# US 12,110,559 B2 · DNA methylation analysis to identify cell type

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- Number: US 12,110,559 B2
- Document ID: US12110559B2
- Status: Granted
- Kind: B2
- Application number: 18/520,939
- Filed: 2023-11-28
- Published: 2024-04-25
- Granted: 2024-10-08
- Earliest claimed: 2021-06-03
- Assignees: Inherent Biosciences, Inc.
- Applicants: Inherent Biosciences, Inc.
- Inventors: Timothy Jenkins; Ryan Barney; Carter Norton
- Web page: https://patentgrep.com/patent/US12110559B2

## CPC

- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 2600/154: Methylation markers
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Disclosed herein are methods of identifying a cell type by its genomic methylation pattern from a biological sample of a human subject. In some cases, the method can comprise identifying sperm and/or prostate cells. Also described herein are methods of identifying cell types for forensic analysis and for the treatment of a disease. The methods disclosed herein may comprise determining the DNA methylation pattern of DNA extracted from a semen sample. Also disclosed herein are methods of treating a disease or condition of a subject.

## Claims

### Claim 1

A method for detecting a sperm cell comprising:
a) obtaining a biological sample from a subject, wherein the biological sample comprises seminal fluid;
b) extracting cell free DNA from the biological sample;
c) detecting a methylation pattern of a region of the extracted cell free DNA from the biological sample with a first computer program, executed on a computer, wherein the methylation pattern comprises the presence or the absence of methylation of at least one region of the extracted cell free DNA, wherein the detecting comprises obtaining methylation data from a presence or an absence of a methylated base by a sodium bisulfite conversion, a sequencing, a differential enzymatic cleavage of DNA, an affinity capture of methylated DNA, an array, or any combination thereof;
d) comparing the methylation pattern of the region of the extracted cell free DNA from the biological sample to a control pattern of DNA methylation with a second computer program, executed on a computer thereby determining the presence or the absence of the sperm cell in the biological sample by the methylation pattern;
e) transferring the result of the presence or the absence of the sperm cell in the biological sample to a user interface on an electronic display via a communication network; and
wherein the at least one region of the extracted cell free DNA comprises chr1: 182921660-182923994, chr1: 18807594-18809765, chr1: 190067323-190068181, chr1:247681102-247681932, chr1:35441734-35443497, chr1:9130738-9132197, chr1:92682167-92683822, chr10:13771107-13771466, chr10:29084707-29085500, chr10:85324700-85325063, chr11:70415188-70416310, chr11:92615215-92617119, chr12:52472758-52474048, chr13:36787282-36789909, chr16:89345548-89346492, chr16:90172237-90173535, chr17:43318045-43319383, chr17:74302589-74304620, chr17:7909776-7910041, chr19:10463037-10465258, chr19:2926081-2927446, chr19:36612371-36612912, chr19:50058328-50060307, chr2: 128158047-128159059, chr20:55684764-55685923, chr22:32749516-32750882, chr22:50008841-50010192, chr3:77145918-77148085, chr3:9027157-9027577, chr4:128650393-128652120, chr4:151500192-151505193, chr4: 189580331-189580876, chr4: 190731443-190732882, chr5:121186479-121188229, chr5:5057256-5057720, chr7:5540163-5541567, chr7:5645728-5648123, chr7:64330493-64331111, chr7:6524925-6525026, chr7:6865886-6867075, chr7:72741781-72743404, chr7:75023589-75024730, chr8:109143682-109144231, chr9: 138590204-138594308, chr9:72129977-72131656, chrX:129471948-129472963, chrX: 134974589-134976103, chrX:31089692-31090940, chrX:40481859-40483213, chrX:42636927-42638572 or any combination thereof.

### Claim 2 (depends on 1)

The method of claim 1, wherein the absence of methylation of the at least one region indicates the presence of the sperm cell in the biological sample.

### Claim 3 (depends on 1)

The method of claim 1, wherein the at least one region comprises a plurality of CpG sites for methylation.

### Claim 4 (depends on 3)

The method of claim 3, wherein at least about 75% of the plurality of CpG sites for methylation are unmethylated.

### Claim 5 (depends on 3)

The method of claim 3, wherein at least about 75% of the plurality of CpG sites for methylation are methylated.

### Claim 6 (depends on 1)

The method of claim 1, wherein the methylation pattern is identified in at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 regions of the extracted cell free DNA.

