Methods of diagnosing and treating medullary cystic kidney disease

Patent metadata

Field	Value
Publication number	US-2015252423-A1
Application number	US-201514618525-A
Country	US
Kind code	A1
Filing date	Feb 10, 2015
Priority date	Aug 10, 2012
Publication date	Sep 10, 2015
Grant date	—

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 diagnosing or determining a predisposition to developing 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), wherein the presence of said point mutation indicates that the subject has or is predisposed to developing MCKD1.

2 . A method of diagnosing a frameshift mutation in a subject comprising analyzing a sample to determine the presence or absence of a frameshift mutation in the GC-rich variable number of tandem repeats (VNTR) sequence, wherein said frameshift mutation produces a mutant 8G homopolymer.

3 . The method of claim 2 , wherein said frameshift mutation produces a truncated MUC-1 polypeptide having a pI greater that the wildtype MUC-1 polypeptide.

4 . An assay for 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) 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 produced 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. detecting the presence of a reaction product containing the cytosine insertion, 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).

5 . The assay of claim 4 , 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).

6 . The assay of claim 4 , wherein the endonuclease is Mwo1.

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

8 . The assay of claim 4 , wherein the detecting is performed by size exclusion chromatography.

9 . The assay of claim 4 , wherein the detecting is performed by mass spectroscopy.

10 . The assay of claim 4 , wherein the reaction product containing the cytosine insertion is about 5,904 daltons.

11 . The assay of claim 4 , wherein a purification step is performed before step (b), (c) and or (d).

12 . A method of treating or alleviating a symptom of MCKD1 comprising, administering to a subject a compound that inhibits the expression for activity of MUC-1.

13 . The method of claim 12 , wherein the compound is an antisense MUC-1 nucleic acid, a MUC-1 specific short-interfering RNA, or a MUC-1 specific ribozyme.

14 . The method of claim 2 , wherein the sample is a nucleic acid sample.

15 . The method of claim 14 , wherein the nucleic acid is DNA, RNA or cDNA.

16 . The method of claim 2 , wherein the sample is any sample containing the truncated MUC-1 polypeptide.

17 . The method of claim 4 , wherein the nucleic acid sample is DNA, RNA or cDNA.

Assignees

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

Inventors

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

Classifications

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6883 Primary

  for diseases caused by alterations of genetic material · CPC title

• C12N15/1138

  against receptors or cell surface proteins · CPC title

• C12Q2600/118

  Prognosis of disease development · CPC title