Search

US-20260125759-A1 - BIOMARKERS ASSOCIATED WITH AGING

US20260125759A1US 20260125759 A1US20260125759 A1US 20260125759A1US-20260125759-A1

Abstract

The present invention discloses a novel epigenetic biomarker for detection of aging. Particularly, the present disclosure assesses methylation of the epigenetic biomarkers to determine the aging status of a subject. The present disclosure also discloses primers and probes used herein.

Inventors

  • Hsieh-Tsung SHEN
  • Ruo-Kai LIN

Assignees

  • EG BioMed Co., Ltd.

Dates

Publication Date
20260507
Application Date
20251031

Claims (13)

  1. 1 . A method for detecting an aging status of a subject, wherein the method comprises: (a) extracting (i) cell-free DNA (cfDNA) from a biological sample of the subject or (ii) genomic DNA from a biological sample of the subject, wherein the cfDNA or the genomic DNA comprises a DENND2B DNA and a reference gene DNA; (b) assaying a methylation level of one or more CpG site of the DENND2B DNA from the cfDNA or the genomic DNA; (c) measuring a relative nucleic acid quantity of the reference gene DNA from the cfDNA or the genomic DNA; and (d) determining an aging status of the subject based on a combination of the methylation level and the relative nucleic acid quantity.
  2. 2 . The method of claim 1 , wherein a pair of DENND2B methylation-specific primers used to assay the methylation level comprises SEQ ID NO: 2 and SEQ ID NO: 3, or sequences having at least 85% identity thereto.
  3. 3 . The method of claim 1 , wherein a DENND2B methylation-specific probe used to assay the methylation level comprises SEQ ID NO: 7, or a sequence having at least 85% identity thereto.
  4. 4 . The method of claim 1 , wherein the reference gene DNA comprises a ACTB DNA.
  5. 5 . The method of claim 4 , wherein a pair of primers used to measure the relative nucleic acid quantity comprises SEQ ID NO: 5 and SEQ ID NO: 6, or sequences having at least 85% identity thereto.
  6. 6 . The method of claim 4 , wherein a probe used to measure the relative nucleic acid quantity comprises SEQ ID NO: 8, or a sequence having at least 85% identity thereto.
  7. 7 . The method of claim 1 , wherein the determining the aging status comprises calculating an aging index by binomial regression based on the methylation level and the relative nucleic acid quantity.
  8. 8 . The method of claim 1 , wherein the assay of step (b) is performed using methylation specific PCR (MSP), quantitative methylation-specific PCR (QMSP), bisulfite conversion, bisulfite sequencing (BS), pyrosequencing, microarrays, or methylation DNA immunoprecipitation PCR (MeDIP-PCR).
  9. 9 . The method of claim 1 , wherein the biological sample comprises a liquid biopsy.
  10. 10 . The method of claim 1 , wherein the biological sample comprises plasma or serum.
  11. 11 . A kit for detecting an aging status of a subject, comprising a pair of primers having the sequences of SEQ ID NOs: 2 and 3 and/or a probe having the sequence of SEQ ID NO: 7 for assaying methylation level of a DENND2B DNA from a biological sample of a subject.
  12. 12 . The kit of claim 11 , further comprising a pair of primers having the sequences of SEQ ID NOs: 5 and 6 and/or a probe having the sequence of SEQ ID NO: 8 for measuring a relative nucleic acid quantity of a reference gene DNA from the biological sample.
  13. 13 . The kit of claim 12 , wherein the reference gene DNA comprises a ACTB DNA.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of and priority to U.S. Provisional Patent Application No. 63/715,284, filed 1 Nov. 2024, the contents of which is incorporated by reference in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which is submitted electronically in.xml format and is hereby incorporated by reference in its entirety. The .xml copy, created on Oct. 31, 2025, is named “US16327_SequenceListing.xml” and is 8,362 bytes in size. FIELD OF THE INVENTION The present disclosure relates to the field of gene biomarkers. Particularly, the present disclosure assesses methylation of the gene biomarkers to determine the aging status of a subject. BACKGROUND OF THE INVENTION Aging refers to the gradual decline of essential physiological functions needed for survival and reproduction as time passes. It is a continuous, natural process that starts in early adulthood and impacts every member of a species. This process results from the accumulation of various molecular and cellular damages over time, causing a slow reduction in both physical and cognitive abilities, an increased vulnerability to diseases, and eventually, death. DNA methylation at the fifth carbon position of cytosine (5mC) within CpG dinucleotides is a stable epigenetic modification that plays a crucial role in mammalian development, cell differentiation, and the preservation of cellular identity by regulating gene expression. Aberrant patterns of DNA methylation have been linked to various diseases and health conditions. However, there is still a need for novel epigenetic biomarkers to assess the aging status of a subject. SUMMARY OF THE INVENTION The present invention discloses a novel epigenetic biomarker for detection of aging. Particularly, the present disclosure assesses methylation of the epigenetic biomarkers to determine the aging status of a subject. The present disclosure also discloses primers and probes used herein. Detection of Aging In one embodiment, the present disclosure provides a method for detecting an aging status of a subject, wherein the method comprises: (a) extracting (i) cell-free DNA (cfDNA) from a biological sample of the subject or (ii) genomic DNA from a biological sample of the subject, wherein the cfDNA or the genomic DNA comprises a DENND2B DNA and a reference gene DNA;(b) assaying a methylation level of one or more CpG site of the DENND2B DNA from the cfDNA or the genomic DNA;(c) measuring a relative nucleic acid quantity of the reference gene DNA from the cfDNA or the genomic DNA; and(d) determining an aging status of the subject based on a combination of the methylation level and the relative nucleic acid quantity. In some embodiments, a pair of DENND2B methylation-specific primers used to assay the methylation level of one or more CpG site of the DENND2B DNA comprises sequences having at least 85% identity to SEQ ID NO: 2 and SEQ ID NO: 3. In some embodiments, the pair of DENND2B methylation-specific primers comprises sequences having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 2 and SEQ ID NO: 3. In one embodiment, the pair of DENND2B methylation-specific primers comprises the sequences of SEQ ID NO: 2 and SEQ ID NO: 3. In another embodiment, the pair of DENND2B methylation-specific primers have the sequences of SEQ ID NO: 2 and SEQ ID NO: 3. In some embodiments, a DENND2B methylation-specific probe used to assay the methylation level of one or more CpG site of the DENND2B DNA comprises a sequence having at least 85% identity to SEQ ID NO: 7. In some embodiments, the DENND2B methylation-specific probe comprises a sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 7. In one embodiment, the DENND2B methylation-specific probe comprises the sequence of SEQ ID NO: 7. In another embodiment, the DENND2B methylation-specific probe has the sequence of SEQ ID NO: 7. In some embodiments, the reference gene DNA comprises a ACTB DNA. In some embodiments, a pair of primers used to measure the relative nucleic acid quantity of the reference gene DNA comprises sequences having at least 85% identity to SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the pair of primers used to measure the relative nucleic acid quantity comprises sequences having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID NO: 5 and SEQ ID NO: 6. In one embodiment, the pair of primers used to measure the relative nucleic acid quantity comprises the sequences of SEQ ID NO: 5 and SEQ ID NO: 6. In another embodiment, th