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Pan-genomics and multi-omics for deciphering genetic variation and accelerating genetic improvement in ruminant livestock.

Livestock reference genomes have transformed the discovery of variants associated with production, reproduction, health, and environmental adaptation. Nevertheless, a single linear reference represents only one mosaic haplotype and incompletely captures sequence diversity within a species, particularly structural variants, copy-number changes, repeat-rich regions, and breed-specific sequences. Pangenomes address this limitation by integrating multiple high-quality assemblies or population-scale variants into a unified sequence or graph representation. Concurrently, multi-omics approaches connect genomic variation with transcriptomic, epigenomic, manuscriptproteomic, metabolomic, and microbiome responses, thereby improving biological interpretation of genotype-phenotype relationships. This review synthesizes recent progress in livestock pangenomics and multi-omics, with emphasis on cattle, goats, sheep, water buffalo, and chickens. It describes advances in long-read and haplotype-resolved sequencing, graph construction, structural-variant discovery and genotyping, functional annotation, and integrative analysis. Recent pangenome studies have uncovered substantial non-reference sequence, reduced reference bias, identified breed- and population-specific structural variants, and resolved candidate variants underlying pigmentation, body size, tail morphology, cashmere production, altitude adaptation, and other economically relevant traits. However, translation into routine breeding remains constrained by uneven population representation, inconsistent structural-variant definitions, limited functional annotation, computational demands, and insufficient validation across environments. Future progress will depend on diverse near-complete assemblies, graph-aware imputation and genomic prediction, long-read transcriptomics, single-cell and spatial omics, rigorous causal validation, and open, interoperable resources. Together, these developments can support more accurate, resilient, and biologically informed livestock improvement. Importantly, current dairy-cattle evidence indicates that pangenome-derived structural variants can substantially improve variant discovery and functional interpretation while yielding only marginal average gains in routine genomic prediction, favoring targeted augmentation rather than wholesale replacement of established SNP-based evaluations.

Animals↗

Case Report: Early infantile drug-resistant epilepsy and gastrointestinal dysmotility associated with a de novo GNAO1 variant and a 16p13.11 microdeletion.

BACKGROUND: The GNAO1 gene is located on the long arm of chromosome 16 (16q13) and is associated with Developmental and Epileptic Encephalopathy 17. It encodes a protein involved in regulating various neurotransmitters and neuronal growth and development. The 16p13.11 microdeletion is a genomic deletion in the p13.11 region of the short arm of chromosome 16, previously reported to predispose individuals to neurodevelopmental disorders. These two regions do not overlap on the chromosome, and the 16p13.11 microdeletion does not involve the GNAO1 gene locus. CASE PRESENTATION: We reports a 1-month-old infant presenting with concurrent GNAO1 (p. G203R) gene variation and a 16p13.11 microdeletion. The patient initially presented with recurrent abdominal distension in the neonatal period, which gradually progressed to include focal seizures and intractable epileptic spasms. Interictal EEG showed multifocal discharges and burst-suppression patterns. Brain MRI revealed no abnormalities. A full gastrointestinal series suggested intestinal obstruction and gastro-esophageal reflux. Improved whole-exome sequencing identified: 1) A single nucleotide variantion: GNAO1, NM_020988.3:exon6: c.607G > A (p.Gly203Arg). 2) A copy number variation: A pathogenic 1.453 Mb deletion in the 16p13.11 region. CONCLUSIONS: We describe the developmental trajectory of an infant with overlapping features of gastrointestinal symptoms and drug-resistant epilepsy, carrying a GNAO1 variant and a 16p13.11 microdeletion. This case provides insights into the contribution of both single nucleotide variant and copy number variant to the phenotype.

16p13.11 microdeletion↗

The influence of GAP promoter variants on hirudin production, average plasmid copy number and cell growth in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has been engineered to synthesize and secrete desulfato-hirudin (hirudin), a thrombin inhibitor from the leech Hirudo medicinalis. The synthetic gene coding for hirudin was expressed constitutively under the control of four size-variants of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) and cloned into a 2 mu based multicopy yeast vector. The constitutive action of the four promoter variants was confirmed by demonstrating that the expression and secretion of hirudin is growth-related. The different efficiencies of the promoter variants not only affected hirudin expression but also led to changes in several cellular parameters, such as cell growth, average plasmid copy number and plasmid stability. The observed changes show that yeast cells establish a specific equilibrium for each promoter variant. We conclude, that the adjustment of cellular parameters in response to the expression levels of a heterologous protein is regulated by two counteracting selective forces: (1) the need for complementation of the auxotrophic host marker by the plasmid-encoded selection gene which, in the case of dLEU2, requires several plasmid copies; and (2) a selective advantage of cells with a lower copy number enabling them to escape the burden of heterologous protein production.

