While balanced reciprocal translocations are relatively common, they often remain clinically silent unless they lead to the disruption of functional genes. In this study, we present the case of a boy exhibiting developmental delay and mild intellectual disability. Initial karyotyping revealed a translocation t(5;6)(q13;q23) between chromosomes 5 and 6 with limited resolution. Optical genome mapping (OGM) enabled a more precise depiction of the breakpoint regions involved in the reciprocal translocation. While the breakpoint region on chromosome 6 did not encompass any known gene, OGM revealed the disruption of the RASGRF2 (Ras protein-specific guanine nucleotide releasing factor 2) gene on chromosome 5, implicating RASGRF2 as a potential candidate gene contributing to the observed developmental delay in the patient. Variations in RASGRF2 have so far not been reported in developmental delay, but research on the RASGRF2 gene underscores its significance in various aspects of neurodevelopment, including synaptic plasticity, signaling pathways, and behavioral responses. This study highlights the utility of OGM in identifying breakpoint regions, providing possible insights into the understanding of neurodevelopmental disorders. It also helps affected individuals in gaining more knowledge about potential causes of their conditions.

Carcinogenesis often involves significant alterations in the cancer genome architecture, marked by large structural and copy number variations (SVs and CNVs) that are difficult to capture with short-read sequencing. Traditionally, cytogenetic techniques are applied to detect such aberrations, but they are limited in resolution and do not cover features smaller than several hundred kilobases. Optical genome mapping and nanopore sequencing are attractive technologies that bridge this resolution gap and offer enhanced performance for cytogenetic applications. These methods profile native, individual DNA molecules, thus capturing epigenetic information. We applied both techniques to characterize a clear cell renal cell carcinoma (ccRCC) tumor\'s structural and copy number landscape, highlighting the relative strengths of each method in the context of variant size and average read length. Additionally, we assessed their utility for methylome and hydroxymethylome profiling, emphasizing differences in epigenetic analysis applicability.

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During the last five decades, chromosome analysis identified recurring translocations and inversions in leukemias and lymphomas, which led to cloning of genes at the breakpoints that contribute to oncogenesis. Such molecular cytogenetic methods as fluorescence in situ hybridization (FISH), copy number (CN) arrays or optical genome mapping (OGM) have augmented standard chromosome analysis. The use of both cytogenetic and molecular methods, such as reverse transcription-polymerase chain reaction (RT-PCR) and next generation sequencing (NGS), including whole-genome sequencing (WGS), discloses alterations that not only delineate separate WHO disease entities but also constitute independent prognostic factors, whose use in the clinic improves management of patients with hematologic neoplasms.

Optical Genome Mapping (OGM) technology has garnered growing interest for the identification of chromosomal structural variations (SVs), particularly complex ones that are implicated in genetic diseases in humans. In this study, we performed genetic diagnostics on a neonatal patient who presented with feeding difficulties, hypotonia, and an atrial septal defect. We utilized a combination of trio-whole exome sequencing and OGM for our analysis. The results revealed an unbalanced translocation between maternal chromosomes 4 and 6 in the proband, ogm[GRch38]t(4:6)(q35.2;q25.3), resulting in a 2.8 Mb deletion at the 4q35 terminal and a 10.2 Mb duplication at the 6q25 terminal. In summary, this study highlights how OGM, in conjunction with other genetic approaches, can unveil the genetic etiology of complex clinical syndromes. Neonatal patients often exhibit low specific phenotypes, underlining the significance of SV detection.

Cytogenetic studies are essential in the diagnosis and follow up of patients with bone marrow failure syndromes (BMFSs), but obtaining good quality results is often challenging due to hypocellularity. Optical Genome Mapping (OGM), a novel technology capable of detecting most types chromosomal structural variants (SVs) at high resolution, is being increasingly used in many settings, including hematologic malignancies. Herein, we compared conventional cytogenetic techniques to OGM in 20 patients with diverse BMFSs. Twenty metaphases for the karyotype were only obtained in three subjects (15%), and no SVs were found in any of the samples. One patient with culture failure showed a gain in chromosome 1q by fluorescence in situ hybridization, which was confirmed by OGM. In contrast, OGM provided good quality results in all subjects, and SVs were detected in 14 of them (70%), mostly corresponding to cryptic submicroscopic alterations not observed by standard techniques. Therefore, OGM emerges as a powerful tool that provides complete and evaluable results in hypocellular BMFSs, reducing multiple tests into a single assay and overcoming some of the main limitations of conventional techniques. Furthermore, in addition to confirming the abnormalities detected by conventional techniques, OGM found new alterations beyond their detection limits.

