Rapid and Inexpensive Screening of Genomic Copy Number Variations Using a Novel Quantitative Fluorescent PCR Method

Martin Stofanko,Sarah H Elsea,Higgor Gonçalves-Dornelas,Heloísa B Pena,Sérgio Danilo Junho Pena,Joan C Han

doi:10.1155/2013/704917

Abstract

Detection of human microdeletion and microduplication syndromes poses significant burden on public healthcare systems in developing countries. With genome-wide diagnostic assays frequently inaccessible, targeted low-cost PCR-based approaches are preferred. However, their reproducibility depends on equally efficient amplification using a number of target and control primers. To address this, the recently described technique called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR) was shown to reliably detect four human syndromes by quantifying DNA amplification in an internally controlled PCR reaction. Here, we confirm its utility in the detection of eight human microdeletion syndromes, including the more common WAGR, Smith-Magenis, and Potocki-Lupski syndromes with 100% sensitivity and 100% specificity. We present selection, design, and performance evaluation of detection primers using variety of approaches. We conclude that MQF-PCR is an easily adaptable method for detection of human pathological chromosomal aberrations.

Highlights

We present the development of Microduplication Quantitative Fluorescent PCR (MQF-PCR) primer pairs to detect several other microdeletion syndromes: Wilms tumour, aniridia, genitourinary anomalies, and mental retardation syndrome (WAGR; OMIM: 194072), Smith-Magenis (OMIM: 182290), Potocki-Lupski (OMIM: 610883), microdeletions at Xp22.3 (OMIM: 308100, 308700), Langer-Giedion (OMIM: 150230), Greig cephalopolysyndactyly (OMIM: 175700), Miller-Dieker lissencephaly (OMIM: 247200), and Saethre-Chotzen (OMIM: 101400) syndromes
In the detection of the WAGR syndrome (Figure 1(b)), we observed in normal controls a ratio close to 1 : 1 of the peak that corresponds to the WAGR critical region at 11p13 and the control peak at 17q22
In order to correct for the observed uneven amplification efficiency, we normalized our data to a reference sample of mixed DNA from 100 individuals that was processed at the same time [5]

Summary

Introduction

Repetitive DNA accounts for more than 50% of human genome [1] and is one of the primary driving forces of evolution of all eukaryotes. The resulting pathological copy number variations (CNVs) [2] include microscopic and submicroscopic deletions and duplications (microdeletions and microduplications) that have been implicated as etiologic factors in many human clinical syndromes. Molecular diagnosis of these aberrations has significantly evolved in resolution from simple metaphase karyotyping to genomewide molecular screens [3, 4]. We developed assays for 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. We present the development of MQF-PCR primer pairs to detect several other microdeletion syndromes: Wilms tumour, aniridia, genitourinary anomalies, and mental retardation syndrome (WAGR; OMIM: 194072), Smith-Magenis (OMIM: 182290), Potocki-Lupski (OMIM: 610883), microdeletions at Xp22.3 (OMIM: 308100, 308700), Langer-Giedion (OMIM: 150230), Greig cephalopolysyndactyly (OMIM: 175700), Miller-Dieker lissencephaly (OMIM: 247200), and Saethre-Chotzen (OMIM: 101400) syndromes

Materials and Methods

Results and Discussion

Conclusion

Disclosure