Noninvasive Fetal Aneuploidy Detection via cfDNA Sequencing
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Solution Overview
Problem
Current methods for diagnosing genetic abnormalities, such as copy number variations, are invasive, lack sensitivity, and are limited by sequencing biases, making them inadequate for noninvasive prenatal diagnostics and cancer monitoring.
Innovation Solution
A statistical approach that sequences nucleic acids from maternal samples to determine fetal chromosomal aneuploidies by calculating single chromosome doses and comparing them to threshold values, using sequence tags and normalizing chromosome sequences to identify copy number variations without the need for cell culture.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional invasive procedures (amniocentesis) are used to obtain cells for karyotype analysis, then diagnostic information can be obtained, but the procedure carries risks and requires invasive sampling
Solution Approach 1:
The patent uses circulating cell-free DNA (cfDNA) as an intermediary substance that carries genetic information from the fetus to the maternal bloodstream. By sequencing and analyzing cfDNA fragments in maternal blood, the invention enables noninvasive detection of fetal chromosomal aneuploidies, eliminating the need for invasive procedures like amniocentesis while maintaining diagnostic reliability
Solution Approach 2:
The patent replaces the mechanical invasive sampling process (needle insertion for amniocentesis) with a molecular biology approach using next-generation sequencing technology. Instead of physically extracting cells, the method sequences DNA fragments already present in maternal circulation, substituting a mechanical intervention with a biochemical analysis
2Measurement precision
If fluorescence in situ hybridization (FISH) is used for molecular-cytogenetic analysis, then copy number variations can be detected, but the method has insufficient sensitivity due to limited cfDNA levels
Solution Approach 1:
The patent segments the genome into numerous small genomic loci that can be independently targeted and sequenced. By dividing the genomic analysis into many small targets across the genome, the method increases the total number of detectable cfDNA fragments, thereby improving sensitivity despite limited cfDNA availability
Solution Approach 2:
The patent changes the detection parameter from whole-chromosome or large-region analysis (as in FISH) to small-locus-specific sequencing. This parameter change allows the method to detect copy number variations with higher sensitivity by accumulating statistical evidence from many small genomic regions rather than relying on limited large-region signals
3Measurement precision
If array-Comparative Genomic Hybridization (array-CGH) is used for genetic analysis, then chromosomal abnormalities can be identified, but the method suffers from sequencing bias inherent in genomic information
Solution Approach 1:
The patent changes from hybridization-based detection (array-CGH) to direct sequencing-based detection. By using next-generation sequencing to directly count and analyze cfDNA fragments at specific genomic loci, the method eliminates the hybridization biases inherent in array-CGH, providing more accurate and unbiased copy number variation detection
Solution Approach 2:
The patent substitutes the hybridization mechanism of array-CGH with a sequencing mechanism. Instead of relying on probe-target hybridization efficiency (which introduces bias), the method uses direct DNA sequencing and fragment counting, replacing a bias-prone mechanical process with a more accurate molecular counting approach
4Reliability
If cell culture methods are used for karyotype analysis, then chromosomal abnormalities can be detected, but the process requires time-consuming cell proliferation
Solution Approach 1:
The patent performs preliminary action by analyzing circulating cell-free DNA that is already present in the maternal bloodstream at the time of sampling. Instead of waiting for cells to proliferate in culture, the method directly sequences and analyzes the genetic material already available, eliminating the time-consuming cell culture step while maintaining diagnostic accuracy
Data Source
AI summary
The invention provides a method for determining copy number variations (CNV) of a sequence of interest in a test sample that comprises a mixture of nucleic acids that are known or are suspected to differ in the amount of one or more sequence of interest. The method comprises a statistical approach that accounts for accrued variability stemming from process-related, interchromosomal and inter-sequencing variability. The method is applicable to determining CNV of any fetal aneuploidy, and CNVs known or suspected to be associated with a variety of medical conditions. CNV that can be determined according to the method include trisomies and monosomies of any one or more of chromosomes 1-22, X and Y, other chromosomal polysomies, and deletions and/or duplications of segments of any one or more of the chromosomes, which can be detected by sequencing only once the nucleic acids of a test sample.