A method for accurate detection of genomic microdeletions using real-time quantitative PCR

Area of Science:

• Genetics
• Molecular Biology
• Genomic Medicine

Background:

• Quantitative Polymerase Chain Reaction (qPCR) is established for gene expression but not widely used for genomic copy number alterations.
• Microdeletions and microduplications are linked to genetic disorders and often detected by Fluorescence In Situ Hybridization (FISH).
• Current cytogenetic techniques may fail to detect suspected constitutional genomic imbalances.

Purpose of the Study:

• To present a novel application of real-time qPCR for detecting chromosomal microdeletions and microduplications.
• To validate this qPCR approach in clinical samples for copy number alterations at 22q11.
• To assess the utility of qPCR as a diagnostic tool for genetic disorders.

Main Methods:

• Developed and applied a real-time qPCR assay using 10 primers spanning the 22q11.2 region.
• Tested the assay on DNA from 12 patients with 22q11 deletion syndrome, 1 patient with 22q trisomy, and 4 controls.
• Utilized in silico modeling for primer design to avoid repetitive DNA regions.

Main Results:

• The qPCR assay accurately detected hemizygous deletions and confirmed FISH results with 100% concordance.
• qPCR allowed for refined mapping of the deletion region at 22q11.
• The assay successfully identified genomic duplication in a chromosome 22 trisomic sample.

Conclusions:

• A novel qPCR method for detecting chromosomal microdeletions and microduplications is presented.
• This qPCR approach offers higher genomic resolution than standard cytogenetic assays.
• Implementing qPCR can replace complex, costly, and time-consuming FISH screening for clinically significant genomic alterations.