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. 2023 Mar;25(3):175-188.
doi: 10.1016/j.jmoldx.2022.12.005.

Multisite Assessment of Optical Genome Mapping for Analysis of Structural Variants in Constitutional Postnatal Cases

M Anwar Iqbal  1 , Ulrich Broeckel  2 , Brynn Levy  3 , Steven Skinner  4 , Nikhil S Sahajpal  5 , Vanessa Rodriguez  6 , Aaron Stence  7 , Kamel Awayda  1 , Gunter Scharer  2 , Cindy Skinner  4 , Roger Stevenson  4 , Aaron Bossler  8 , Peter L Nagy  9 , Ravindra Kolhe  10
Affiliations

Multisite Assessment of Optical Genome Mapping for Analysis of Structural Variants in Constitutional Postnatal Cases

M Anwar Iqbal et al. J Mol Diagn. 2023 Mar.

Abstract

This study compares optical genome mapping (OGM) performed at multiple sites with current standard-of-care (SOC) methods used in clinical cytogenetics. This study included 50 negative controls and 359 samples from individuals (patients) with suspected genetic conditions referred for cytogenetic testing. OGM was performed using the Saphyr system and Bionano Access software version 1.7. Structural variants, including copy number variants, aneuploidy, and regions of homozygosity, were detected and classified according to American College of Medical Genetics and Genomics guidelines. Repeated expansions in FMR1 and contractions in facioscapulohumeral dystrophy 1 were also analyzed. OGM results were compared with SOC for technical concordance, clinical classification concordance, intrasite and intersite reproducibility, and ability to provide additional, clinically relevant information. Across five testing sites, 98.8% (404/409) of samples yielded successful OGM data for analysis and interpretation. Overall, technical concordance for OGM to detect previously reported SOC results was 99.5% (399/401). The blinded analysis and variant classification agreement between SOC and OGM was 97.6% (364/373). Replicate analysis of 130 structural variations was 100% concordant. On the basis of this demonstration of the analytic validity and clinical utility of OGM by this multisite assessment, the authors recommend this technology as an alternative to existing SOC tests for rapid detection and diagnosis in postnatal constitutional disorders.

