Fast detection of deletion breakpoints using quantitative PCR.

Gulshara Abildinova,Leon Raskin,Helena Tuchinsky,Albert Pinhasov,Zhanara Abdrakhmanova,Elimelech Nesher

doi:10.1590/1678-4685-gmb-2015-0159

Abstract

The routine detection of large and medium copy number variants (CNVs) is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories.

Highlights

The number of reported single nucleotide variants and small indels has grown significantly since completion of the Human Genome Project (Naidoo et al, 2011)
multiple ligation probe amplification (MLPA) analysis of two boys (6 and 8 years old) with symptoms of DMD identified a deletion between exons 44 and 51 of the DMD gene that was characterized by the absence of the probe signal at the expected positions for exons 45-50 (Figure 1)
We demonstrated the usefulness of our strategy by identifying the breakpoints of a hemizygotic deletion in exons 45-50 of the DMD gene

Summary

Introduction

The number of reported single nucleotide variants and small indels has grown significantly since completion of the Human Genome Project (Naidoo et al, 2011). The list of medium and large germline insertions, deletions, inversions and translocations is far from complete (MacDonald et al, 2014). The Database of Genomic Variants (MacDonald et al, 2014) established to catalogue copy number variations (CNVs) larger than 50 bp contains millions of CNVs with median size alterations of 1-10 kb. For the majority of CNVs, the database provides the confidence interval where the breakpoints likely reside, but no exact deletion or insertion breakpoints are known. Variation in the size of a deletion in Williams syndrome involves different genes that contribute to distinct phenotypes of this multisystem disorder (Ibn-Salem et al, 2014)

Methods

Results

Conclusion