Optical Genome Mapping for Cryptic Chromosomal Rearrangements Identification in Clinical Practice

To editor: Balanced chromosomal rearrangements (BCRs), predominantly reciprocal translocations and inversions represent disease-related structural variations (SVs) that directly disrupt gene structure and function. Moreover, BCRs predispose offspring to unbalanced rearrangements, resulting in adverse pregnancy outcomes.1 The timely and accurate identification of carriers is crucial for minimizing the risk of associated adverse pregnancy outcomes, assessing recurrent risk, and planning appropriate management. Conventional cytogenetic karyotyping serves as the standard first-tier approach for BCR detection, yielding a rate of 2%–4% in the recurrent miscarriage population.2 However, its limited resolution (typically around 5–10 Mb) poses challenges in detecting cryptic BCRs with smaller or similarly banded segments, as well as complex SVs involving multiple chromosomes. Optical genome mapping (OGM), an emerging technology from Bionano Genomics, has a high resolution for detecting nearly all types of genomic SVs and has been recognized as a revolution in next-generation cytogenomic analysis.3,4 A growing number of studies have recently demonstrated the efficiency and superiority of OGM in various clinical settings, including both prenatal and postnatal congenital conditions, as well as various hematological and other malignancies.5–7 Here, we explore the value of OGM in identifying cryptic BCRs using a case series from our clinical practice. This study was approved by the ethics committee of the Nanjing Drum Tower Hospital affiliated with Nanjing University Medical School (no. 2021-464-02). Case presentation Three couples of Chinese origin, nonconsanguineous and healthy, were enrolled in this study due to a history of adverse pregnancy outcomes. The female of the first couple was gravida 1, para 1 (G1P1). She previously gave birth to a seemingly healthy boy after an uneventful pregnancy, but he was noted to have delayed motor development milestones after early infancy. The boy did not exhibit language development until the age of five; at this point, he was diagnosed with developmental delay and intellectual disability. The female of the second couple was G5P1. Her prior pregnancies included an abortion that was not medically indicated (first pregnancy), the live birth of a girl diagnosed with moderate intellectual disability at 6 years of age (second pregnancy), two spontaneous miscarriages at 7–8 weeks' gestation, and an intrauterine fetal demise at 18 weeks (fifth pregnancy). The couple underwent karyotyping after the fourth pregnancy, but the findings were negative: the girl did not undergo genetic testing nor were the products of the conception analyzed. The female of the third couple experienced spontaneous miscarriages during her first and second pregnancies, which prompted an evaluation for recurrent miscarriages that included karyotyping. The findings, however, were negative. Her third pregnancy ended in intrauterine fetal demise at 34 weeks' gestation. A terminal deletion and a terminal duplication on separated chromosomes, potentially deriving from parental BCRs were found on whole exome sequencing of peripheral blood in the proband of the first family, as well as on chromosome microarray analysis of products of the conception of the most recent pregnancies in the second and third families (Table 1). These individuals all attended the Obstetrics and Gynecology Medical Center of Nanjing Drum Tower Hospital affiliated with Nanjing University Medical School, seeking guidance concerning future pregnancies. Genetic counseling was offered before any testing and written, informed consent was provided prior to testing initiation. Table 1 - Offspring copy number variation, optical genome mapping, and fluorescence in situ hybridization results. Offspring CNVs OGM Couple Gender of the carrier Location CN Length (Mb) Structural Variation Breakpoints (GRCh38/hg38) FISH 1 Female 2q37.3-qter 3 5.1 46,XX,t(2;10)(q37.3;p15.3) chr2::237,006,752-237,025,670 t(2;10)(2p+,10p+;2q+,10q+) 10p15.3-pter 1 2.6 chr10:2,612,834-2,622,310 2 Female 9q34.3-qter 1 2.9 46,XX,t(9;22)(q34.3;q13.2) chr9:135,192,382-135,199,824 t(9;22)(9p+,22q+;9q+) 22q13.2-qter 3 8.0 chr22:42,686,810-42,715,235 3 Female 4q34.3-qter 1 10.7 46,XX,t(4;12)(q34.3;p12.3)* chr4:179,355,987-179,361,221 t(4;12)(4p+,12p+;4q+,12q+) chr4:180,005,910-180,009,061 12p12.3-pter 3 16.1 chr12:16,068,858-16,074,606 *The derivative chromosome 12 consists of a 4q34.3-4qter (10.1 Mb) with inverted direction, a 4q34.3 (0.6 Mb) with forward direction, and a 12p12.3-12qter segment.CNV: Copy number variation; CN: Copy number; FISH: Fluorescence in situ hybridization; Mb: Megabyte; OGM: Optical genome mapping. For BCRs identification, Bionano OGM (Bionano Genomics, Inc., USA), fluorescence in situ hybridization (FISH) and G-banded karyotyping were performed simultaneously on samples obtained from individuals of the couples. In brief, G-banded karyotyping (450–550 bands) of peripheral blood lymphocytes was performed according to standard protocol. Fluorescent-labeled bacterial artificial chromosome probes designed according to the offspring rearrangements were prepared, and FISH analysis was conducted on lymphocytes at Be Creative Lab (Beijing) Co. Ltd. For OGM, ultra-high molecular weight DNA was extracted, labeled, and processed for analysis of the Bionano Genomics Saphyr® platform according to manufacturer protocols (Bionano Genomics, Inc. at Juno Genomics Lab (Hangzhou) Co. Ltd.). Genome map assembly was performed using Bionano Solve™ (v3.7) software; OGM-specific pipelines were managed using Bionano Access™ (v1.7) software, with a direct alignment of consensus maps to the reference human genome (GRCh38/hg38).3,4 As shown in Table 1 and Figures 1 and 2, OGM and FISH findings identified