# US 2013/0157958 A1 · Diagnosis of cowden and cowden-like syndrome by detection of decreased killin expression

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- Number: US 2013/0157958 A1
- Document ID: US20130157958A1
- Status: Published application
- Kind: A1
- Application number: 13/721,696
- Filed: 2012-12-20
- Published: 2013-06-20
- Earliest claimed: 2011-12-20
- Assignees: The Cleveland Clinic Foundation
- Inventors: Charis Eng
- Web page: https://patentgrep.com/patent/US20130157958A1

## CPC

- C12Q 1/6883: for diseases caused by alterations of genetic material
- A61K 31/706: containing six-membered rings with nitrogen as a ring hetero atom
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 2600/154: Methylation markers
- C12Q 2600/158: Expression 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
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

A method of diagnosing Cowden syndrome (CS) and Cowden-like Syndrome (CLS) is described. The method includes diagnosing CS and CLS in a subject by identifying a decrease in expression of the KILLIN gene, or by identifying hypermethylation of the KILLIN promoter region. Kits for diagnosing CS and CLS by identifying subjects having KILLIN promoter region hypermethylation and primers specific for a methylated KILLIN promoter region are also described.

## Claims

### Claim 1

A method of diagnosing Cowden syndrome and Cowden-like Syndrome, the method comprising the steps of:
(a) obtaining a biological sample from a subject;
(b) determining the level of expression of the KILLIN gene in the biological sample; and
(c) comparing the level of expression of the KILLIN gene in the biological sample to a control value for KILLIN gene expression;
wherein a lower level of expression of the KILLIN gene in the biological sample relative to the KILLIN expression control value provides a diagnosis that the subject has a substantially increased risk of having Cowden syndrome or Cowden-like syndrome.

### Claim 2 (depends on 1)

The method of claim 1, wherein the level of KILLIN gene expression is measured by detecting the methylation of a KILLIN promoter region, wherein hypermethylation of the KILLIN promoter region indicates a lower level of KILLIN gene expression.

### Claim 3 (depends on 2)

The method of claim 2, wherein the KILLIN promoter region comprises a nucleotide sequence having at least about 85% sequence identify to SEQ ID NO: 1.

### Claim 4 (depends on 2)

The method of claim 2, wherein the KILLIN promoter region comprises a nucleotide sequence having at least about 95% sequence identify to SEQ ID NO: 1.

### Claim 5 (depends on 2)

The method of claim 2, wherein the biological sample is blood.

### Claim 6 (depends on 2)

The method of claim 2, wherein the step of detecting methylation of the KILLIN promoter region includes the step of bringing the KILLIN promoter region into contact with sodium bisulfate under conditions suitable to modify unmethylated cytosine of the KILLIN promoter region into uracil.

### Claim 7 (depends on 2)

The method of claim 2, wherein the step of detecting methylation of the KILLIN promoter region is conducted using combined bisulfate restriction analysis.

### Claim 8 (depends on 7)

The method of claim 7, wherein the combined bisulfate restriction analysis includes use of PCR primers SEQ ID NO: 12 and SEQ ID NO: 13.

### Claim 9 (depends on 1)

The method of claim 1, wherein the subject is human.

### Claim 10 (depends on 1)

The method of claim 1, wherein the subject has one or more symptoms of Cowden syndrome.

### Claim 11 (depends on 10)

The method of claim 10, wherein the one or more symptoms of Cowden syndrome are selected from breast cancer, thyroid cancer, and kidney cancer.

### Claim 12 (depends on 1)

The method of claim 1, further comprising the step of providing a therapeutic intervention for a subject identified as having a substantially increased risk of having Cowden syndrome or Cowden-like syndrome.

### Claim 13

A kit for diagnosing Cowden Syndrome and Cowden-like Syndrome, comprising:
a carrier compartmentalized to include a plurality of containers and to receive a DNA sample including the KILLIN promoter region from a subject therein;
the carrier comprising a first container including sodium bisulfate, and the solvents and reagents necessary to selectively convert unmethylated cytosine of the DNA sample into uracil;
a second container containing a PCR primer pair corresponding to the methylated base sequence of the KILLIN promoter region and the solvents and reagents necessary to obtain an amplified base sequence;
a third container containing a labeled probe complementary to the amplified base sequence; and means for detecting the labeled probe to quantitatively analyze the amount of methylation of the KILLIN promoter region; and a KILLIN promoter region control.

### Claim 13 (depends on 13)

The kit of claim 13, wherein the PCR primer pair is SEQ ID NO: 12 and SEQ ID NO: 13.

### Claim 14 (depends on 13)

The kit of claim 13, wherein the kit further comprises instructions for use of the kit to compare the amount of methylation of the KILLIN promoter region in the DNA sample to a KILLIN promoter region control, wherein hypermethylation of the KILLIN promoter region indicates a diagnosis of Cowden syndrome or Cowden-like Syndrome for the subject.

### Claim 14

A pair of PCR primer sequences for DNA methylation analysis comprising a forward PCR primer and a reverse PCR primer, wherein the primers include from 20 to 25 nucleotides and are effective to amplify SEQ ID NO: 3 using the polymerase chain reaction.

### Claim 15 (depends on 14)

The PCR primer sequences of claim 14, wherein the forward PCR primer is SEQ ID NO: 12 and the reverse PCR primer is SEQ ID NO: 13.

### Claim 16

A method for treating a subject having Cowden syndrome or Cowden-like syndrome by administering to the subject a therapeutically effective amount of a DNA methyltransferase inhibitor.

### Claim 17 (depends on 16)

The method of claim 16, wherein the method further comprises administering a histone deacetylase inhibitor to the subject.

### Claim 18 (depends on 16)

The method of claim 16, wherein the subject has been found to lack germline PTEN mutation.

### Claim 19 (depends on 16)

The method of claim 16, wherein the subject is human.