Cellular and genomic approaches for exploring structural chromosomal rearrangements

Abstract

Human chromosomes are arranged in a linear and conserved sequence order that undergoes further spatial folding within the three-dimensional space of the nucleus. Although structural variations in this organization are an important source of natural genetic diversity, cytogenetic aberrations can also underlie a number of human diseases and disorders. Approaches for studying chromosome structure began half a century ago with karyotyping of Giemsa-banded chromosomes and has now evolved to encompass high-resolution fluorescence microscopy, reporter-based assays, and next-generation DNA sequencing technologies. Here, we provide a general overview of experimental methods at different resolution and sensitivity scales and discuss how they can be complemented to provide synergistic insight into the study of human chromosome structural rearrangements. These approaches range from kilobase-level resolution DNA fluorescence in situ hybridization (FISH)-based imaging approaches of individual cells to genome-wide sequencing strategies that can capture nucleotide-level information from diverse sample types. Technological advances coupled to the combinatorial use of multiple methods have resulted in the discovery of new rearrangement classes along with mechanistic insights into the processes that drive structural alterations in the human genome.

Keywords: Chromosome rearrangements; Cytogenetics; FISH; Genome instability; Karyotype; Structural variants.

Figure 1.

Historical overview of approaches for detecting and studying human chromosome structure.

Figure 2.. Microscopy approaches for the detection of structural chromosomal rearrangements.

a) Giemsa-banded karyotype derived from an acute lymphoblastic leukemia patient exhibiting two rearrangements. b) Rearrangements detected with DNA paint probes targeting chromosome 15 in colorectal cancer cells following exposure to 2 Gy ionizing radiation. c) Multi-color chromosome painting of a colorectal cancer cell line carrying a rearrangement between chromosomes 10, 13, and 14. d) Schematics of interphase nuclei hybridized to the indicated FISH probes to resolve different types of DNA copy number and structural alterations.

Figure 3.. Detection of rare rearrangement events following DNA double-strand break induction.

a) Schematic to detect intra-chromosomal deletions. A stop cassette flanked by two experimentally-induced double-strand break (DSB) sites is inserted between a promoter and GFP coding sequences. After DSB induction, a deletion will result in GFP expression that can be detected by flow cytometry. b) Schematic to detect intra-chromosomal inversions. GFP coding sequence is placed in an inverted orientation relative to the promoter. After DSB induction, a correct inversion event will result in GFP expression that can be detected by flow cytometry. c) Schematic to detect a translocation. A neomycin resistance gene is split within an intron, each part targeted to two non-homologous chromosomes. DSB induction can result in an inter-chromosomal translocation that brings two separate parts of the neo gene together. The percentage of G418-resistant clones after selection reflects the relative translocation frequency. d) Schematic to detect balanced translocations. Two DSBs are induced at different genomic sites, and non-reciprocal or reciprocal translocations generated after DSB repair can be detected by PCR.

Figure 4.. DNA sequencing technologies for rearrangement detection.

Comparison between a) short-read sequencing using paired-end analysis of discordant and split reads, and b) single-molecule long-read sequencing technology.