MUC1 VNTR Frameshift Mutation Diagnostic Assay
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Solution Overview
Problem
Current methods fail to accurately diagnose medullary cystic kidney disease type 1 (MCKD1) due to its variable severity, lack of extrarenal manifestations, and the difficulty in identifying the causative genetic mutation, hindering both diagnosis and research.
Innovation Solution
A frameshift mutation in the GC-rich variable number of tandem repeats (VNTR) sequence of the MUC1 gene is identified, and a diagnostic assay is developed to detect a cytosine insertion, allowing for the detection of a predisposition to MCKD1 by analyzing nucleic acid samples using endonuclease digestion, PCR, and probe extension reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional diagnostic methods are used for MCKD1, then the diagnosis process is simple, but the diagnostic accuracy is low due to variable severity and lack of characteristic manifestations
Solution Approach 1:
The diagnostic approach is segmented into multiple independent steps: endonuclease digestion to cleave wild-type sequences, PCR amplification of specific regions, and probe extension reactions. This segmentation allows each step to target specific molecular features, thereby improving diagnostic accuracy while keeping each individual step relatively simple and manageable.
Solution Approach 2:
The endonuclease digestion step performs preliminary action by selectively cleaving wild-type MUC1 sequences before detection. This preliminary processing creates distinct molecular patterns that make subsequent detection of mutant sequences more accurate and reliable, addressing the diagnostic accuracy challenge.
2Reliability
If the MUC1 gene VNTR region is analyzed to detect frameshift mutations, then the diagnostic reliability is improved, but the difficulty of detecting and measuring increases
Solution Approach 1:
The patent introduces several intermediary elements to facilitate detection: restriction endonucleases as intermediaries to cleave DNA at specific sites, PCR primers as intermediaries to amplify the VNTR region, and probes as intermediaries to hybridize with mutant sequences. These intermediaries transform the difficult-to-detect frameshift mutation into detectable molecular patterns, improving reliability while managing detection complexity.
Solution Approach 2:
The patent replaces direct sequencing methods with a molecular biology-based detection system using endonuclease digestion, PCR, and probe extension. This substitution transforms the detection mechanism from direct observation of sequence changes to indirect detection through enzymatic reactions and hybridization, making the detection process more reliable for frameshift mutations.
3Reliability
If living related kidney donation is pursued without genetic testing, then the donation process is faster, but the risk of undetected disease transmission increases
Solution Approach 1:
The diagnostic assay performs preliminary action by detecting the frameshift mutation in potential donors before the donation decision is made. This preliminary genetic screening provides reliable disease status information upfront, enabling informed decisions about donor suitability without delaying the overall donation process significantly.
Solution Approach 2:
The patent enables skipping lengthy and complex comprehensive genetic analyses by targeting specifically the MUC1 gene VNTR region where the frameshift mutation occurs. This focused approach rushes through the essential diagnostic step efficiently, providing rapid and reliable results for donor screening without the time loss associated with broader genetic testing.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The assay effectively differentiates between wild-type and mutant MUC1 sequences, providing a reliable method for diagnosing MCKD1 and potentially informing preventative or therapeutic approaches by targeting the MUC1 gene expression.
Implementation Method 1
performing an endonuclease digestion on a nucleic acid sample, wherein said endonuclease selectively cleaves a wild-type MUC1 nucleic acid sequence and not a mutant MUC1 sequence
Implementation Method 2
performing a PCR reaction on said first plurality of nucleic acid fragments to produced a plurality of amplified nucleic acid products
Implementation Method 3
performing a probe extension reaction on the second plurality of nucleic acid fragments using a probe that specifically binds upstream of the cytosine insertion
Data Source
AI summary
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).