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Identification and Validation of Heart-Specific <t>Methylated</t> CpG Sites in PIH1D1 (A) Workflow illustrating the selection criteria for heart-specific methylated CpG sites (HSMCs). CpG sites were filtered by comparing methylation levels in the left atrium to those in 24 other tissues. Sites with methylation levels below 10% in noncardiac tissues were first selected. CpG sites showing a methylation difference >10% between the left atrium and the average of other tissues were further classified as HSMCs. (B) A total of 33 CpG sites met these criteria and were classified as HSMCs. Heatmap showing the methylation β-values of these 33 HSMCs across 25 different human tissues. (C) Scatter plot showing the β-values of the left atrium (x-axis) compared with the average β-values of 24 other tissues (y-axis) with SD error bars. The orange dot highlights PIH1D1 , representing a CpG site of interest for further investigation. (D) <t>DNA</t> methylation β-values of PIH1D1 (cg02184280) across The Cancer Genome Atlas (TCGA) and The Genomic Data Commons (GDC) data sets, including pan-cancer and breast cancer cohorts. β-values remain below 10% in primary tumors, adjacent normal tissues, and metastatic samples, with higher hypomethylation specificity observed in breast cancer patients. (E) Schematic representation highlighting the CpG site cg02184280 (purple) and the design of methylation-specific PCR (MSP) primers (red) and bisulfite pyrosequencing primers (orange). (F) Gel-based MSP shows amplicons in primary human cardiomyocytes (HCM), induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs), and AC16 cells but not in GES or breast cancer cell lines. Amplicons were also observed in doxorubicin-treated iPSC-CMs, indicating the specificity of the detected CpG sites to cardiac tissue methylation patterns. In vitro methylated human DNA (IVD) was used as a positive control to validate the specificity and efficiency of the methylation-specific primers. “M” indicates detection of methylated PIH1D1 , while a product in the “U” lane indicates unmethylated PIH1D1 .
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Identification and Validation of Heart-Specific <t>Methylated</t> CpG Sites in PIH1D1 (A) Workflow illustrating the selection criteria for heart-specific methylated CpG sites (HSMCs). CpG sites were filtered by comparing methylation levels in the left atrium to those in 24 other tissues. Sites with methylation levels below 10% in noncardiac tissues were first selected. CpG sites showing a methylation difference >10% between the left atrium and the average of other tissues were further classified as HSMCs. (B) A total of 33 CpG sites met these criteria and were classified as HSMCs. Heatmap showing the methylation β-values of these 33 HSMCs across 25 different human tissues. (C) Scatter plot showing the β-values of the left atrium (x-axis) compared with the average β-values of 24 other tissues (y-axis) with SD error bars. The orange dot highlights PIH1D1 , representing a CpG site of interest for further investigation. (D) <t>DNA</t> methylation β-values of PIH1D1 (cg02184280) across The Cancer Genome Atlas (TCGA) and The Genomic Data Commons (GDC) data sets, including pan-cancer and breast cancer cohorts. β-values remain below 10% in primary tumors, adjacent normal tissues, and metastatic samples, with higher hypomethylation specificity observed in breast cancer patients. (E) Schematic representation highlighting the CpG site cg02184280 (purple) and the design of methylation-specific PCR (MSP) primers (red) and bisulfite pyrosequencing primers (orange). (F) Gel-based MSP shows amplicons in primary human cardiomyocytes (HCM), induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs), and AC16 cells but not in GES or breast cancer cell lines. Amplicons were also observed in doxorubicin-treated iPSC-CMs, indicating the specificity of the detected CpG sites to cardiac tissue methylation patterns. In vitro methylated human DNA (IVD) was used as a positive control to validate the specificity and efficiency of the methylation-specific primers. “M” indicates detection of methylated PIH1D1 , while a product in the “U” lane indicates unmethylated PIH1D1 .
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Identification and Validation of Heart-Specific <t>Methylated</t> CpG Sites in PIH1D1 (A) Workflow illustrating the selection criteria for heart-specific methylated CpG sites (HSMCs). CpG sites were filtered by comparing methylation levels in the left atrium to those in 24 other tissues. Sites with methylation levels below 10% in noncardiac tissues were first selected. CpG sites showing a methylation difference >10% between the left atrium and the average of other tissues were further classified as HSMCs. (B) A total of 33 CpG sites met these criteria and were classified as HSMCs. Heatmap showing the methylation β-values of these 33 HSMCs across 25 different human tissues. (C) Scatter plot showing the β-values of the left atrium (x-axis) compared with the average β-values of 24 other tissues (y-axis) with SD error bars. The orange dot highlights PIH1D1 , representing a CpG site of interest for further investigation. (D) <t>DNA</t> methylation β-values of PIH1D1 (cg02184280) across The Cancer