Methods of diagnosing and treating medullary cystic kidney disease

Patent metadata

Field	Value
Publication number	US-10370716-B2
Application number	US-201514618525-A
Country	US
Kind code	B2
Filing date	Feb 10, 2015
Priority date	Aug 10, 2012
Publication date	Aug 6, 2019
Grant date	Aug 6, 2019

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).

First claim

What is claimed is:

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.

2. 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).

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

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

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

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

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

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

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.

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

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

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

13. 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).

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

15. 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.

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

17. The method of claim 1 or 9 , wherein the MUC-1 inhibitor is a flavone having the structure

or a salt thereof, wherein
R 1 is H, —OH, ═O, substituted or unsubstituted alkyl(C 1-8 ), alkoxy(C 1-8 ), haloalkyl(C 1-8 ), substituted phenyl or unsubstituted phenyl, wherein if R 1 is ═O, C 7 -C 8 is a double bond;
R 2 is H, —OH, alkyl(C 1-8 ), substituted phenyl, unsubstituted phenyl, phenyl, phenyl thiazole, imidazole, pyrazole or furan;
R 3 is H, —OH, ═O, halogen, haloalkyl(C 1-8 ), substituted or unsubstituted alkyl(C 1-8 ), substituted phenyl or unsubstituted phenyl, wherein if R 3 is ═O, C 8 -C 9 is a double bond;
R 4 is H or —OH;
R 5 is H, —OH, substituted or unsubstituted alkyl(C 1-8 ) or alkoxy(C 1-8 ), or OR 8 , wherein R 8 is alkyl(C 1-8 ), an ester or an amide;
R 6 is H, —OH, substituted or unsubstituted alkyl(C 1-8 ) or alkoxy(C 1-8 ), or OR 8 , wherein R 8 is alkyl(C 1-8 ), an ester or an amide; and
R 7 is H, —OH, or substituted or unsubstituted alkyl(C 1-8 ), with the proviso that R 1 and R 3 cannot both be ═O.

18. 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.

Assignees

• Broad Inst Inc
• Massachusetts Gen Hospital
• Inst Carlos Slim De La Salud A C

Inventors

• Kirby Andrew
• Gnirke Andreas
• Blumenstiel Brendan
• Defelice Matthew
• Daly Mark

Classifications

• C12Q2600/118

  Prognosis of disease development · CPC title

• C12Q1/6883 Primary

  for diseases caused by alterations of genetic material · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12N15/1138

  against receptors or cell surface proteins · CPC title