The proband was an 8-year-old girl with multiple anomalies, including glaucoma, corneal opacities, deafness, tooth deformity, abnormal tongue, hypoglycemia, foot varus, and aberrant hip joint. She also presented with intellectual disability due to loss of language function and lack of response to normal communication, as well as a lag in growth, with initial standing at 3 years old and independent walking at 7 years old. The proband’s mother also showed several similar deformities but generally had milder phenotypes, including occasional nystagmus, pectus carinatum, low verbal skills, and intelligence. Notably, the proband’s grandmother was healthy but had experienced four abnormal pregnancies. Her first child died a month after birth, and the following three pregnancies were spontaneously aborted. The proband’s father, grandfather, and other grandparents showed no abnormalities. A pedigree chart of the family is shown in Figure 1A. The proband underwent eye and orthopedic surgeries prior to genetic testing. In addition, self-care rehabilitation was routinely conducted, but only slight symptomatic improvement was observed. The proband’s mother did not receive any therapeutic intervention. Written informed consent was obtained from all participants, and this study was approved by the ethics board of Ningbo Women and Children’s Hospital (No. EC 2020-048). This study conformed to the provisions of the Declaration of Helsinki as revised in 2013.

Genomic DNA was isolated from the peripheral blood of the proband and her parents using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, United States) in accordance with the manufacturer’s protocol. Trio-WES was performed using the HiSeq 2,500 sequencing platform (Illumina, San Diego, CA, United States). Raw fastq data (including CNVs) were filtered using fastp (v0.23.1) and annotated based on the human reference genome (hg19). BAM files were generated by re-ordering using Burrows–Wheeler Aligner (0.7.17-r1194-dirty). The Genome Analysis Toolkit (GATK, v4.20) was used to obtain single nucleotide polymorphisms (SNP) and insertions and deletions, and ANNOVAR and VEP were carried out for variation annotations. Pathogenicity classification and interpretation of sequence variants were performed in accordance with the guidelines of the American College of Medical Genetics and Genomics (ACMG). Sanger sequencing was performed to confirm the identified variants. CNVs were obtained using the eXome Hidden Markov model, and copy number was estimated using Lattice-Aligned Mixture Models (clamms). The pathogenicity classification of the identified CNVs was consistent with the consensus recommendations of ACMG and ClinGen in 2021 (Riggs et al., 2021).

The Chromed CK cell synchronization kit (Shanghai Lechen Biotechnology Co. Ltd., Item No: CM-SK-80), which included high-resolution reagent solutions A (Fudr) and B (Thymidine), was used in this experiment. High-resolution karyotyping was performed in accordance with the manufacturer’s instructions. Briefly, 2 mL of anticoagulated peripheral blood (heparin) was collected from the proband’s grandparents. T lymphocytes in the peripheral blood were first stimulated into childish lymphocytes by plant lectins (PHA). After 72 h of cell culture, Solution A was added. After 16–18 h, the cells were treated with solution B. This process was maintained for 3–5 h, after which the cells were harvested using colchicine. After the experimental procedures of preparation, baking, drying, and sealing, the automatic scanning system GSL120 (Leica Biosystems Richmond, Inc., 5,205 Route 12 P. 0. Box 528 Richmond, Illinois 60071, United States) was used to automatically capture and record karyotypes. The karyotyping results were consistent with the International Nomenclature System for Human Cytogenetics (ISCN 2020).

OGM experiments were performed in accordance with the manufacturer’s instructions. Briefly, 2 mL of anticoagulant peripheral blood (EDTA) was collected from the proband’s grandparents. UHMW gDNA was extracted using the Blood and Cell DNA Isolation Kit v2 (Bionano Genomics, San Diego, CA, United States). Then, UHMW gDNA molecules were labeled with the DLS (Direct Label and Stain) DNA Labeling Kit (Bionano Genomics, San Diego, CA, United States of America). The DNA was quantified using a Qubit dsDNA Assay BR Kit with a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). The labeled UHMW gDNA was loaded onto a Saphyr chip for linearization and imaged using a Saphyr instrument (Bionano Genomics, San Diego, CA, United States). Bionano Access v1.7 software was used for bioinformatics analysis, which included de novo assembly and comparison with a reference genome map (hg19).

A PubMed search was performed using the following keywords: “OGM” and “SVs”. Studies concerning the clinical application of OGM for detecting chromosomal SVs in human samples were downloaded and systematically reviewed. Detailed information, including the aim of clinical research, results and clinical efficacy evaluation, publication date, and PMID, is summarized in Table 1.

To unveil the genetic basis of the phenotype and ascertain the clinical diagnosis, we first performed trio-WES on the proband and her parents. The proband carried a heterozygous duplication of 5,397.10 Kbp in the 5p15.33–5p15.32 region of chromosome 5 and a heterozygous deletion of 4,655.47 Kbp in the 6p25.3–6p25.1 region of chromosome 6 (Figures 1B, C), both of which were inherited from her mother. These unbalanced rearrangements were not detected in the father of the proband (Figure 1C). A duplication in chromosome 5, which is a non-polymorphic variant, was not reported in the DGV database. The DECIPHER database described a similar case with clinical phenotypes including cerebral hemorrhage, hearing impairment, neurodevelopmental delay, and strabismus (Patient ID: 339222). In accordance with the ACMG guidelines, the proband’s duplication was classified as having unknown clinical significance.