While short-read sequencing currently dominates genetic research and diagnostics, it frequently falls short of capturing certain structural variants (SVs), which are often implicated in the etiology of neurodevelopmental disorders (NDDs). Optical genome mapping (OGM) is an innovative technique capable of capturing SVs that are undetectable or challenging-to-detect via short-read methods. This study aimed to investigate NDDs using OGM, specifically focusing on cases that remained unsolved after standard exome sequencing. OGM was performed in 47 families using ultra-high molecular weight DNA. Single-molecule maps were assembled de novo, followed by SV and copy number variant calling. We identified 7 variants of interest, of which 5 (10.6%) were classified as likely pathogenic or pathogenic, located in BCL11A, OPHN1, PHF8, SON, and NFIA. We also identified an inversion disrupting NAALADL2, a gene which previously was found to harbor complex rearrangements in two NDD cases. Variants in known NDD genes or candidate variants of interest missed by exome sequencing mainly consisted of larger insertions (> 1kbp), inversions, and deletions/duplications of a low number of exons (1-4 exons). In conclusion, in addition to improving molecular diagnosis in NDDs, this technique may also reveal novel NDD genes which may harbor complex SVs often missed by standard sequencing techniques.

Bilateral perisylvian polymicrogyria is the most common form of regional polymicrogyria within malformations of cortical development, constituting 20% of all malformations of cortical development. Bilateral perisylvian polymicrogyria is characterized by an excessive folding of the cerebral cortex and abnormal cortical layering. Notable clinical features include upper motoneuron dysfunction, dysarthria and asymmetric quadriparesis. Cognitive impairment and epilepsy are frequently observed. To identify genetic variants underlying bilateral perisylvian polymicrogyria in Finland, we examined 21 families using standard exome sequencing, complemented by optical genome mapping and/or deep exome sequencing. Pathogenic or likely pathogenic variants were identified in 5/21 (24%) of families, of which all were confirmed as de novo. These variants were identified in five genes, i.e. DDX23, NUS1, SCN3A, TUBA1A and TUBB2B, with NUS1 and DDX23 being associated with bilateral perisylvian polymicrogyria for the first time. In conclusion, our results confirm the previously reported genetic heterogeneity of bilateral perisylvian polymicrogyria and underscore the necessity of more advanced methods to elucidate the genetic background of bilateral perisylvian polymicrogyria.

BACKGROUND: Despite recent advances in prenatal genetic diagnosis, medical geneticists still face considerable difficulty in interpreting the clinical outcome of copy-number-variant duplications and defining the mechanisms underlying the formation of certain chromosomal rearrangements. Optical genome mapping (OGM) is an emerging cytogenomic tool with proved ability to identify the full spectrum of cytogenetic aberrations.
METHODS: Here, we report on the use of OGM in a prenatal diagnosis setting. Detailed breakpoint mapping was used to determine the relative orientations of triplicated and duplicated segments in two unrelated foetuses harbouring chromosomal aberrations: a de novo 15q23q24.2 triplication and a paternally inherited 13q14.2 duplication that overlapped partially with the RB1 gene.
RESULTS: OGM enabled us to suggest a plausible mechanism for the triplication and confirmed that the RB1 duplication was direct oriented and in tandem. This enabled us to predict the pathogenic consequences, refine the prognosis and adapt the follow-up and familial screening appropriately.
CONCLUSIONS: Along with an increase in diagnostic rates, OGM can rapidly highlight genotype-phenotype correlations, improve genetic counselling and significantly influence prenatal management.

Structural variants drive tumorigenesis by disrupting normal gene function through insertions, inversions, translocations, and copy number changes, including deletions and duplications. Detecting structural variants is crucial for revealing their roles in tumor development, clinical outcomes, and personalized therapy. Presently, most studies rely on short-read data from next-generation sequencing that aligns back to a reference genome to determine if and, if so, where a structural variant occurs. However, structural variant discovery by short-read sequencing is challenging, primarily because of the difficulty in mapping regions of repetitive sequences. Optical genome mapping (OGM) is a recent technology used for imaging and assembling long DNA strands to detect structural variations. To capture the structural variant landscape more thoroughly in the human genome, we developed an integrated pipeline that combines Bionano OGM and Illumina whole-genome sequencing and applied it to samples from 29 pediatric B-ALL patients. The addition of OGM allowed us to identify 511 deletions, 506 insertions, 93 duplications/gains, and 145 translocations that were otherwise missed in the short-read data. Moreover, we identified several novel gene fusions, the expression of which was confirmed by RNA sequencing. Our results highlight the benefit of integrating OGM and short-read detection methods to obtain a comprehensive analysis of genetic variation that can aid in clinical diagnosis, provide new therapeutic targets, and improve personalized medicine in cancers driven by structural variation.