BCRs associated with offspring imbalances in all three couples; the breakpoints closely aligned with offspring rearrangements. All BCRs remained undetectable on G-banding. Sizes of translocated segments ranged from 2.6 to 8.0 Mb in couples 1 and 2. Translocated segments over 10 Mb in size, however, could also remain undetectable and could thus be defined as "large cryptic genomic rearrangements."8 In the third couple, translocated segments at chromosomes 4 and 12 were as large as 10.7 and 16.1 Mb, respectively, and were similarly not detected on karyotyping. The OGM technique involves labeling ultra-high molecular weight DNA with fluorophores via the action of the methyltransferase DLE-1 at the recognition motif CTTAAG, producing 14–15 labels per 100 kb; the resolution for detecting both balanced and unbalanced SVs is as small as 30 kb.3,4 Here, we found that the high resolution of OGM makes it a promising method for detecting cryptic BCRs; our findings were in agreement with those of two other recent studies.9,10Figure 1: Results forcryptic BCRs in couple 1 (A–E) and 2 (F–H). A Mapping of recombinant chromosome 2 (chr2, upper panel) and chromosome 10 (chr10, lower panel). B Model of the rearrangement. C Circos plot fromOGM. D FISH image, arrows indicating occurrence of derivative chromosome 10 (der(10)) and 2 (der(2)) (left and right panels, with colored signals indicating chromosome ends). E Karyotyping of chr2 and chr10 (red arrows representing OGM-predicted breakpoints). F Mapping of recombinant chr9 and chr22 (left), circos plot from OGM indicates translocation (right). G FISH image showing occurrence of der (22) and der(9) (left and right panels respectively, with colored signals indicating chromosome ends).H Karyotyping of chr9 and chr22 (red arrows representing OGM-predicted breakpoints). BCRs: Balanced chromosomal rearrangements; FISH: Fluorescence in situ hybridization; OGM: Optical genome mapping.Figure 2: OGM, FISH, and karyotyping results for cryptic BCR in couple 3. A OGM mapping showed chr4 (upper panel) and chr12 (middle panel) with breakpoints. Lower panel showed a 4q34.3 segment on der(12) included both forward and inverted segments. B Circos plot from OGM. C Schematic representation of chr4 (upper panel) and der(12) (lower panel) breakpoints. D FISH image (arrows) indicating der(12) (left panel) and der(4) (right panel) occurrences. E Karyotyping of chr4 and chr12, with predicted breakpoints marked by red arrows. BCR: Balanced chromosomal rearrangement; FISH: Fluorescence in situ hybridization; OGM: Optical genome mapping.Detailed information about breakpoints is another superior characteristic of OGM when compared to karyotyping and FISH.3,4 Although breakpoints identified by OGM were not directly validated using other methods, consistency with offspring rearrangements nevertheless underscored the reliability of OGM in identifying SV breakpoints. The breakpoints at 22q11.23 of the carrier in the second couple likely disrupted the gene A4GALT (OMIM 607922), which is associated with an autosomal recessive phenotype.11 No other OMIM genes were disrupted based on breakpoint location. Furthermore, OGM revealed that the derivative chromosome 12 consisted of an inverted 4q34.3-4qter (10.1 Mb), a 4q34.3 (0.6 Mb) in the forward direction unexpectedly, and a 12p12.3-12qter segment (Fig. 2A, C), suggesting that an insertional translocation was involved in the cryptic BCR.12 Insertional translocations are rare events that require at least three breaks, with two on the donor chromosome and one at the insertion site of the acceptor chromosome.12 Studies have shown that a large proportion of rearrangements, even apparent BCRs, encompass complexity not clearly visualized in routine karyotyping.13 Because of its capacity to provide a comprehensive, high-resolution overview of genomic structures, OGM can help resolve such genomic complexity. Here, the value of OGM was highlighted in a clinical context, not only considering our described cases but also those of high-risk couples without knowledge of potential imbalances. For couples at high risk of SVs based on histories of adverse pregnancy outcomes or known issues with heredity, genetic evaluation of the proband is of great significance to guide clinical management. However, establishing a diagnosis of the proband is often challenging. For example, although both the second and third couples experienced adverse pregnancy outcomes prior to their most recent attempt at pregnancy, genetic testing was not conducted on either of the probands. Although karyotyping was considered in these circumstances, the next pregnancy was attempted under the misleading influence of negative findings. FISH is a reliable alternative for the detection of cryptic SVs and our findings certainly underscored its efficacy in identifying relevant BCRs. Nevertheless, as FISH is conducted using probes designed based on offspring imbalances, this technique is less than ideal for highly accurate and comprehensive analyses at the genome-wide level. Unlike FISH, which requires specific probes, OGM utilizes a large number of DNA probes evenly distributed throughout the genome, eliminating the need for specificity and significantly enhancing BCR detection.3,4 Taken together, OGM should be considered as a first-line technique for comprehensively evaluating cryptic SVs in high-risk couples thus helping establish a timely diagnosis of cryptic SVs and allowing for optimal decision-making when a certain genetic abnormality was not considered. Conclusion Our findings highlight the superiority of OGM in detecting cryptic BCRs, especially when SVs are not suspected. To further explore the value of OGM in detecting cryptic SVs, additional studies evaluating large samples of both high-risk couples (i.e., individuals experiencing recurrent miscarriages) as well as the general population are required.