Optical genome mapping enables constitutional chromosomal aberration detection

Abstract

Chromosomal aberrations including structural variations (SVs) are a major cause of human genetic diseases. Their detection in clinical routine still relies on standard cytogenetics. Drawbacks of these tests are a very low resolution (karyotyping) and the inability to detect balanced SVs or indicate the genomic localization and orientation of duplicated segments or insertions (copy number variant [CNV] microarrays). Here, we investigated the ability of optical genome mapping (OGM) to detect known constitutional chromosomal aberrations. Ultra-high-molecular-weight DNA was isolated from 85 blood or cultured cells and processed via OGM. A de novo genome assembly was performed followed by structural variant and CNV calling and annotation, and results were compared to known aberrations from standard-of-care tests (karyotype, FISH, and/or CNV microarray). In total, we analyzed 99 chromosomal aberrations, including seven aneuploidies, 19 deletions, 20 duplications, 34 translocations, six inversions, two insertions, six isochromosomes, one ring chromosome, and four complex rearrangements. Several of these variants encompass complex regions of the human genome involved in repeat-mediated microdeletion/microduplication syndromes. High-resolution OGM reached 100% concordance compared to standard assays for all aberrations with non-centromeric breakpoints. This proof-of-principle study demonstrates the ability of OGM to detect nearly all types of chromosomal aberrations. We also suggest suited filtering strategies to prioritize clinically relevant aberrations and discuss future improvements. These results highlight the potential for OGM to provide a cost-effective and easy-to-use alternative that would allow comprehensive detection of chromosomal aberrations and structural variants, which could give rise to an era of "next-generation cytogenetics."

Keywords: CNV microarray; FISH; OGM; breakpoint characterization; chromosomal aberration; constitutional aberrations; cytogenetics; karyotyping; optical genome mapping; structural variants.

Conflict of interest statement

Bionano Genomics provided a portion of the reagents used for this manuscript. Other than this, the authors declare no competing interests.

Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Description of the study population and the detected aberrations (A) Main reason for referral. (B) Distribution of the different cytogenetic and molecular tests used for diagnosis. (C) Distribution of chromosomal aberrations as assessed by standard of care genetic investigations. (D) CNV detection and filtering. Average number of CNVs detected per sample before and after applying an FCN filtering (2.8 for duplications). (E) SV detection and filtering. Average number of SVs detected per sample before and after applying sequential filtering steps: rare variants only (not found in a control database including 204 human samples), rare variants larger than 20 kb, and rare variants larger than 20 kb and that overlap with genes. Error bars in (D) and (E) represent standard deviations.

Figure 2

Results from representative examples of different chromosomal aberrations (A) Sample 1. Left: CNV microarray data showing an 8p22p21.3 deletion (18,825,888–19,364,764 bp). Middle: genome-wide circos plot showing chromosome 8. The CNV profile represented by the blue line shows the deletion (black circle). Right: sample genome map against reference, showing the deletion and affected genes (hg19). (B) Sample 18. Left: karyogram showing a reciprocal translocation between chromosomes 5 and 8, t(5;8)(p13.1;p11.2). Middle: circos plot showing chromosomes 5 and 8 connected with a pink line, representing the translocation. Right: genome map, of which the left part maps to chromosome 8 and the right part to chromosome 5. (C) Sample 15. Left: karyogram showing an inversion on chromosome 13 (red arrow). Middle: circos plot showing the inversion represented by a pink line connecting two distal regions on chromosome 13. Right: genome map that is partly inverted when compared to the reference. One of the breakpoints is most likely interrupting KLHL1. (D) Sample 39. Left: karyogram showing one chromosome X and a small marker. Middle: circos plot of chromosome X. The CNV plot in blue shows a large loss of material on most of the p- and q-arms, and the pink line connecting the distal regions of the non-deleted segment indicates the presence of a ring chromosome. Right: different genome maps (dark blue bars on top and below the reference) indicating the presence of the ring chromosome. The assembled genome map below the reference shows that the left part (light green bar) maps to a region upstream of the centromere, whereas the right part (light blue bar) maps to a region downstream of the centromere.

Figure 3

Isodicentric Y chromosomes show specific assembly map patterns (A) G-banding with trypsin-Giemsa (GTG) and reverse banding using heat and Giemsa (RHG) of X and Y chromosomes of sample 57. (B) FISH for sample 57 via fluorescently labeled probes TelXp/Yp (green) and RP11-209I11 (red, located at Yq11.223). (C) Genome maps of Y chromosomes of samples 69 (normal chromosome Y), 55, 57, 27, and 79. Dotted red boxes indicate where isodicentric Y chromosomes have no coverage when compared to non-isodicentric Y chromosomes (the top genome map) corresponding to the deleted segment. Most likely the genome maps of the four isochromosomes by optical genome mapping point to three different breakpoints (deletion starts), (sample 55, 21.0 Mb; samples 57 and 79, 20.1 Mb; and sample 27, 19.9 Mb). The first three breakpoints coincide with palindrome 5 (hg19, chrY: 19,567,684–20,063,140 bp), which may be involved in the mediation of the isochromosomes or cause issues in a more precise assembly with the current optical genome mapping. The deletion in sample 55 locates in direct proximity of palindrome 4 (hg19, chrY: 20,612,099–20,802,247 bp).

Figure 4

Examples of inversions and translocations interrupting well-known disease-causing genes (A) Inversion inv(18)(q22.1q12.3), disrupting SETBP1 in sample 47. (B) Translocation t(9;17)(p13.3;q21.31), interrupting KANSL1 in sample 49. (C) Translocation t(20;21)(q11.22;q22.13), interrupting DYRK1A and PIGU in sample 54.

Figure 5

Examples of recurrent CNVs involved in well-recognized microdeletion/microduplication syndromes (A) A 22q11.21 deletion from sample 2. (B) A 16p12.2 deletion from sample 76. (C and D) Two distinct cases of 17p12 duplication from samples 8 (C) and 43 (D) in which the SV call reveals identical breakpoint locations. Sample maps also identify the location and orientation of the duplicated segments as tandem duplications.

Figure 6

Complex rearrangements (A) Sample 28: (from left to right) karyogram showing the translocation t(3;6) and the derivative chromosomes 4 and 5; CNV-microarray data showing two de novo deletions on chromosomes 4 and 5; and optical genome mapping circos plot of chromosomes 3, 4, 5, 6, and 13. Optical genome mapping data confirm all known aberrations and show the presence of additional translocations t(3;4) and t(4;6) plus an inversion on chromosome 13. (B) Sample 66: (from left to right) karyogram showing a three-way translocation 46,XY,t(3;13;5)(p11.1;p12;p14); CGH pane showing multiple deletions on chromosome 3; circos plot of chromosomes 3 and 5 (chr13 not represented in this plot because of the centromeric localization of the breakpoint) showing multiple intrachromosomal translocations on chromosome 3 and a translocation between chromosomes 3 and 5; and genome maps of chromosome 3 showing multiple rearrangements on chromosome 3, suggesting a chromoanagenesis.