Genomic copy number variation

Unravelling its etiology and role in human disease

General Background
Microarray-based high-resolution genome profiling technologies have taken the resolution to detect genomic copy number variation (CNV) to a level 100-1000 times higher than achievable by conventional karyotyping. The current resolution of commercially available oligonucleotide-based microarrays allows the detection of all CNVs larger than 50 kb in size. We, and others, have successfully applied these high-resolution genome-wide technologies in clinical genetics. This has led to (i) a significant increase in diagnostic yield for patients with unexplained mental retardation (MR), (ii) the identification of genes causative for sporadic malformation syndromes, and (iii) the identification of novel microdeletion syndromes. In addition to these pathogenic CNVs, extensive DNA copy number variation has recently been identified in healthy individuals (benign CNVs). These CNVs are anticipated to contribute to normal phenotypic variation as well as to susceptibility to multifactorial disease.
As yet, little is known about the occurrence of CNVs. It is, however, known that certain genomic architectural features, such as low-copy repeats (LCRs), may facilitate CNV formation. Due to extensive sequence homology, LCRs can mediate meiotic misalignment, followed by a molecular mechanism referred to as non-allelic homologous recombination (NAHR). Most clinically recognizable microdeletion syndromes result from NAHR between LCRs and are, therefore, termed genomic disorders. As such, these CNVs do not represent random events but, instead, reflect the underlying genomic architectural features. For the majority of recently identified rare pathogenic CNVs, however, the mechanism is unknown. Since genomic disorders taught us that CNVs do not occur in random genomic locations, it may be expected that additional genomic architectural features play a key role in the formation of rare pathogenic CNVs. The spectrum of these genomic features is, however, poorly explored. It is expected that these features include non-B DNA structures, common fragile sites, recombination hotspots, evolutionary conserved breakpoint regions, and various recombination-associated motifs.

Identification and characterization of (novel) genomic architectural features and molecular mechanisms underlying structural variation to better understand genomic (in)stability.

Project 1
  • In-depth molecular analysis of CNV breakpoints of rare pathogenic CNVs for genomic architectural features
  • Custom-made tiling resolution arrays for breakpoint mapping, followed by breakpoint-spanning PCRs
  • Bioinformatic analyses for architectural features such as low-copy repeats, smaller inverted repeats, mirror repeats, CG-tracts and sequence motifs
  • Deduct molecular mechanisms based on the breakpoint analyses and genomic features identified

Project 2
  • Optimize next generation sequencing protocol for the detection of genomic structural variation
  • Compare SV variants from next generation sequencing with CNV data obtained by 2.7M Affymetrix arrays and determine the specificity and sensitivity of NGS data
  • Optimize CNV detection using NGS data from fragment runs
  • Analyze MR-associated CNVs using Roche 454 sequencing
  • Use next generation sequencing data together with the approaches of project 1 (see above) to identify molecular mechanisms for CNV formation

Project description
Molecular and mechanistic characterization of CNVs associated with mental retardation
Recently, a model for CNV formation has implicated mitotic replication-based mechanisms, such as (alternative) non-homologous end joining and fork stalling and template switching, in the etiology of human pathogenic CNVs. The extent to which such mitotic mechanisms contribute to rare pathogenic CNVs remains to be determined. In addition, it is unexplored whether genomic architectural features such as repetitive elements or sequence motifs associated with DNA breakage stimulate the formation of rare pathogenic CNVs. To this end, we have sequenced breakpoint junctions of 30 rare pathogenic microdeletions and eight tandem duplications, representing the largest series of such CNVs examined to date in this much detail. For these breakpoints we determined various genomic architectural features, including the presence of microhomology, repetitive elements and sequence junction motifs. Our results suggest that rare pathogenic microdeletions and tandem duplications do not occur at random genome sequences, but are stimulated and potentially catalyzed by various genomic architectural features.

In-depth analysis of structural variation to better understand genomic (in)stability
For several clinical syndromes, it is known that the presence of a structural variant, such as an inversion, may predispose to the generation of microdeletions and microduplications. For example, the 17q21.31 microdeletion is associated with the presence of a 900-kb inversion in one of the parents. This region of the genome, thus, is more prone to genomic instability compared to other regions of the genome. As such, studying the nature of these inversions is of utmost importance to understand genome (in)stability.
Major advances in DNA sequencing technologies, collective termed next-generation sequencing (NGS) technologies, are now enabling the comprehensive analysis of whole genomes for all types of structural variation, including the previously undetectable balanced inversions. As such, NGS technologies are currently used to explore how genomic architecture predispose structural variation and increased genome instability.

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