Analysis of GAPDH expression was performed as an internal control. Serial dilutions of first-strand cDNA preparations from tumor samples and a normal breast tissue sample were used as templates for PCR. The DNA methylation measurement (qPCR) for each locus in each tumor sample is indicated (- sparse, + intermediate and ++ dense methylation).

Mentions: To address the association between hypermethylation and transcription repression, we performed RT-PCR analyses of the GHSR gene (Fig. 7). Four breast IDC samples (>90% neoplastic cellularity) were analyzed for both DNA methylation and transcription. Expression of GHSR1a (see Discussion) was undetectable in all four tumor samples, while expression was detected at 1∶10 dilution of the normal breast cDNA. Consistent with the high sensitivity of hypermethylation at the GHSR locus, all four tumor samples demonstrated intermediate to dense DNA methylation at this locus.


Identification of novel high-frequency DNA methylation changes in breast cancer.

Ordway JM, Budiman MA, Korshunova Y, Maloney RK, Bedell JA, Citek RW, Bacher B, Peterson S, Rohlfing T, Hall J, Brown R, Lakey N, Doerge RW, Martienssen RA, Leon J, McPherson JD, Jeddeloh JA - PLoS ONE (2007)

Correlation between DNA hypermethylation and gene expression.Transcription of GHSR1a was analyzed by RT-PCR. RT-PCR was performed using gene-specific primer pairs designed to flank intronic sequence so that the contribution of contaminating genomic DNA could be excluded. Analysis of GAPDH expression was performed as an internal control. Serial dilutions of first-strand cDNA preparations from tumor samples and a normal breast tissue sample were used as templates for PCR. The DNA methylation measurement (qPCR) for each locus in each tumor sample is indicated (- sparse, + intermediate and ++ dense methylation).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2117343&req=5

pone-0001314-g007: Correlation between DNA hypermethylation and gene expression.Transcription of GHSR1a was analyzed by RT-PCR. RT-PCR was performed using gene-specific primer pairs designed to flank intronic sequence so that the contribution of contaminating genomic DNA could be excluded. Analysis of GAPDH expression was performed as an internal control. Serial dilutions of first-strand cDNA preparations from tumor samples and a normal breast tissue sample were used as templates for PCR. The DNA methylation measurement (qPCR) for each locus in each tumor sample is indicated (- sparse, + intermediate and ++ dense methylation).
Mentions: To address the association between hypermethylation and transcription repression, we performed RT-PCR analyses of the GHSR gene (Fig. 7). Four breast IDC samples (>90% neoplastic cellularity) were analyzed for both DNA methylation and transcription. Expression of GHSR1a (see Discussion) was undetectable in all four tumor samples, while expression was detected at 1∶10 dilution of the normal breast cDNA. Consistent with the high sensitivity of hypermethylation at the GHSR locus, all four tumor samples demonstrated intermediate to dense DNA methylation at this locus.

Bottom Line: Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis.The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively.Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date.

View Article: PubMed Central - PubMed

Affiliation: Orion Genomics, St. Louis, Missouri, United States of America. jordway@oriongenomics.com

ABSTRACT
Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.

Show MeSH
Related in: MedlinePlus