Integration of Newer Genomic Technologies into Clinical Cytogenetics Laboratories

Abstract

Over the past several decades, clinical cytogenetics has branched out from the use of light microscopy and examination of banded chromosomes to embrace multiple newer techniques, including fluorescence in situ hybridization (FISH), multiple generations of microarray designs, as well as the newest technologies, namely, optical genome mapping (OGM) and genomic proximity mapping (GPM). While these newer technologies have had an increasingly molecular genetic focus over time, they are still rooted in the field of cytogenetics, the genetics of the single cell. This review provides a brief overview of the earliest, as well as the most recent, techniques available to clinical cytogenetics laboratories for both constitutional and neoplastic testing and discusses some advantages and disadvantages of each.

Keywords: chromosomal microarray; cytogenetics; fluorescence in situ hybridization (FISH); genomic proximity mapping; optical genome mapping.

Figure 1

Comparison of G-banding, FISH, and SNP array (CMA) results from a patient with B-cell acute lymphoblastic leukemia (B-ALL). (A) Karyogram showing the classic t(9;22) rearrangement. (B) Interphase FISH showing BCR::ABL1 positivity with dual-color/dual-fusion FISH signals in upper panel and, in lower panel showing IKZF1 FISH with partial loss of IKZF1, with one diminished red signal. (C) CMA showing two adjacent IKZF1 deletions, which is consistent with the diminished red signals observed by FISH. (D) CMA showing CDKN2A and CDKN2B with heterozygous (Left) and homozygous deletions (Right). Note lack of copy-number abnormality at 9q34 with balanced t(9;22) rearrangement. These results are consistent with B-ALL, IKZF1(+) and poor prognostic risk.

Figure 2

(A) Experimental workflow of optical genome mapping (OGM). (B) Identification of distinct variant classes by OGM (Figure provided courtesy of Bionano. © 2021–2025 Bionano Genomics, Inc. Used by permission. All rights reserved).

Figure 3

OGM corrected previously incorrect interpretations due to low resolution of karyotyping in two cases. (A) In a multiple myeloma case, FISH testing shows a gain of one copy of FGFR3 on 4p [23/50 cells]; (B) OGM revealed that the gain observed by FISH was by a t(4;5)(p16.3; p13.3), with the breakpoint at 4p16.3 that overlaps the region of FISH dual-fusion probe used for detecting FGFR3 gain. The breakpoint of the translocation did not disrupt the FGFR3 gene. (C) In a CLL case, karyotyping detected dic(21;21)(q22; p11.2)[12/20], and interphase FISH with RUNX1 probe showed gain of RUNX1. (D) OGM showed the dic(21;21) as most likely a der(21)ins(21;1)(q22.12; q?21.1q23.1)inv dup(q?21.1q23.1) with a copy number gain at the breakpoint on chromosome 1 (Adapted from [28] with Open Access).

Figure 4

Principles of genomic proximity mapping (GPM). (A) Cellular samples are collected from patients and subjected to crosslinking while still intact, freezing native three-dimensional chromatin conformation in place prior to proximity ligation and library generation. (B) The frequency that pairs of sequences physically interact is governed primarily by their distance along the linear length of a chromosome. Using this information, the CytoTerra (Phase Genomics) variant callers can identify abnormalities in chromosome structure. (C) A visual guide to how classes of chromosome aberrations appear on the GPM sequence interaction matrix. Genomic coordinates are mirrored on X and Y axes while sequence interaction frequency is represented with increasing intensity on the heat map. Using a combination of interaction frequency and sequencing coverage depth, GPM can identify every major class of structural variation (Figure provided courtesy of Phase Genomics, Inc.).

Figure 5

GPM results for a patient with multiple constitutional inter-chromosomal rearrangements, including ins(4;7) and t(7;11). (A) Genome-wide differential heatmap showing interactions between chromosomes 4 and 7, circled upper left, and chromosomes 7 and 11, circled mid-right. Red regions indicate increased interactions, with blue regions indicating reduced interactions. (B) Corresponding chromosome-specific heatmaps and idiograms for the 4;7 and 7;11 rearrangements. The ins(4;7) is indicated in the upper panel by increased interaction intensity between chromosome 4p and chromosome 7q in the Hi-C heatmap, with the t(7;11) in the lower panel showing increased interaction intensity between 7q and 11q. Representative idiograms are shown on the right (Adapted from [3] with Open Access).