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Figures

Figure 1
Figure 1
Sample cohort. A: The pie chart shows the variant classes according to standard-of-care (SOC) testing that were assessed for concordance evaluation in this study. B: The techniques used in SOC testing. n = 404 (B). CMA, chromosomal microarray analysis; FISH, fluorescence in situ hybridization; FSHD1, facioscapulohumeral dystrophy 1; NGS, next-generation sequencing.
Figure 2
Figure 2
Success rates, analytical quality control (QC) metrics, postanalytical QC metrics, and structural variation (SV) calling. First-pass success rate is defined as data meeting approximate QC standards with a single round of laboratory work (extraction, labeling, and chip flow cell). The overall success rate is defined as data meeting approximate QC standards in which one or more rounds of laboratory work were required. The results of analytical and postanalytical QC metrics, as defined in Materials and Methods, are shown. Average unique SV counts by type are provided in Table 1. F, female; M, male; CNV, copy number variation; FSHD1, facioscapulohumeral dystrophy 1; FXS, fragile X syndrome.
Figure 3
Figure 3
Representative examples of concordant microduplication and microdeletions. A: A case with a 4.3-Mbp deletion at chromosome (Chr) 10q11.22q11.23 (Chr10:45,794,475-50,135,024; hg38) identified and highlighted (red) in the copy number variation (CNV) track. The deletion was also captured by Sample Map 1, generated by the de novo assembly algorithm. The bottom shows a diagram of the chromosomal structures predicted by the assembled maps illustrated by blocks, with arrowhead indicating direction. B: The concordant chromosomal microarray analysis (CMA) result of the Chr10 deletion (Chr10:46,900,000-51,800,000; hg19) from the same sample (visualized by NxClinical software version 6.0; BioDiscovery, a Bionano Genomics Company, El Segundo, CA). C and D: A case with 22q11.2 low copy repeat (LCR) D-F (distal type I) deletion of 2.2-Mbp deletion (Chr22:21312519-23542560; hg38) as shown in the CNV track and de novo assembly (Sample Map 1). C: At the bottom, LCR D-F deletion is illustrated by the loss of blocks B through E. A complex configuration of the LCR22 D segment was also noted in the predicted genotype 2. D: The CMA result (generated by ChAS software version 4.3.0.71; ThermoFisher Scientific, Santa Clara, CA) shows the concordant Chr 22q11.2 deletion (Chr22:21,922,619-23,654,064; hg19). E: A case with a 19.7-Mbp copy number gain of Chr 7q21.3q31.2 (Chr7:96198684-115928803; hg38) is highlighted in blue, and the tandem duplication of this region is depicted by Sample Map 1 and illustrated at the bottom (F). The concordant CMA result of the Chr7 duplication (Chr7:95,731,159-115,400,069; hg19; NxClinical software version 6.0). BAF, B allele frequency; Ref, reference.
Figure 4
Figure 4
Representative examples of concordant structural variations (SVs). A: Left panel: A gain of chromosome (Chr) X is shown. Right panel: The whole-genome view copy number variation (CNV) track depicting the gain of Chr X. B: Left panel: Rearrangement between Chr11 and Chr22 from an individual with Emmanuel syndrome [47, XX,+der(22)t(11;22)(q23;q11)] is shown in the circos plot. Right panel: The unbalanced translocation was captured by Sample Map 1, also showing the copy number gain resulting from a der(22) chromosome (CNV tracks support the gains of Chr11 and Chr22). C: Left panel: A circos plot showing a balanced translocation detected between chromosomes 4q27 and 15q11.2, as indicated by a magenta line between Chr4 and Chr15. Right panel: The same translocation event was captured by Sample Map 1. D: An insertional translocation event of a 419-kbp region of 1p35.1-p34.3 (Chr1:34203802-34622531; hg38; partially including C1orf94 gene) is inserted into Xq21.31 (ChrX:88,741,968-88,777,061; hg38; near CPXCR1 gene). Ref, reference.
Figure 5
Figure 5
Representative examples of concordant structural variations (SVs). A: A 275-kbp tandem duplication (red arrows) was identified involving multiple genes, including PPARG, TSEN2, MKRN2, and RAF1, shown in Sample Map 1. B: A 345-kbp intragenic deletion involving exons 1 to 14 of the KMT2C gene is shown in Sample Map 1. C: A 29.8-kbp single-exon deletion involving exon 2 of the PAFAH1B1 gene was identified and indicated (red line) in the SV track. D: A 174-kbp hemizygous deletion involving exon 45 of the DMD gene is shown in Sample Map 1. Chr, chromosome; CNV, copy number variation; Ref, reference.
Figure 6
Figure 6
A: Representative novel finding by optical genome mapping: a 5.5-kb deletion of 12q23.3, likely including exon 20 of the POLR3B gene [ogm(GRCh38) 12q23.3(106446738_106464666)x1]. The predicted 5.5-kb deletion is located within the 17.9-kb window indicated in the structural variation (SV) track. This gene is associated with an autosomal recessive leukodystrophy syndrome that matches the patient phenotype. B: Trio exome sequencing data revealed a maternally inherited sequence variant [exon 13; c.1244T>C(p.Met413Thr)] and could confirm a paternally inherited exon 20 deletion. Also, RNA sequencing results support the exon 20 deletion. Chr, chromosome; CNV, copy number variation; Ref, reference; SeqVar, sequencing variants.
Supplemental Figure S1
Supplemental Figure S1
Replicate analysis. A: Breakpoint variability of position 1 and position 2 for deletions (breakpoints = 30, samples = 15, and cases = 4), duplications (breakpoints = 18, samples = 9, and cases = 2), and intrachromosomal translocations (breakpoints = 34, samples = 17, and cases = 2) of the structural variation (SV) algorithm. B: Breakpoint variability of position 1 and position 2 for deletions (breakpoints = 118, samples = 59, and cases = 10) and duplications (breakpoints = 42, samples = 21, and cases = 5) of the copy number variation (CNV) algorithm. Medial SD is indicated by the horizontal line, and value is written.
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