### Claim 7 (depends on 1)

The method of claim 1, wherein the methylation pattern is identified in at least 3 regions of the extracted cell free DNA.

### Claim 8 (depends on 1)

The method of claim 1, wherein the methylation pattern is identified in at least 5 regions of the extracted cell free DNA.

### Claim 9 (depends on 1)

The method of claim 1, wherein the control pattern of DNA methylation is from a sperm cell.

### Claim 10 (depends on 1)

The method of claim 1, wherein the method further comprises performing a treatment on the subject, wherein the treatment comprises a vasectomy reversal, a vasectomy, a microsurgical testicular sperm extraction (microTESE), a testicular sperm extraction (TESE), a transurethral resection of the ejaculatory ducts (TURED), a circumcision, a surgical correction for scarring, a testosterone replacement, or any combination thereof.

### Claim 11

A forensic analysis method of identifying a sperm cell from an article comprising:
a) extracting cell free DNA from the article;
b) detecting a methylation pattern of a region of the cell free DNA with a first computer program, executed on a computer, wherein the methylation pattern comprises the presence or the absence of methylation of at least one region of DNA of the cell free DNA, wherein the detecting comprises obtaining methylation data from a presence or an absence of a methylated base by a sodium bisulfite conversion, a sequencing, a differential enzymatic cleavage of DNA, an affinity capture of methylated DNA, an array, or any combination thereof;
c) comparing the methylation pattern of the region of the extracted cell free DNA from the article to a control pattern of methylated DNA associated with a sperm cell with a second computer program, executed on a computer;
d) determining the presence or absence of the sperm cell from the article with a third computer program, executed on a computer, wherein the methylation pattern indicates the presence or absence of the sperm cell from the article;
e) transferring the result of the presence or the absence of the sperm cell from the article to a user interface on an electronic display via a communication network, which is a method for forensic analysis; and
wherein the at least one region of the extracted cell free DNA comprises chr1:182921660-182923994, chr1: 18807594-18809765, chr1: 190067323-190068181, chr1:247681102-247681932, chr1:35441734-35443497, chr1:9130738-9132197, chr1:92682167-92683822, chr10:13771107-13771466, chr10:29084707-29085500, chr10:85324700-85325063, chr11:70415188-70416310, chr11:92615215-92617119. chr12:52472758-52474048, chr13:36787282-36789909, chr16:89345548-89346492, chr16:90172237-90173535, chr17:43318045-43319383, chr17:74302589-74304620, chr17:7909776-7910041, chr19: 10463037-10465258, chr19:2926081-2927446, chr19:36612371-36612912, chr19:50058328-50060307, chr2:128158047-128159059, chr20:55684764-55685923, chr22:32749516-32750882, chr22:50008841-50010192, chr3:77145918-77148085, chr3:9027157-9027577, chr4: 128650393-128652120, chr4: 151500192-151505193, chr4:189580331-189580876, chr4:190731443-190732882, chr5:121186479-121188229, chr5:5057256-5057720, chr7:5540163-5541567. chr7:5645728-5648123, chr7:64330493-64331111, chr7:6524925-6525026, chr7:6865886-6867075, chr7:72741781-72743404, chr7:75023589-75024730 chr8: 109143682-109144231, chr9: 138590204-138594308, chr9:72129977-72131656. chrX: 129471948-129472963, chrX:134974589-134976103, chrX:31089692-31090940, chrX:40481859-40483213, chrX:42636927-42638572 or any combination thereof.

### Claim 12 (depends on 11)

The method of claim 11, wherein the article comprises an article of evidence.

### Claim 13 (depends on 12)

The method of claim 12, wherein the article of evidence comprises a clothing, a bed sheet, a weapon, or a tool.

### Claim 14 (depends on 12)

The method of claim 12, wherein the article of evidence is obtained from a sexual assault forensic exam.

### Claim 15 (depends on 11)

The method of claim 11, wherein the absence of methylation of the at least one region indicates the presence of the sperm cell from the article.

### Claim 16 (depends on 11)

The method of claim 11, wherein the methylation pattern comprises the presence or the absence of methylation of at least three regions of the cell free DNA.

### Claim 17 (depends on 11)

The method of claim 11, wherein the methylation pattern is identified in at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 regions of the extracted cell free DNA.