The authors highlight that while current SOC methods, such as karyotyping, chromosomal microarray (CMA), and whole-exome sequencing (WES), are proficient at detecting deletions and sequence variants, they frequently fall short in providing precise breakpoint information for duplications and balanced SVs. This limitation can severely impede clinical assessment, especially when patient phenotypes are unclear or when family segregation analysis is not feasible. The study posits OGM as a promising and efficient solution to unlock this clinically relevant information.
Background and Limitations of Conventional Methods Genetic testing in clinical practice currently employs a diverse array of SOC methods. However, these techniques often struggle with comprehensive characterization of SVs. For instance, they may fail to resolve the breakpoints of copy-number neutral (balanced) aberrations and copy-number gains, which is a significant disadvantage when accurate variant classification is critical. This issue is particularly pronounced in prenatal diagnostics or for diseases with non-specific phenotypes, where clinical data might be insufficient. While segregation analysis of close relatives can sometimes provide tentative clinical assessments, this approach is limited to pathogenic variants causing observable phenotypes and requires informative carriers. This excludes numerous scenarios, such as cases involving egg or sperm donation, unavailable relatives, or late-onset, low-penetrance, or recessive (especially X-linked recessive) diseases, all of which necessitate precise topological characterization of the SV.
Study Objective and Methods The primary objective of this retrospective study was to systematically evaluate OGM as an additional tool in the routine diagnostic pipeline to gain crucial clinical insights into SVs that SOC methods could not fully characterize.
The study involved a cohort of prenatal and postnatal samples collected over a one-year period in 2023 at the Institute of Medical Genetics. Sample materials included blood, amniotic fluid, or chorionic villi specimens (CVS), based on the clinical indication. Initial genetic tests were performed using SOC methods such as WES, CMA, or karyotyping.
For OGM, ultra-high-molecular-weight (UHMW) DNA was extracted from 1.0 to 1.5 million viable cells using Bionano Prep kits. The DNA was labeled with DLE-1 enzyme (Bionano Genomics) using a Direct Label and Stain kit, then loaded onto a Saphyr Chip® and imaged on a Bionano Saphyr system. Data collection aimed for over 300 Gbp of UHMW DNA, corresponding to more than 80x coverage of the genomic reference. While automated SV calling algorithms were used, the study primarily relied on direct visual assessment by an experienced human evaluator for regions of interest, often bypassing automated calls to avoid potential errors.
Follow-up analyses using OGM were specifically considered under certain criteria: (1) to differentiate true SVs from potential detection artifacts, (2) to assess the SV’s clinical relevance, or (3) to estimate the risk of its recurrence in the patient’s offspring.
Results: OGM’s Impact on Challenging Cases Out of 3021 patients referred for genetic analysis in 2023, follow-up analyses for potentially pathogenic abnormalities were requested for 41 cases using secondary SOC methods. However, for a small subset of seven patient cases (P1–P7), conventional follow-up approaches proved unfeasible, necessitating the use of OGM. In all seven cases, OGM proved crucial. The data obtained either allowed direct interpretation of the corresponding SV or enabled region-specific downstream analysis to achieve that goal. These cases presented highly individual genetic and clinical constellations, yet all qualified for OGM for the same reason: to investigate potential gene disruptions by observed or suspected SVs, either by pinpointing the location and orientation of duplicated material or by characterizing breakpoints in copy-number neutral aberrations.
The study detailed three instructive cases (P1-P3) and briefly mentioned others (P4-P7):
- Case P1: Localization of Additional Genetic Material
- Initial Findings: A male fetus, sonographically inconspicuous, showed two X-chromosomal duplications by CMA: a 193 kbp region in Xp22.31 (involving STS gene) and a 654 kbp region in Xp21.2p21.1 (involving DMD gene), both with potential severe implications. MLPA confirmed maternal origin.
- OGM Contribution: OGM was performed to localize the duplicated material. It suggested a complex rearrangement on the X chromosome’s p-arm, involving consecutive insertion of material from both duplications downstream of the STS gene [352, 354a]. The automated SV caller missed one breakpoint, and the CNV caller did not detect the smaller duplication due to its size.
- Subsequent Targeted Sequencing: Long-range PCR followed by Sanger sequencing was performed to identify exact breakpoints. This revealed a homologous base at one breakpoint (non-homologous end joining) and a 714 bp LINE (Long Interspersed Nuclear Element) region at the other, fusing STS intron 10 with DMD intron 55.
