# US 10,370,716 B2 · Methods of diagnosing and treating medullary cystic kidney disease

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- Number: US 10,370,716 B2
- Document ID: US10370716B2
- Status: Granted
- Kind: B2
- Application number: 14/618,525
- Filed: 2015-02-10
- Published: 2015-09-10
- Granted: 2019-08-06
- Earliest claimed: 2012-08-10
- Assignees: The Broad Institute, Inc.; The General Hospital Corporation; Instituto Carlos Slim De La Salud, A.C.
- Applicants: The Broad Institute, Inc.; The General Hospital Corporation; Instituto Carlos Slim de la Salud, A.C.
- Inventors: Andrew Kirby; Andreas Gnirke; Brendan Blumenstiel; Matthew Defelice; Mark Daly
- Web page: https://patentgrep.com/patent/US10370716B2

## CPC

- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12N 15/1138: {against receptors or cell surface proteins}
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/156: Polymorphic or mutational 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
- 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 invention features a highly sensitive assay for detecting frameshift mutations for high throughput use. Also provided herein are methods for diagnosing or determining a predisposition for developing medullary cystic kidney disease type 1 (MCKD1) in a subject by detecting a frameshift mutation in the GC-rich variable number of tandem repeats (VNTR) sequence of the mucin 1 gene (MUC-1).

## Claims

### Claim 1

A method of treating a medullary cystic kidney disease type 1 (MCKD1) comprising detecting a point mutation resulting in insertion of a cytosine in the GC-rich variable number of tandem repeats (VNTR) sequence of the coding region of the mucin 1 gene (MUC-1) in a nucleic acid sample, wherein the presence of said point mutation indicates that the subject has or is predisposed to developing MCKD1;
said method comprising:
a. performing an endonuclease digestion on a nucleic acid sample; wherein said endonuclease selectively cleaves a wild-type MUC-1 nucleic acid sequence and not a mutant MUC-1 sequence having a cytosine insertion in the GC-rich variable number of tandem repeats (VNTR) region to produce a first plurality of nucleic acid fragments;
b. performing a PCR reaction on said first plurality of nucleic acid fragments to produce a plurality of amplified nucleic acid products;
c. performing a second endonuclease digestion on the plurality of amplified nucleic acid products, wherein said endonuclease selectively cleaves a wild-type MUC-1 nucleic acid sequence and not a mutant MUC-1 sequence having a cytosine insertion in the GC-rich variable number of tandem repeats (VNTR) region to produce a second plurality of nucleic acid fragments;
d. performing a probe extension reaction on said second plurality of nucleic acid fragments using a probe that specifically binds upstream of the cytosine insertion to produce a plurality of reaction products; and
e. measuring the mass or size of the reaction product and calculating the difference in mass or size from the wild type sequence, thereby determining a cytosine insertion in the GC-rich variable number of tandem repeats (VNTR) sequence of the coding region of the mucin 1 gene (MUC-1), and,
f. administering to a subject in need thereof a MUC-1 inhibitor, wherein the MUC-1 inhibitor is a MUC-1 nucleic acid, a MUC-1 specific short-interfering RNA, a MUC-1 specific ribozyme, a MUC-1 specific peptide inhibitor or a MUC-1 specific small molecule, wherein the MUC-1 inhibitor reduces MUC-1 production in a target cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the endonuclease specifically cleaves a nucleic acid having the nucleic acid sequence GCCCCCCCAGC (SEQ ID NO: 1) and not a mutant sequence having the nucleic acid sequence GCCCCCCCCAGC (SEQ ID NO: 2).

### Claim 3 (depends on 1)

The method of claim 1, wherein the endonuclease is Mwol.

### Claim 4 (depends on 1)

The method of claim 1, wherein said probe comprises the nucleic acid sequence CGGGCTCCACCGCCCCCCC (SEQ ID NO:3).

### Claim 5 (depends on 1)

The method of claim 1, wherein the detecting is performed by mass spectroscopy.

### Claim 6 (depends on 1)

The method of claim 1, wherein the reaction product containing the cytosine insertion is about 5,904 daltons.

