Simple, Rapid and Inexpensive Quantitative Fluorescent PCR Method for Detection of Microdeletion and Microduplication Syndromes

Martin Stofanko,Pricila Silva Cunha,Higgor Gonçalves-Dornelas,Heloísa B Pena,Angela M Vianna-Morgante,Sérgio Danilo Junho Pena,Osman El-Maarri

doi:10.1371/journal.pone.0061328

Martin Stofanko, Pricila Silva Cunha + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0061328

Copy DOI

Abstract

Because of economic limitations, the cost-effective diagnosis of patients affected with rare microdeletion or microduplication syndromes is a challenge in developing countries. Here we report a sensitive, rapid, and affordable detection method that we have called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR). Our procedure is based on the finding of genomic regions with high homology to segments of the critical microdeletion/microduplication region. PCR amplification of both using the same primer pair, establishes competitive kinetics and relative quantification of amplicons, as happens in microsatellite-based Quantitative Fluorescence PCR. We used patients with two common microdeletion syndromes, the Williams-Beuren syndrome (7q11.23 microdeletion) and the 22q11.2 microdeletion syndromes and discovered that MQF-PCR could detect both with 100% sensitivity and 100% specificity. Additionally, we demonstrated that the same principle could be reliably used for detection of microduplication syndromes, by using patients with the Lubs (MECP2 duplication) syndrome and the 17q11.2 microduplication involving the NF1 gene. We propose that MQF-PCR is a useful procedure for laboratory confirmation of the clinical diagnosis of microdeletion/microduplication syndromes, ideally suited for use in developing countries, but having general applicability as well.

Highlights

Microdeletion and microduplication syndromes comprise a large group of human diseases that arise from imbalance in the transcription of genes due to partial loss or gain of genetic material of, typically, less than 5 Mb [1]
We show that the principle of the HGQ-PCR method of Lee et al [17] can be used as a simple, rapid, DNA quality independent and internally controlled PCR-based detection test for sub-microscopic genome rearrangements that we have called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR)
We propose that MQF-PCR is a very useful procedure for laboratory confirmation of the clinical diagnosis of microdeletion/microduplication syndromes

Summary

Introduction

Microdeletion and microduplication syndromes comprise a large group of human diseases that arise from imbalance in the transcription of genes due to partial loss or gain of genetic material of, typically, less than 5 Mb [1]. But frequently encompass mental retardation, autism, physical dysmorphism and/or organ malformations. Many microdeletion or microduplication syndromes can be diagnosed on the basis of phenotype alone, but molecular confirmation is essential for correct and reliable clinical and genetic prognoses. A variety of techniques can be used, ranging from microscopic methods, such as Fluorescence in situ Hybridization (FISH), to molecular methods involving PCR and genome-wide microarrays [2,3]. The most important nontargeted approaches are Array Comparative Genomic Hybridization (aCGH) and Single Nucleotide Polymorphism (SNP) arrays

Methods

Results

Conclusion