- Clinical Outcome: The presence of an intact DMD copy and the insertion of duplicated material outside gene-containing regions led to the classification of both duplications as likely benign. This assessment was confirmed by a healthy male relative carrying the aberration, and the pregnancy continued successfully.
- Case P2: Breakpoint Clarification of a Balanced SV
- Initial Findings: Fetal karyotyping suggested a paracentric inversion of the q-arm on chromosome 7 (q11.2q22), but CMA showed no CNVs in the region. While inherited inversions are often benign, similar SVs on chromosome 7 have been linked to hematological malignancies.
- OGM Contribution: OGM was used to characterize the inversion. It depicted an inversion involving cytobands 7q11.23 and 7q22.1, with breakpoints mapping to large regions of homologous segmental duplications (0.75 and 3.25 Mbp). Low effective coverage from OGM (25x instead of >80x recommended) likely hindered automated identification.
- Parental OGM and FISH: Parental karyotyping revealed maternal inheritance. OGM on maternal DNA with adequate quality metrics confirmed the inversion’s breakpoints and allowed automated identification. FISH analysis also confirmed the inversion, consistent with OGM data.
- Clinical Outcome: The breakpoint intervals were sufficiently defined to rule out disruption of known genes, classifying the variant as likely benign. The pregnancy continued to term, resulting in a healthy girl.
- Case P3: Confirmation of a Cryptic SV
- Initial Findings: WES of a newborn girl with suspected hereditary pseudohypoaldosteronism showed no coverage of exon 13 of the SCNN1B gene, leading to suspicion of a cryptic SV. Previous PCR attempts to confirm the SV were unsuccessful.
- OGM Contribution: Years later, OGM became available. Fetal OGM data revealed a homozygous paracentric inversion on chromosome 16 (16p13.13 and 16p12.2). The distal breakpoint interval overlapped with SCNN1B exon 13, strongly suggesting gene disruption. The automated SV caller identified it as two separate intrachromosomal fusions. Parental OGM showed the mother was heterozygous and the father homozygous for the inversion, consistent with his phenotype.
- Subsequent Long-Read Sequencing: PCR based on OGM-identified breakpoint intervals yielded an amplicon. Long-read sequencing of this amplicon definitively determined the exact breakpoint within exon 13, confirming the disruptive and pathogenic effect of the inversion. FISH analysis also confirmed the inversion.
- Clinical Outcome: The confirmed breakpoint knowledge enabled the development of a customized PCR test for convenient family carrier testing.
The study also noted other cases where OGM proved useful: P4, a translocation breakpoint located approximately 300 kbp upstream of the FOXL2 gene, not completely ruling out impairment of regulatory elements; and cases P5-P7, where OGM demonstrated that suspected aberrations found by fetal karyotyping were likely artifacts or benign, reducing unnecessary patient distress.
Discussion and Future Directions The study concludes that OGM offers crucial information not obtainable through conventional SOC methods, specifically concerning the precise locations of SV breakpoints. It serves as a low-threshold method for following up on uncertain results, thereby alleviating patient distress. The authors propose a diagnostic strategy where OGM is primarily utilized as a follow-up analysis for well-defined questions, rather than a first-line method. This approach leverages OGM’s strengths to complement existing SOC methods, leading to more clinically actionable reports.
Clinical scenarios that particularly benefit from OGM include cases with a lack of informative family history, situations where carrier analysis is unfeasible (e.g., egg/sperm donation, unavailable relatives, or suspected late-onset/low-penetrance/recessive diseases), and challenging prenatal settings where unclear results can cause significant parental distress. In the prenatal cases presented, OGM provided reports that facilitated parental decision-making and were perceived as reassuring, leading to the continuation of all pregnancies.
However, the study also acknowledges certain limitations of OGM. It shows reduced sensitivity in detecting mosaic chromosomal abnormalities, events in regions of low complexity, and segmental duplications that lead to non-allelic recombination (e.g., Robertsonian translocations). Furthermore, OGM technologies cannot currently detect variations within centromeres or the short arms of acrocentric chromosomes. While long-read sequencing technologies are emerging as a potential solution for these challenging regions, their widespread clinical availability is still developing. The authors suggest that the core OMKar algorithm, which underlies OGM’s analysis, is agnostic to specific sequencing technologies and could potentially be adapted to other platforms as more datasets become available.
In conclusion, this retrospective study strongly advocates for OGM as a valuable tool for enhancing the accuracy and comprehensiveness of constitutional genetic diagnostics. By providing detailed characterization of complex SVs, particularly breakpoint locations, OGM significantly complements existing SOC methods, enabling more precise diagnoses and guiding clinical management, especially in challenging prenatal cases.