### Claim 7 (depends on 1)

The method of claim 1, wherein a purification step is performed before step (b), (c) and or (d).

### Claim 8 (depends on 1)

The method of claim 1, wherein the nucleic acid sample is DNA, RNA or cDNA.

### Claim 9

A method of treating a medullary cystic kidney disease type 1 (MCKD 1) comprising
detecting a point mutation resulting in insertion of a cytosine in the GC-rich variable number of tandem repeats (VNTR) sequence of the coding region of the mucin 1 gene (MUC-1) in a nucleic acid sample, wherein the presence of said point mutation indicates that the subject has or is predisposed to developing MCKD1 and
administering to a subject in need thereof a MUC-1 inhibitor, wherein the MUC-1 inhibitor is a MUC-1 nucleic acid, a MUC-1 specific short-interfering RNA, a MUC-1 specific ribozyme, a MUC-1 specific peptide inhibitor or a MUC-1 specific small molecule,
wherein the MUC-1 inhibitor reduces MUC-1 production in a target cell.

### Claim 10 (depends on 9)

The method of claim 9, wherein the detecting is performed by mass spectroscopy.

### Claim 11 (depends on 9)

The method of claim 9, wherein the nucleic acid sample is DNA, RNA or cDNA.

### Claim 12 (depends on 9)

The method of claim 9 further comprising amplifying a nucleic acid comprising the MUC-1 GC-rich variable number of tandem repeats (VNTR) region.

### Claim 13 (depends on 12)

The method of claim 12 further comprising measuring the mass or size of the amplified nucleic acid and calculating the difference in mass or size from the wild type sequence, thereby determining a cytosine insertion in the GC-rich variable number of tandem repeats (VNTR) sequence of the coding region of the mucin 1 gene (MUC-1).

### Claim 14 (depends on 12)

The method of claim 12, wherein the amplified nucleic acid containing the cytosine insertion is about 5,904 daltons.

### Claim 15 (depends on 1, 9)

The method of claim 1 or 9, wherein the MUC-1 inhibitor is an antisense MUC-1 nucleic acid, a MUC-1 specific short-interfering RNA, or a MUC-1 specific ribozyme.

### Claim 16 (depends on 1, 9)

The method of claim 1 or 9, wherein the MUC-1 inhibitor is Go-201 or Go-203.

### Claim 17 (depends on 1, 9)

The method of claim 1 or 9, wherein the MUC-1 inhibitor is a flavone having the structure
or a salt thereof, wherein
R1 is H, —OH, ═O, substituted or unsubstituted alkyl(C1-8), alkoxy(C1-8), haloalkyl(C1-8), substituted phenyl or unsubstituted phenyl, wherein if R1 is ═O, C7-C8 is a double bond;
R2 is H, —OH, alkyl(C1-8), substituted phenyl, unsubstituted phenyl, phenyl, phenyl thiazole, imidazole, pyrazole or furan;
R3 is H, —OH, ═O, halogen, haloalkyl(C1-8), substituted or unsubstituted alkyl(C1-8), substituted phenyl or unsubstituted phenyl, wherein if R3 is ═O, C8-C9 is a double bond;
R4 is H or —OH;
R5 is H, —OH, substituted or unsubstituted alkyl(C1-8) or alkoxy(C1-8), or OR8, wherein R8 is alkyl(C1-8), an ester or an amide;
R6 is H, —OH, substituted or unsubstituted alkyl(C1-8) or alkoxy(C1-8), or OR8, wherein R8 is alkyl(C1-8), an ester or an amide; and
R7 is H, —OH, or substituted or unsubstituted alkyl(C1-8), with the proviso that R1 and R3 cannot both be ═O.

### Claim 18 (depends on 17)

The method of claim 17, wherein the flavone is Morin, Apigenin, Kaempferol, Fisetin, PD98059, 7-(benzyloxy)-4-(trifluoromethyl)-2H-chromen-2-one or 7-[(3-oxobutan-2-yl)oxy]-4-phenyl-2H-chromen-2-one, or a salt thereof.