Resolving a complex structural variant case study

Recent research has demonstrated that complex structural variations (cxSVs) — which involve three or more breakpoint junctions — are considerably more abundant and diverse than previously appreciated, with an average of 14 cxSVs detected in developmental disorder samples¹. The advent of high-throughput genomic technologies has made the identification of cxSVs possible; however, detailed breakpoint analysis is still challenging, even for modern sequencing and microarray platforms.

At the University of Cambridge, UK, Alba Sanchis-Juan and colleagues are studying the association of cxSVs with Mendelian disease². Using short-read sequencing technology they screened 1,342 samples from individuals with neurodevelopmental or retinal disorders to identify cxSVs that overlapped with clinically-important, disease-associated genes. Of the four samples identified, the nature of the variant could be resolved in all but one sample using a combination of short-read sequencing, Sanger sequencing and microarray analysis. Data for the unresolved sample, which came from a female neonate who presented with foetal bradycardia (low heart rate), suggested two alternative models of cxSV structure, in which either one or two disrupted copies of the gene CDKL5, associated with early infantile epileptic encephalopathy, were present (Figure 1).

To characterise the true nature and likely pathogenicity of the cxSV, the team turned to the long read sequencing capabilities offered by nanopore technology.

‘Long-read whole genome sequencing is the best technology to look at breakpoint analysis in repetitive regions’³.

Using a low-pass whole genome sequencing approach on the MinION, a minimum of 4-fold coverage was achieved for all breakpoints, with an average read length of 8kb. The long reads allowed phasing of the aberrant locus to be determined, identifying paternal inheritance. The phased reads also established the presence of an intact and disrupted copy of the CDKL5 gene, providing evidence to support the proposed model 1 (Figure 1). Furthermore, nanopore sequencing confirmed all the novel breakpoint junctions that could not be elucidated using Sanger sequencing due to the repetitive nature of the DNA, which precluded the design of specific primers. Subsequent gene expression analysis demonstrated that both CDKL5 alleles are expressed in the sample, indicating that the variant is likely benign.

Figure 1: Accurate resolution of a cxSV in a neonate sample using nanopore sequencing. Short read sequencing technology detected a duplicationinversion-duplication event that overlapped with CDKL5 but was unable to resolve between two models of cxSV structure and formation. Long nanopore sequencing reads allowed accurate phasing and breakpoint resolution – confirming the existence of one intact and one disrupted copy of CDKL5 and supporting the proposed Model 1. Blue = duplications; green = inversions. Image adapted from Sanchis-Juan et al.³

This case study was taken from the clinical white paper.