The deletion on chromosome 6 contained FOXC1 with a haploinsufficiency score of 3. Heterozygous mutation in FOXC1 could lead to Anterior segment dysgenesis 3 (MIM number: 601631) and Axenfeld–Rieger syndrome type 3 (RIEG3, MIM number: 602482). ASGD and ASMD are heterogeneous developmental disorders that affect the anterior segment of the eye, including the cornea, iris, lens, trabecular meshwork, and Schlemm’s canal. The clinical features of ASGD include iris hypoplasia, enlarged or reduced corneal diameter, corneal vascularization and opacity, posterior embryotoxin, corectopia, polycoria, abnormal iridocorneal angle, ectopia lentis, and anterior synechiae between the iris and posterior corneal surface (Cheong et al., 2016). The clinical phenotypes of RIEG3 are highly heterogeneous and include zygomatic planar process, ocular hypertelorism, sensorineural deafness, glaucoma, eyeball protrusion, posterior embryonic ring, denture, microdentin, vermium hypoplasia, atrial septal defect, patent ductus arteriosus, iris hypoplasia, pupil dislocation, concave nasal ridge, and midface retroretraction (Gould et al., 1997; Cunningham et al., 1998). The deletion in the 6p25.3–6p25.1 region, which is a non-polymorphic variant, was not included in the DGV database. Conversely, the DECIPHER database enrolled a crowd of cases presenting with multiple anomalies and carrying similar pathogenic/likely pathogenic CNVs (Patient ID:308608, 332098, 401710, etc.). Combined with the proband’s clinical symptoms, the deletion in the 6p25.3–6p25.1 region was classified as pathogenic.

High-resolution karyotype analysis was performed to trace the origin of the unbalanced rearrangements between chromosomes 5 and 6 in the proband’s grandparents. However, the grandmother and grandfather both exhibited normal karyotypes (Figures 2A, B). Given that OGM has a 10,000-fold higher resolution than karyotyping, the grandparents’ peripheral blood was further subjected to OGM. As expected, no abnormalities were found in the grandfather (Figure 2D), whereas a cryptic BT between chromosomes 5 and 6 was uncovered in the grandmother (Figure 2C), which may explain the 5.4 Mb 5p terminal duplication and 4.7 Mb 6p terminal deletion in the proband and her mother. According to the breakpoints on the chromosomes, chromosome 5 was separated into Chr5-A (5 pter-5p15.31, 6.301 M) and Chr5-B (5p15.31-5 qter, 174.599 M), and chromosome 6 was separated into Chr6-A (6 pter-6p25.1, 5.025 M) and Chr6-B (6p25.1-6 qter, 166.075 M) (Figure 3A). The derived chromosome 5 comprised Chr6-A and Chr5-B, and the derived chromosome 6 comprised Chr5-A and Chr6-B (Figure 3B).

Most studies have validated a 100% clinical concordance of OGM with traditional cytogenetic analysis and identified additional cryptic variations that remain beyond the purview of current technologies (Table 1). OGM has been extensively used to characterize SVs for the diagnosis of hematological neoplasms. The study by Luhmann et al. (2021) indicated that 12 pediatric acute lymphoblastic leukemia samples were analyzed using OGM, and all genomic aberrations identified by other techniques were also detected using OGM. Their results showed that OGM was superior to well-established techniques for the resolution of complex structures and had a higher sensitivity for the detection of copy number alterations (Luhmann et al., 2021). Sahajpal & Mondal et al. also demonstrated that OGM outperforms SOC tests and may be a first-tier cytogenomic test for hematologic malignancies (Sahajpal et al., 2022b). Recently, Levy’s group reported in a real-world setting the performance of OGM in a cohort of 100 acute myeloid leukemia (AML) cases that were previously characterized by KT alone or KT and FISH or CMA. Their multicenter study indicates that OGM effectively recovers clinically relevant SVs and CNVs found by SOC methods and reveals an additional 13% of cases that had been missed by the routine methods (Levy et al., 2023). Moreover, the application and clinical utility of OGM have been reported in other solid neoplasms, including multiple myeloma (Gupta et al., 2015), solid tumor oligodendroglioma (Ray et al., 2013), and prostate cancer (Shim et al., 2022).

Aside from its superiority in the precise diagnosis of oncology, OGM also has greater efficiency in detecting postnatal/prenatal constitutional genetic disorders. A previous multi-site assessment of OGM in constitutional postnatal cases indicated that the technical concordance for OGM to detect previously reported SOC results was 99.5%, and the replicate analysis of 130 SVs was 100% concordant (Iqbal et al., 2023). Additionally, high-resolution OGM has been successfully used to detect cryptic reciprocal translocation in 11 couples with normal karyotypes and abortions/affected offspring with unbalanced rearrangements (Zhang et al., 2022). Another study used a combination of OGM and linkage-based karyomapping for the rapid prenatal diagnoses of facioscapulohumeral muscular dystrophy 1 (Zheng et al., 2020). A commentary by Sahajpal et al. in 2021 highlighted the potential of OGM as a standard of care in prenatal genetic testing (Sahajpal et al., 2021). Furthermore, Sahajpal & Jill et al. performed OGM in 52 patients with severe COVID-19 to identify rare/unique SVs as decisive predisposition factors associated with COVID-19. This study is the first to systematically assess the potential role of SVs in the pathogenesis of COVID-19 severity (Sahajpal et al., 2022a). Systematic information on OGM application and clinical utility for detecting chromosomal SVs was collected and summarized in Table 1.