Genome Atlas (TCGA) and The Genomic Data Commons (GDC) data sets, including pan-cancer and breast cancer cohorts. β-values remain below 10% in primary tumors, adjacent normal tissues, and metastatic samples, with higher hypomethylation specificity observed in breast cancer patients. (E) Schematic representation highlighting the CpG site cg02184280 (purple) and the design of methylation-specific PCR (MSP) primers (red) and bisulfite pyrosequencing primers (orange). (F) Gel-based MSP shows amplicons in primary human cardiomyocytes (HCM), induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs), and AC16 cells but not in GES or breast cancer cell lines. Amplicons were also observed in doxorubicin-treated iPSC-CMs, indicating the specificity of the detected CpG sites to cardiac tissue methylation patterns. In vitro methylated human DNA (IVD) was used as a positive control to validate the specificity and efficiency of the methylation-specific primers. “M” indicates detection of methylated PIH1D1 , while a product in the “U” lane indicates unmethylated PIH1D1 .
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Identification and Validation of Heart-Specific <t>Methylated</t> CpG Sites in PIH1D1 (A) Workflow illustrating the selection criteria for heart-specific methylated CpG sites (HSMCs). CpG sites were filtered by comparing methylation levels in the left atrium to those in 24 other tissues. Sites with methylation levels below 10% in noncardiac tissues were first selected. CpG sites showing a methylation difference >10% between the left atrium and the average of other tissues were further classified as HSMCs. (B) A total of 33 CpG sites met these criteria and were classified as HSMCs. Heatmap showing the methylation β-values of these 33 HSMCs across 25 different human tissues. (C) Scatter plot showing the β-values of the left atrium (x-axis) compared with the average β-values of 24 other tissues (y-axis) with SD error bars. The orange dot highlights PIH1D1 , representing a CpG site of interest for further investigation. (D) <t>DNA</t> methylation β-values of PIH1D1 (cg02184280) across The Cancer Genome Atlas (TCGA) and The Genomic Data Commons (GDC) data sets, including pan-cancer and breast cancer cohorts. β-values remain below 10% in primary tumors, adjacent normal tissues, and metastatic samples, with higher hypomethylation specificity observed in breast cancer patients. (E) Schematic representation highlighting the CpG site cg02184280 (purple) and the design of methylation-specific PCR (MSP) primers (red) and bisulfite pyrosequencing primers (orange). (F) Gel-based MSP shows amplicons in primary human cardiomyocytes (HCM), induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs), and AC16 cells but not in GES or breast cancer cell lines. Amplicons were also observed in doxorubicin-treated iPSC-CMs, indicating the specificity of the detected CpG sites to cardiac tissue methylation patterns. In vitro methylated human DNA (IVD) was used as a positive control to validate the specificity and efficiency of the methylation-specific primers. “M” indicates detection of methylated PIH1D1 , while a product in the “U” lane indicates unmethylated PIH1D1 .
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Identification and Validation of Heart-Specific Methylated CpG Sites in PIH1D1 (A) Workflow illustrating the selection criteria for heart-specific methylated CpG sites (HSMCs). CpG sites were filtered by comparing methylation levels in the left atrium to those in 24 other tissues. Sites with methylation levels below 10% in noncardiac tissues were first selected. CpG sites showing a methylation difference >10% between the left atrium and the average of other tissues were further classified as HSMCs. (B) A total of 33 CpG sites met these criteria and were classified as HSMCs. Heatmap showing the methylation β-values of these 33 HSMCs across 25 different human tissues. (C) Scatter plot showing the β-values of the left atrium (x-axis) compared with the average β-values of 24 other tissues (y-axis) with SD error bars. The orange dot highlights PIH1D1 , representing a CpG site of interest for further investigation. (D) DNA methylation β-values of PIH1D1 (cg02184280) across The Cancer Genome Atlas (TCGA) and The Genomic Data Commons (GDC) data sets, including pan-cancer and breast cancer cohorts. β-values remain below 10% in primary tumors, adjacent normal tissues, and metastatic samples, with higher hypomethylation specificity observed in breast cancer patients. (E) Schematic representation highlighting the CpG site cg02184280 (purple) and the design of methylation-specific PCR (MSP) primers (red) and bisulfite pyrosequencing primers (orange). (F) Gel-based MSP shows amplicons in primary human cardiomyocytes (HCM), induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs), and AC16 cells but not in GES or breast cancer cell lines. Amplicons were also observed in doxorubicin-treated iPSC-CMs, indicating the specificity of the detected CpG sites to cardiac tissue methylation patterns. In vitro methylated human DNA (IVD) was used as a positive control to validate the specificity and efficiency of the methylation-specific primers. “M” indicates detection of methylated PIH1D1 , while a product in the “U” lane indicates unmethylated PIH1D1 .

