Granted Patent B2

US 12,227,810 · App. 18/430,156 · Granted Feb 18, 2025

Inventors: Sanford D. Markowitz (Pepper Pike, OH); Helen Moinova (Beachwood, OH); Amitabh Chak (University Heights, OH); Joseph Willis (Shaker Heights, OH); Thomas LaFramboise (Shaker Heights, OH)

Assignee: Case Western Reserve University

C12Q1/6886C12Q1/6827C12Q1/6853G01N33/574C12Q2600/154C12Q2600/156

Abstract

Claims (20)

1. A method for classifying an esophageal sample from a human subject as a methylated sample, comprising:

providing an esophageal sample obtained from the subject and treating with bisulfite to generate a plurality of bisulfite converted SqBE18 nucleic acid sequences;

amplifying the plurality of bisulfite converted SqBE18 nucleic acid sequences to generate amplicons for each bisulfite converted SqBE18 nucleic acid, wherein each amplicon is a read and wherein the amplicon comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NOs: 8318, 8360, 8332, and/or 8374, or a fragment thereof, said fragment comprising at least 50 nucleotides in length;

measuring the number of methylated cytosines of the CpG dinucleotides in each amplicon of the bisulfite converted SqBE18 nucleic acid sequence, wherein a read is classified as a methylated read when at least 70% of the cytosines in the CpG dinucleotides of the individual amplicon of the bisulfite converted SqBE18 nucleic acid sequence are methylated; and,

calculating a percentage of total reads that are methylated reads from the amplifying and measuring steps wherein the esophageal sample obtained from the human subject has at least 0.1% of the total reads that are methylated reads, whereby the esophageal sample obtained from the subject is classified as a methylated sample.

2. The method of claim 1 , further comprising sequencing each of the amplicons.

3. The method of claim 1 , wherein the amplified portion comprises 21 dinucleotides that correspond to or are derived from 21 CpG dinucleotides present in the native non-bisulfite converted SqBE18 genomic sequence.

4. The method of claim 1 , wherein the amplified portion comprises the nucleotide sequence of SEQ ID NOs: 8318, 8360, 8332 and/or 8374.

5. The method of claim 1 , wherein the esophageal sample obtained from the subject is classified as a methylated sample when 1% to 3.5% of the reads are classified as methylated reads.

6. The method of claim 4 , wherein the esophageal sample obtained from the subject is classified as a methylated sample when 1% to 3.11% of the reads are classified as methylated reads.

7. The method of claim 1 , wherein the esophageal sample obtained from the subject is classified as a methylated sample when at least 0.5% of the reads are classified as methylated reads.

8. The method of claim 1 , wherein the esophageal sample obtained from the subject is classified as a methylated sample when at least 0.76% of the reads are classified as methylated reads.

9. The method of claim 1 , wherein the amplicon comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NOs: 8318, 8360, 8332 and/or 8374.

10. The method of claim 1 , further comprising obtaining the esophageal sample by cytology brushing.

11. The method of claim 1 , further comprising obtaining the esophageal sample by balloon.

12. The method of claim 1 , wherein the amplifying is performed using PCR.

13. The method of claim 1 , wherein the amplifying comprises use of methylation-specific primers.

14. The method of claim 1 , wherein the amplifying comprises use of methylation indifferent PCR primers.

15. The method of claim 14 , wherein the amplifying comprises use of primers that are both methylation indifferent and bisulfite specific.

16. The method of claim 1 , wherein the measuring the number of methylated cytosines of the CpG dinucleotides in each amplicon is performed using sequencing wherein each cytosine base in an original template is counted as methylated or unmethylated.

Assignments (1)

Continuity (4)

References Cited (95)