Base Sequence↗

Evaluating the pathogenic significance of unique chromosomal variants in craniosynostosis using patient-derived induced pluripotent stem cells and mouse modelling.

PURPOSE: Unravelling causal links between unique structural/copy-number variants (SV/CNV) and associated phenotypes is essential for correct genetic counselling. We investigated two families in which patients with craniosynostosis had SV/CNV potentially dysregulating a fibroblast growth factor (FGF)-encoding gene; a 730 kb dup(4)(q21.21) including FGF5; and a complex 568 kb interspersed 13q12.11 duplication, located 841 kb from FGF9. METHODS: We combined bioinformatic predictions of altered topologically-associating domain (TAD) structure, with experimental analysis (RNA- and ATAC- [assay for transposase-accessible chromatin] sequencing) of patient induced pluripotent stem cell lines (iPSCs) differentiated to neural crest (NCC) and osteoprogenitor (OPC) identities. For the dup(4)(q21.21) we generated a mouse bearing an equivalent rearrangement using CRISPR-Cas9 targeting. RESULTS: TAD analysis suggested potential dysregulation of the FGF5/FGF9 gene by bringing it into a novel genomic milieu. The RNA- and ATAC-seq assays demonstrated FGF5/FGF9 upregulation (2.7-18x) and local opening of chromatin, in 3/4 cell lines. For the dup(4)(q21.21), a causal role was supported by the mouse model, whereas interpretation of the 13q12.11 SV is confounded by a co-existing FOXP2 pathogenic variant. CONCLUSION: Patient iPSC-differentiated NCC and OPC lines, combined with TAD-based modelling to generate testable functional hypotheses, provide valuable functional evidence when evaluating causation of unique SV/CNV in craniosynostosis.

copy-number variant↗

Inverted triplications formed by iterative template switches generate structural variant diversity at genomic disorder loci.

The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a complex genomic rearrangement (CGR). Although it has been identified as an important pathogenic DNA mutation signature in genomic disorders and cancer genomes, its architecture remains unresolved. Here, we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the DNA of 24 patients identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted structural variant (SV) haplotypes. Using a combination of short-read genome sequencing (GS), long-read GS, optical genome mapping, and single-cell DNA template strand sequencing (strand-seq), the haplotype structure was resolved in 18 samples. The point of template switching in 4 samples was shown to be a segment of ∼2.2-5.5 kb of 100% nucleotide similarity within inverted repeat pairs. These data provide experimental evidence that inverted low-copy repeats act as recombinant substrates. This type of CGR can result in multiple conformers generating diverse SV haplotypes in susceptible dosage-sensitive loci.

Humans↗

Prenatal SNP-array chromosomal microarray analysis in 3,549 pregnancies: indication-specific yields and clinical implications.

BACKGROUND: SNP-based chromosomal microarray analysis (CMA) is widely used in invasive prenatal diagnosis, yet real-world performance across contemporary referral pathways, especially in the NIPT era, remains incompletely characterized. METHODS: We retrospectively analyzed 3,549 prenatal invasive samples tested by SNP array, and evaluated diagnostic yield overall and by referral indication and ultrasound phenotype. RESULTS: In total, we identified 398 pathogenic or likely pathogenic (P/LP) variants across 386 fetuses, resulting in an overall diagnostic yield of 10.9% (386/3,549). These findings comprised 223 aneuploidies and 175 pathogenic CNVs. In contrast, variants of uncertain significance (VOUS) were detected in 12.0% (426/3,549) of cases. Diagnostic yields were heavily stratified by indication: yields peaked in NIPT high-risk referrals (38.9%) and were intermediate in ultrasound-based cases (~ 11%), but dropped significantly in the advanced maternal age (AMA; 4.2%) and serum screening (~ 5-6%) groups. Conversely, VOUS rates remained remarkably stable across all referral categories. Sub-analysis of ultrasound abnormalities revealed that multisystem anomalies conferred the highest risk (27.3%), driven predominantly by aneuploidies; among soft markers, increased nuchal translucency (NT) emerged as the strongest predictor of chromosomal pathology. CONCLUSIONS: In our cohort, SNP-array identified clinically actionable findings in 10.9% of cases. NIPT enriched diagnostic yields, particularly for aneuploidies, and NT thickness was strongly associated with pathogenic findings. These results support an indication-based approach to genomic testing, with NIPT as a triage tool for aneuploidy and CMA for high-risk populations, while improving VOUS counseling.