Journal: JACC: Basic to Translational Science

Article Title: Methylated PIH1D1 as a Heart-Specific Biomarker for Anthracycline-Induced Cardiac Remodeling in Breast Cancer Patients

doi: 10.1016/j.jacbts.2026.101510

Figure Lengend Snippet: Identification and Validation of Heart-Specific Methylated CpG Sites in PIH1D1 (A) Workflow illustrating the selection criteria for heart-specific methylated CpG sites (HSMCs). CpG sites were filtered by comparing methylation levels in the left atrium to those in 24 other tissues. Sites with methylation levels below 10% in noncardiac tissues were first selected. CpG sites showing a methylation difference >10% between the left atrium and the average of other tissues were further classified as HSMCs. (B) A total of 33 CpG sites met these criteria and were classified as HSMCs. Heatmap showing the methylation β-values of these 33 HSMCs across 25 different human tissues. (C) Scatter plot showing the β-values of the left atrium (x-axis) compared with the average β-values of 24 other tissues (y-axis) with SD error bars. The orange dot highlights PIH1D1 , representing a CpG site of interest for further investigation. (D) DNA methylation β-values of PIH1D1 (cg02184280) across The Cancer Genome Atlas (TCGA) and The Genomic Data Commons (GDC) data sets, including pan-cancer and breast cancer cohorts. β-values remain below 10% in primary tumors, adjacent normal tissues, and metastatic samples, with higher hypomethylation specificity observed in breast cancer patients. (E) Schematic representation highlighting the CpG site cg02184280 (purple) and the design of methylation-specific PCR (MSP) primers (red) and bisulfite pyrosequencing primers (orange). (F) Gel-based MSP shows amplicons in primary human cardiomyocytes (HCM), induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs), and AC16 cells but not in GES or breast cancer cell lines. Amplicons were also observed in doxorubicin-treated iPSC-CMs, indicating the specificity of the detected CpG sites to cardiac tissue methylation patterns. In vitro methylated human DNA (IVD) was used as a positive control to validate the specificity and efficiency of the methylation-specific primers. “M” indicates detection of methylated PIH1D1 , while a product in the “U” lane indicates unmethylated PIH1D1 .

Article Snippet: In vitro methylated human DNA (ZYMO) was used as a positive control for methylation.

Techniques: Biomarker Discovery, Methylation, Selection, DNA Methylation Assay, Derivative Assay, In Vitro, Positive Control