SNPs and real-time quantitative PCR method for constitutional allelic copy number determination, the VPREB1 marker case

Abstract

Background22q11.2 microdeletion is responsible for the DiGeorge Syndrome, characterized by heart defects, psychiatric disorders, endocrine and immune alterations and a 1 in 4000 live birth prevalence. Real-time quantitative PCR (qPCR) approaches for allelic copy number determination have recently been investigated in 22q11.2 microdeletions detection. The qPCR method was performed for 22q11.2 microdeletions detection as a first-level screening approach in a genetically unknown series of patients with congenital heart defects. A technical issue related to the VPREB1 qPCR marker was pointed out.MethodsA set of 100 unrelated Italian patients with congenital heart defects were tested for 22q11.2 microdeletions by a qPCR method using six different markers. Fluorescence In Situ Hybridization technique (FISH) was used for confirmation.ResultsqPCR identified six patients harbouring the 22q11.2 microdeletion, confirmed by FISH. The VPREB1 gene marker presented with a pattern consistent with hemideletion in one 3 Mb deleted patient, suggestive for a long distal deletion, and in additional five non-deleted patients. The long distal 22q11.2 deletion was not confirmed by Comparative Genomic Hybridization. Indeed, the VPREB1 gene marker generated false positive results in association with the rs1320 G/A SNP, a polymorphism localized within the VPREB1 marker reverse primer sequence. Patients heterozygous for rs1320 SNP, showed a qPCR profile consistent with the presence of a hemideletion.ConclusionsThough the qPCR technique showed advantages as a screening approach in terms of cost and time, the VPREB1 marker case revealed that single nucleotide polymorphisms can interfere with qPCR data generating erroneous allelic copy number interpretations.

MALDI-TOF-MS Assay to Detect the Hemizygous 22q11.2 Deletion in DNA from Dried Blood Spots

“…Other PCR methods that use DNA extracted from whole blood or cell lines, including real-time quantitative PCR, quantitative interspecies competitive PCR, competitive fluorescent multiplex short tandem repeat polymorphism assay, and multiplex ligation-dependent probe amplification, have been shown to identify the hemizygous deletion of 22q11.2 compared with a reference gene (18–25). These approaches require complex algorithms to compensate for variable amplification efficiency of the reference and target sequences, which could introduce imprecision and affect interpretation of results.…”

PCR Assay Using 22q11.2 Highly Polymorphic Markers for Exclusion of 22q Microdeletion: Technical Optimization and Application in North Africa

“…Recent advances in molecular biology have led to the discovery of a progressively increasing number of microdeletional and microduplicational syndromes through the search for imbalances in the regions delimited by the LCRs using CGH array [21,22]. The same advances have made it possible to broaden the spectrum of techniques enabling their detection and screening on a large scale, namely CGH array, real-time PCR, and the MLPA technique [20,23].…”

Accurate, Fast and Cost-Effective Diagnostic Test for Monosomy 1p36 Using Real-Time Quantitative PCR

“…However, the high cost of this test limits its broad adoption, especially in developing countries [ 13 ]. In face of this reality, we found in qPCR technique the requirements of cost-effectiveness and easy execution for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries [ 13 – 18 ]. Thus, in this study, we report the development of a qPCR assay for the detection of copy number changes in the 1p36 region using two target genes, PRKCZ and SKI , which are deleted in the majority of patients with monosomy 1p36.…”

“…Since a characteristic phenotype has been defined, monosomy 1p36 is amenable to targeted molecular diagnosis. For that, we felt that real-time quantitative PCR (qPCR) met the requirements of cost-effectiveness and easy execution [ 13 – 18 ]. Thus, we report the development of a sensitive, rapid, and affordable qPCR diagnostic method for monosomy 1p36, using two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI .…”