Humans↗

Novel and High-Throughput Method of Isolating Single Fetal Cells Using FACS for NIPT.

OBJECTIVE: To evaluate fluorescence activated cell sorting (FACS) as a method of single-cell isolation of rare circulating fetal cells from maternal blood for use in cell-based non-invasive prenatal testing (cbNIPT). METHOD: Blood samples (30 mL) were collected from 75 'low-risk' pregnant women (gestational age 10-15 weeks). Fetal cells were enriched and stained using magnetic activated cell sorting. Following enrichment, single fetal cells were sorted in individual PCR tubes by FACS. After cell lysis, verification of fetal cell origin was performed using short tandem repeat (STR) analysis with the GlobalFiler PCR Amplification kit. RESULTS: An average of 13.7 cells were sorted using FACS. STR analysis identified 8.2 fetal cells on average, representing 60.2% of the sorted cells. The four-step single-cell isolation procedure facilitated an overall enrichment of approximately 16-million-fold. One sample did not render any fetal cell, corresponding to 1.3% of the samples. CONCLUSION: FACS, which is typically used for segregation of large populations of cells, can be used for single-cell isolation of rare fetal cells in an automated setup. This not only helps in making cell isolation faster and high throughput but also provides fetal cells for a more comprehensive genetic analysis of the fetus.

Humans↗

A Novel Complete F8 Tandem Duplication Causing Elevated Factor VIII Activity and Associated with Venous Thromboembolism.

Background Coagulation factor VIII (FVIII) is a critical component of the intrinsic coagulation pathway. While elevated FVIII levels are an established risk factor for venous thromboembolism (VTE), genetic variants in the F8 gene directly causing such elevations remain scarce. Here, we report a novel complete F8 tandem duplication identified in a female patient with splanchnic venous thrombosis (SVT). Methods We performed genetic testing using a thrombophilia panel targeting 35 genes involved in thrombosis and haemostasis to detect both point variants and copy number variations (CNVs). Family co-segregation analysis and phenotypic assays for FVIII and von Willebrand factor (VWF) were conducted. The structural basis of the identified F8 copy number gain was elucidated using optical genome mapping (OGM). Full-length F8 mRNA amplification, quantitative PCR, plasma FVIII Western blotting, and X-chromosome inactivation analysis were performed to assess the functional consequences of the duplication. Thrombin generation test (TGT) was employed to assess the hypercoagulable state. Results Genetic testing identified three copies of all 26 exons of the F8 gene in the proband, which was also detected in her mother (CNVs = 3) and son (CNVs = 2). One-stage clotting and chromogenic assays confirmed persistently elevated FVIII activity in the proband and her mother, accompanied by increased FVIII antigen levels. The OGM analysis confirmed a 229 kb tandem duplication including the F8 gene on one of the proband's X chromosomes. The junction regions exhibited high sequence homology and were rich in repetitive sequences, which precluded precise breakpoint mapping. Full-length F8 mRNA amplification revealed no aberrant transcripts, whereas quantitative PCR showed increased F8 mRNA expression in all carriers. Plasma FVIII Western blotting indicated FVIII heavy and light chains of expected molecular weights with increased band intensity in carriers. X-chromosome inactivation analysis in female carriers showed no significant skewing. TGT in two available carriers showed increased thrombin generation compared with a normal control at both low (1 pM) and high (5 pM) tissue factor concentrations. Conclusion We identified a novel complete F8 tandem duplication associated with increased FVIII expression and a hypercoagulable phenotype in a female patient with SVT. These findings support F8 gene dosage gain as a rare gain-of-function mechanism contributing to elevated FVIII levels and thrombophilia, while variation in VWF levels and acquired risk factors may modify thrombotic penetrance.

coagulation factor VIII↗

Conservation and divergence in multigene families: alternatives to selection and drift.

