Directional genomic hybridization for chromosomal inversion discovery and detection

Abstract

Chromosomal rearrangements are a source of structural variation within the genome that figure prominently in human disease, where the importance of translocations and deletions is well recognized. In principle, inversions—reversals in the orientation of DNA sequences within a chromosome—should have similar detrimental potential. However, the study of inversions has been hampered by traditional approaches used for their detection, which are not particularly robust. Even with significant advances in whole genome approaches, changes in the absolute orientation of DNA remain difficult to detect routinely. Consequently, our understanding of inversions is still surprisingly limited, as is our appreciation for their frequency and involvement in human disease. Here, we introduce the directional genomic hybridization methodology of chromatid painting—a whole new way of looking at structural features of the genome—that can be employed with high resolution on a cell-by-cell basis, and demonstrate its basic capabilities for genome-wide discovery and targeted detection of inversions. Bioinformatics enabled development of sequence- and strand-specific directional probe sets, which when coupled with single-stranded hybridization, greatly improved the resolution and ease of inversion detection. We highlight examples of the far-ranging applicability of this cytogenomics-based approach, which include confirmation of the alignment of the human genome database and evidence that individuals themselves share similar sequence directionality, as well as use in comparative and evolutionary studies for any species whose genome has been sequenced. In addition to applications related to basic mechanistic studies, the information obtainable with strand-specific hybridization strategies may ultimately enable novel gene discovery, thereby benefitting the diagnosis and treatment of a variety of human disease states and disorders including cancer, autism, and idiopathic infertility.Electronic supplementary materialThe online version of this article (doi:10.1007/s10577-013-9345-0) contains supplementary material, which is available to authorized users.

Telomere length and chromosomal instability for predicting individual radiosensitivity and risk via machine learning

“…The possibility of improving assessment of chromosomal instability and associated risk for development of secondary cancers following radiation therapy [14][15][16] was also explored utilizing dGH, which facilitated inversion detection at higher resolution than traditional cytogenetic assays 57,69 . Indeed, inversions were observed at higher frequencies than other types of CAs both before and after radiation therapy (Fig 7A), consistent with prior reports 57,69 . Groups of patients with increased frequencies of chromosomal inversions and fragments (deletions), previously proposed signatures of radiation-induced cancers 55 , were also observed three months post-IMRT (Fig 8A/D).…”

“…We hypothesized that the increased efficiency of dGH for detecting inversions would facilitate scoring fewer metaphase spreads (n=30/time point/patient) than traditional cytogenetic techniques 56 , while still retaining superb sensitivity to individual chromosomal instability, and thus the ability to infer patients at higher risks for secondary cancers. Many significant differences in frequencies of IR-induced rearrangements were observed (Fig 7A-D), with inversions occurring at the highest frequencies, consistent with expectations 57,69 . Interestingly, overall average frequencies of inversions at three months post-IMRT were comparable to the in vitro irradiated samples (Fig 7A).…”

“…Directional Genomic Hybridization (dGH) is a cytogenomics, fluorescence-based methodology for high-resolution detection of chromosome aberrations (CAs) missed even by sequencing 68 , particularly inversions 57,69 . We hypothesized that the increased efficiency of dGH for detecting inversions would facilitate scoring fewer metaphase spreads (n=30/time point/patient) than traditional cytogenetic techniques 56 , while still retaining superb sensitivity to individual chromosomal instability, and thus the ability to infer patients at higher risks for secondary cancers.…”

“…directional Genomic Hybridization (dGH), image acquisition, data processing Protocol: High-resolution detection of chromosome aberrations (inversions, translocations) was performed with directional Genomic Hybridization (dGH) whole chromosome (Cy3) and subtelomere (Cy5) paints to chromosomes 1, 2, and 3 (KromaTiD Inc.) as previously described 57 . Briefly, slides were submersed in Hoechst 33258 (Millipore Sigma #B1155) for 15 min, photolyzed for 35 min using a SpectroLinker UV Crosslinker (365 nm UV), and treated with exonuclease III (New England Biolabs #M0206L) for 30 min.…”

“…Cytogenetic analysis however, is both time and labor intensive, often requiring that hundreds or even thousands of cells be scored, limiting its clinical utility 56 . We speculated that inclusion of an additional type of CA, specifically inversions, now possible using the strand-specific cytogenomic methodology of directional Genome Hybridization (dGH) 57 , might serve to reduce the number of cells required, while also informing potential risks for secondary cancers.…”