# US 11,993,820 B2 · Methods of detecting MLH1 methylation

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- Number: US 11,993,820 B2
- Document ID: US11993820B2
- Status: Granted
- Kind: B2
- Application number: 17/234,297
- Filed: 2021-04-19
- Published: 2021-08-19
- Granted: 2024-05-28
- Earliest claimed: 2016-08-10
- Assignees: Quest Diagnostics Investments Incorporated
- Applicants: Quest Diagnostics Investments LLC
- Inventors: Kevin Qu; Feras Hantash; Amber Donahue
- Web page: https://patentgrep.com/patent/US11993820B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C07K 14/82: Translation products from oncogenes
- C12N 9/16: acting on ester bonds (3.1)
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 2600/112: Disease subtyping, staging or classification
- C12Q 2600/154: Methylation markers
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/16: Primer sets for multiplex assays
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present technology relates to methods for excluding Lynch syndrome as a possible diagnosis in patients suffering from colorectal cancers or endometrial cancers. These methods are based on detecting the methylation status of the MLH1 promoter ‘C’ region in colorectal and endometrial cancer patients using an improved and highly sensitive methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay.

## Claims

### Claim 1

A method for detecting methylation of a promoter in a DNA sample comprising:
(a) incubating a DNA sample with a methylation-sensitive restriction enzyme, wherein a first target nucleic acid sequence in a promoter in the sample comprises a recognition site for the methylation-sensitive restriction enzyme, and a second target nucleic acid sequence in a control gene in the sample comprises a recognition site for the methylation-sensitive restriction enzyme;
(b) incubating the sample with a plurality of probes for querying a plurality of target nucleic acids in the sample, wherein the plurality of probes comprises
(i) a first locus specific probe comprising a first target specific region complementary to the first target nucleic acid sequence in the promoter, wherein the sequence of the first locus specific probe comprises a methylation sensitive restriction site; and
(ii) a second locus specific probe comprising a second target specific region complementary to the second target nucleic acid sequence in the control gene, wherein the sequence of the second locus specific probe does not comprise a methylation sensitive restriction site, and wherein the first locus specific probe and second locus specific probe are detectably labelled;
(c) hybridizing the plurality of probes to the plurality of target nucleic acids in the sample to form a plurality of hybridization complexes;
(d) amplifying the plurality of hybridization complexes to produce a plurality of amplicons, wherein amplification is carried out with a plurality of primer sets comprising:
(i) a first forward primer comprising a region that is complementary to a nucleic acid sequence located 5′ from the first target nucleic acid sequence in the promoter;
(ii) a first reverse primer comprising a region that is complementary to a nucleic acid sequence located 3′ from the first target nucleic acid sequence in the promoter;
(iii) a second forward primer comprising a region that is complementary to a nucleic acid sequence located 5′ from the second target nucleic acid sequence in the control gene; and
(iv) a second reverse primer comprising a region that is complementary to a nucleic acid sequence located 3′ from the second target nucleic acid sequence in the control gene; and
(e) detecting the plurality of amplicons, wherein detecting an amplicon comprising the first target nucleic acid sequence in the promoter indicates methylation of the first target nucleic acid sequence in the promoter in the sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the plurality of probes further comprises a third locus specific probe comprising
a third target specific region complementary to a third target nucleic acid sequence in the control gene, wherein the third target nucleic acid sequence does not contain a recognition site for the methylation-sensitive restriction enzyme; and
wherein the third locus specific probe is detectably labelled.

### Claim 3 (depends on 2)

The method of claim 2, wherein the first locus specific probe, the second locus specific probe and the third locus specific probe are detectably labelled with distinct fluorophores.

### Claim 4 (depends on 3)

The method of claim 3, wherein the fluorophores are selected from the group consisting of FAM, CY5 and HEX.

### Claim 5 (depends on 1)

The method of claim 1, wherein the plurality of primer sets further comprises
a third forward primer comprising a region that is complementary to a nucleic acid sequence located 5′ from the third target nucleic acid sequence; and
a third reverse primer comprising a region that is complementary to a nucleic acid sequence located 3′ from the third target nucleic acid sequence.

### Claim 6 (depends on 1)

The method of claim 1, wherein the methylation-sensitive restriction enzyme is HhaI.

### Claim 7 (depends on 1)

The method of claim 1, wherein the sample is a formalin fixed paraffin-embedded tissue sample.

### Claim 8 (depends on 1)

The method of claim 1, wherein the DNA sample comprises genomic DNA.

### Claim 9 (depends on 1)

The method of claim 1, wherein the sample is whole blood.

### Claim 10 (depends on 1)

The method of claim 1, wherein the sample is derived from a subject diagnosed with colorectal or endometrial cancer.

### Claim 11 (depends on 10)

The method of claim 10, wherein the subject diagnosed with colorectal or endometrial cancer is positive for BRAF V600E.

### Claim 12 (depends on 10)

The method of claim 10, wherein the subject diagnosed with colorectal or endometrial cancer is positive for microsatellite instability (MSI).

### Claim 13 (depends on 1)

The method of claim 1, wherein the sample is derived from a subject suspected of having Lynch syndrome.

### Claim 14 (depends on 13)

The method of claim 13, wherein the subject suspected of having Lynch syndrome displays tumors in one or more of colon, rectum, endometrium, stomach, ovary, urinary tract, and small intestine.

### Claim 15 (depends on 14)

The method of claim 14, wherein the tumors of the subject suspected of having Lynch syndrome show a loss of MLH1 protein expression via immunohistochemical (IHC) methods.

### Claim 16 (depends on 14)

The method of claim 14, wherein the tumors of the subject suspected of having Lynch syndrome are positive for MSI.