It is generally assumed that conservation and divergence of DNA signify function (selection) and no function (drift), respectively. This assumption is based on the view that a mutation is a unique event on a single chromosome, the fate of which depends on selection or drift. Knowledge of the rates, units and biases of widespread mechanisms of non-reciprocal DNA exchange, in particular within multigene families, provides alternative explanations for conservation and divergence, notwithstanding biological function. Such mechanisms of DNA turnover cause continual fluctuations in the copy-number of variant genes in an individual and, hence, promote the gradual and cohesive spread of a variant gene throughout a family (homogenization) and throughout a population (fixation). The dual processes (molecular drive) of homogenization and fixation are inextricably linked. Data are presented of the expected stages of transition in the spread of variant repeats by molecular drive in some non-genic families of DNA, seemingly not under the influence of selection. When a molecularly driven change in a given gene family is accompanied by the coevolution (mediated by selection) of other DNA, RNA or protein molecules that interact with the gene family then biological function is observed to be maintained despite sequence divergence. Conversely, the mechanics of DNA turnover and a turnover bias in favour of ancestral sequences can dramatically retard the rate of sequence change, in the absence of function. Examples of the maintenance of function by molecular coevolution and conservation of sequences in the absence of function, are drawn mainly from the rDNA multigene family.

Animals↗

[Restriction fragment length polymorphism of the 5'-region of the bovine ribosomal spacer repeat].

The restriction map of bovine 28S rRNA gene and adjacent 5'-spacer region was determined. The high level of intragenomic and population length polymorphism of EcoRI-BamHI restriction fragment was demonstrated to originated from the 3'-end of 28S rDNA and 5'-spacer of rDNA repeat. This polymorphism is more pronounced than the ones revealed in human and murine rDNA repeats and could be compared with genomic fingerprints obtained by M13 or minisatellite DNA hybridization probes. From family blot-panel analysis we concluded that in progeny only parental sets of length variants were inherited and that the copy number of definite variants does not change in the progeny as well. From these results it was proposed that the definite sets of linked in genome length variants are inherited independently from each other.

Alleles↗

Minimal 16q genomic loss implicates cadherin-11 in retinoblastoma.

Retinoblastoma is initiated by loss of both RB1 alleles. Previous studies have shown that retinoblastoma tumors also show further genomic gains and losses. We now define a 2.62 Mbp minimal region of genomic loss of chromosome 16q22, which is likely to contain tumor suppressor gene(s), in 76 retinoblastoma tumors, using loss of heterozygosity (30 of 76 tumors) and quantitative multiplex PCR (71 of 76 tumors). The sequence-tagged site WI-5835 within intron 2 of the cadherin-11 (CDH11) gene showed the highest frequency of loss (54%, 22 of 41 samples tested). A second hotspot for loss (39%, 9 of 23 samples tested) was detected within intron 2 of the cadherin-13 (CDH13) gene. Furthermore, deletion of the exons of CDH11 and/or WI-5835 was shown by quantitative multiplex PCR in 17 of 30 (57%) of previously untested tumors. Immunoblot analyses revealed that 91% (20 of 22) retinoblastoma exhibited either a complete loss or a decrease of the intact form of CDH11 and 8 of 13 showed a prevalent band suggestive of the variant form. Copy number of WI-5835 for these samples correlated with CDH11 protein expression. CDH11 staining was evident in the inner nuclear layer in early mouse retinal development and in small transgenic murine SV40 large T antigen-induced retinoblastoma tumors, but advanced tumors frequently showed loss of CDH11 expression by reverse transcription-PCR, suggestive of a role for CDH11 in tumor progression or metastasis. CDH13 protein and mRNA were consistently expressed in all human and murine retinoblastoma compared with normal adult human retina. Our analyses implicate CDH11, but not CDH13, as a potential tumor suppressor gene in retinoblastoma.

Animals↗

[Study of polymorphism in human rRNA gene clusters. Population and familial analysis of basic types of RFLP].

The frequency and average amount of copy number per genome were defined for standard and a number of new variants of BamHI 5'-NTS RFLP from populations of Moscow, Riga and individuals with Down syndrome. It was demonstrated that the populations studied differ neither in population frequency nor in the average amount of copy number of the variants. New variants were detected in the EcoRI 3'-NTS RFLP system and their amplification, as well as discordance among MZ twins. Possible target for methylation in the HindII site of 3' end of 28S rRNA gene was revealed. Analysis of data obtained demonstrated inefficiency of using the RFLP systems in systematic mapping of NOR-chromosomes. Our data also suggested a possible role of amplification of one copy repeated unit rRNA genes in their evolution.

