Fast detection of deletion breakpoints using quantitative PCR

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.

Complexity of skeletal muscle degeneration: multi-systems pathophysiology and organ crosstalk in dystrophinopathy

“…1 , primary genetic abnormalities in the DMD gene on the short arm of the X-chromosome include small and large deletions, small and large insertions, large duplications, missense point mutations, nonsense point mutations, splice site mutations and mid-intronic mutations [ 28 , 99 ]. Diagnostic testing of these diverse primary abnormalities in the DMD gene can be routinely performed with a variety of genetic techniques [ 297 ], such as (i) diverse types of polymerase chain reaction assays [ 2 ] that mostly focus on the analysis of potential deletions [ 149 ], (ii) comparative genomic hybridisation array technology that can predict whether genetic changes may disrupt the reading frame [ 204 ], (iii) multiplex ligation-dependent probe amplification methods which are capable of swiftly assessing the copy number of exons and related genetic changes [ 292 ] and (iv) next-generation sequencing for the analysis of nonsense or missense types of point mutations, as well as small deletions [ 243 , 255 ]. Genomic sequencing has also been applied to the detailed genetic characterisation of female carriers of the mutated DMD gene [ 368 ].…”