Down Syndrome↗

Optical genome mapping improves clinical interpretation of constitutional copy-number gains and reduces their VUS burden.

PURPOSE: Genomic structure of copy-number gains is critical for their clinical interpretation but cannot be determined by chromosomal microarray (CMA) analysis, which does not provide information about chromosomal location and orientation of multiplied regions. We thus hypothesized that in CMA testing gains have higher probability than losses to be classified as variants of uncertain significance (VUS) and that structural information from optical genome mapping (OGM) may improve their interpretation. METHODS: Using a χ2 test, we assessed the association between classification of copy-number variants as VUS and their type (gains vs losses) in a cohort of 4073 CMA cases. Thirty-three VUS gains involving disease-associated genes were characterized by OGM to evaluate if OGM data enable their more conclusive clinical interpretation. RESULTS: The proportion of variants reported as VUS compared with likely pathogenic/pathogenic was significantly higher for gains than losses, confirming their increased VUS burden. OGM successfully determined genomic structure for all 33 copy-number gains, showing that 26 of 33 were tandem duplications and 7 of 33 were complex rearrangements. Structural information facilitated clinical interpretation in majority of the cases; it supported benign nature for 27 of 33 gains and was inconclusive or supported pathogenic role for 6 of 33. An estimated 20% of reported VUS gains would not have been reportable if we had OGM data. CONCLUSION: We illustrate a specific advantage of OGM compared with CMA: in addition to detecting both copy-number variants and balanced rearrangements, OGM improves clinical interpretation of copy-number gains by providing structural information and is thus expected to significantly decrease their VUS burden.

Humans↗

Gene dose-dependent maturation and receptor editing of B cells expressing immunoglobulin (Ig)G1 or IgM/IgG1 tail antigen receptors.

Conserved differences between the transmembrane and cytoplasmic domains of membrane immunoglobulin (Ig)M and IgG may alter the function of antigen receptors on naive versus memory B cells. Here, we compare the ability of these domains to signal B cell allelic exclusion and maturation in transgenic mice. A lysozyme-binding antibody was expressed in parallel sets of mice as IgM, IgG1, or a chimeric receptor with IgM extracellular domains and transmembrane/cytoplasmic domains of IgG1. Like IgM, the IgG1 or chimeric IgM/G receptors triggered heavy chain allelic exclusion and supported development of mature CD21(+) B cells. Many of the IgG or IgM/G B cells became CD21(high) and downregulated their IgG and IgM/G receptors spontaneously, resembling memory B cells and B cells with mutations that exaggerate B cell antigen receptor signaling. Unlike IgM-transgenic mice, "edited" B cells that carry non-hen egg lysozyme binding receptors preferentially accumulated in IgG and IgM/G mice. This was most extreme in lines with the highest transgene copy number and diminished in variant offspring with fewer copies. The sensitivity of B cell maturation to transgene copy number conferred by the IgG transmembrane and cytoplasmic domains may explain the diverse phenotypes found in other IgG-transgenic mouse strains and may reflect exaggerated signaling.

Animals↗

The interleukin-1 receptor antagonist gene: a single-copy variant of the intron 2 variable number tandem repeat (VNTR) polymorphism.

A variable number tandem repeat (VNTR) polymorphism exists within intron 2 of the human interleukin-1 receptor antagonist gene, consisting of perfect repeats of an 86-bp sequence. Five allelic variants have been identified wherein the number of repeats varies from two to six. This is the first report of a rare, single-copy allele designated IL1RN*0.

Adult↗

Variability of human rRNA genes: inheritance and nonrandom chromosomal distribution of structural variants of nontranscribed spacer sequences.

Human rRNA genes contain variable regions, one of which is located in nontranscribed spacers (NTSs) closely downstream from the 3'-end of the transcribed region. This polymorphism may be detected by means of blot hybridization analysis as a set of distinct restriction fragments corresponding to this part of the rRNA genes. We have analyzed DNA of 51 individuals and found eight structural NTS variants of this region; two of these were common to all individuals analyzed, and six others were found in different combinations and with different frequencies. The copy number of each variant also differed but was not less than 10-20 copies per cell. The analysis of DNA isolated from leukocytes of the members of 11 families indicated that some of the structural variants (of the NTS region) are inherited as a single Mendelian locus. We propose that rRNA genes that belong to one particular structural variant form clusters on separate chromosomes. To test this proposition, we developed a combined method, including AgNO3-staining of chromosomes, in situ hybridization, and DNA analysis with methylation-sensitive restrictases, and used it for study of persons who had methylated rRNA genes located on AgNO3-negative nucleolar organizers. It was found that in three of four cases methylated genes really belonged to one structural variant. This approach may be used for detailed localization of separate classes of NTS structural variants of human rRNA genes.

Blotting, Southern↗

DNA copy number changes in epithelioid sarcoma and its variants: a comparative genomic hybridization study.

Epithelioid sarcoma is a distinctive, rare soft tissue sarcoma that typically involves the distal extremities in young adults, and shows epithelioid morphology and immunohistochemical markers of epithelial differentiation. The genetic background of epithelioid sarcoma is poorly understood, and knowledge of it could give insights into the pathogenesis of this tumor and its possible relationship with other malignant tumors. In this study, we analyzed DNA copy number changes in 30 epithelioid sarcomas by comparative genomic hybridization. DNA was extracted from microdissected samples of formaldehyde-fixed and paraffin-embedded tumors with a minimum of 60% of tumor cells in each sample. Sixteen tumors (53%) showed DNA copy number changes at one to six different genomic sites. The majority of the changes were gains, seen in 14 tumors, whereas 10 tumors showed losses. The most common recurrent gains were at 11q13 (five cases), 1q21-q23 (four cases), 6p21.3 (three cases), and 9q31-qter (three cases). High-level amplifications were detected once in 6p21.3-p21.1 and once in 9q32-qter. Recurrent losses were seen at 9pter-p23 (three cases), 13q22-q32 (three cases), 1p13-p22 (two cases), 3p12-p14 (two cases), 4q13-q33 (two cases), 9p21 (two cases), and 13q32-qter (two cases). The most common recurrent gain at 11q13 was seen in both classic cases and angiomatoid and rhabdoid variants supporting the relationship of these variants with the classic epithelioid sarcoma. Expression of cyclin D1 gene, located in 11q13, was immunohistochemically detected in nine of 15 cases including three of five cases with gain of 11q13, suggesting its involvement in epithelioid sarcoma. The observed comparative genomic hybridization changes give targets for future genetic studies on epithelioid sarcoma.

Adolescent↗

Optical genome mapping enhanced by refined variant interpretation in pediatric acute lymphoblastic leukemia.

Reliable detection of structural variants (SVs) and copy number variations (CNVs) is crucial in the contemporary diagnostics of pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, limitations of commonly used conventional and molecular cytogenetic methods may hinder the accurate genetic characterization of patients. Optical genome mapping (OGM) offers a reliable alternative by enabling high-resolution, genome-wide detection of CNVs and SVs. Chromosomal aberrations were screened using OGM in 51 children with B-ALL. The results were compared with those of karyotyping, fluorescence in situ hybridization (FISH), digital multiplex ligation-dependent probe amplification (digitalMLPA), and targeted RNA sequencing (RNA-seq). OGM data showed high congruency with karyotyping and FISH findings, detecting clinically relevant variants beyond G-banding results and unraveling a complex KMT2A fusion undetected by FISH. Gene fusions involved in complex ETV6::RUNX1 translocations, but not detected by RNA-seq, were confirmed using FISH. Normalization of OGM copy number values with DNA-index-improved concordance with FISH-derived copy numbers in near-tri/tetraploid cases. In the peripheral regions of OGM variants (fringe-zones), a novel evaluation strategy called 'FriZone' was applied, which significantly improved the concordance between OGM and digitalMLPA. In addition, a co-segregation analysis revealed strong associations between ETV6::RUNX1 fusion and deletions of ETV6, RAG2, and NR3C2. OGM uncovered complex rearrangements undetected by widely used methods in 15% of cases, improving genetic classification and risk stratification in 10% of the patients. The FriZone analysis and normalization by DNA-index provide a refined, more accurate approach to OGM variant interpretation, facilitating the efficient application of OGM in clinical diagnostics. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Humans↗