Thanks to a long-read sequencing (LRS) approach, in this study, we have reported a molecularly solved case of a proband with a clinical diagnosis of Cornelia de Lange syndrome (CDLS), which is a multisystemic disorder whose causative molecular defects involve cohesin complex genes, with NIPBL located at 5p13.2 accounting for approximately 50%-60% of CDLS cases. The first-tier tests revealed an abnormal karyotype 46,XY,t(5;15)(p13;q25)dn and a preserved NIPBL sequencing. Copy number variants (CNVs) at the translocation breakpoints, in disease genes, or in probably pathogenic loci were excluded by a-CGH analysis. Through fluorescence in situ hybridization (FISH) analysis on derivative chromosome 5, the breakpoint was relocated 3 Mb far from NIPBL 5\'UTR, which seemed fully maintained as FISH-probe mapping to the gene showed no split signals. Moreover, tri-color FISH revealed an apparently balanced paracentric inversion including NIPBL on derivative 5. Based on the strong clinical suspicion, we evaluated the NIPBL transcript by RT-qPCR that revealed a normal amount of transcript till exon 22 and a halved amount of the transcript from exon 23 to 3\'UTR, indicating the expression of a truncated transcript probably leading to a defective protein. Despite RT-qPCR confirmed the patient\'s CDLS clinical diagnosis, the molecular mechanism underlying this event remained to be an unsolved challenge for years. The LRS approach with nanopore technologies was able to fill the gap in this complex scenario and highlighted a chromothripsis event marked out at 5p13.2 by 36 breaks clustered in a 7.3-Mb region. The NIPBL gene was disrupted by 16 breaks and the resulting fragments were relocated in different positions and orientations. LRS confirmed the previous findings, and it has been proven to be crucial to define the complex chromosomal rearrangement in this patient which escaped current diagnostic investigations. Its application in the clinical practice will contribute to solve the unsolved.

BACKGROUND: Ependymomas are the third most common central nervous system tumor in the pediatric population; however, spinal ependymomas in children are rare. Ependymomas affecting the spinal cord most frequently occur in adults of 20-40 years of age. The current World Health Organization classification system for ependymomas is now composed of ten different entities based on histopathology, location, and molecular studies, with evidence that the new classification system more accurately predicts clinical outcomes.
METHODS: We present the case of a 16-year-old Caucasian female patient with a history of type 2 neurofibromatosis with multiple schwannomas, meningioma, and spinal ependymoma. Chromosome analysis of the harvested spinal ependymoma tumor sample revealed a 46,XX,-6,+7,-22,+mar[16]/46,XX[4] karyotype. Subsequent OncoScan microarray analysis of the formalin-fixed paraffin-embedded tumor sample confirmed + 7, -22 and clarified that the marker chromosome represents chromothripsis of the entire chromosome 6 with more than 100 breakpoints. Fluorescent in situ hybridization and microarray analysis showed no evidence of MYCN amplification. The final integrated pathology diagnosis was spinal ependymoma (central nervous system World Health Organization grade 2 with no MYCN amplification.
CONCLUSIONS: This case adds to the existing literature of pediatric patients with spinal ependymomas and expands the cytogenetic findings that may be seen in patients with this tumor type. This case also highlights the value of cytogenetics and microarray analysis in solid tumors to provide a more accurate molecular diagnosis.

Gene fusion events have been linked to oncogenesis in many cancers. However, gene fusions in meningioma are understudied compared to somatic mutations, chromosomal gains/losses, and epigenetic changes. Fusions involving B-raf proto-oncogene, serine/threonine kinase (BRAF) are subtypes of oncogenic BRAF genetic abnormalities that have been reported in certain cases of brain tumors, such as pilocytic astrocytomas. However, BRAF fusions have not been recognized in meningioma. We present the case of an adult female presenting with episodic partial seizures characterized by déjà vu, confusion, and cognitive changes. Brain imaging revealed a cavernous sinus and sphenoid wing mass and she underwent resection. Histopathology revealed a World Health Organization (WHO) grade 1 meningioma. Genetic profiling with next generation sequencing and microarray analysis revealed an in-frame BRAF::PTPRN2 fusion affecting the BRAF kinase domain as well as chromothripsis of chromosome 7q resulting in multiple segmental gains and losses including amplifications of cyclin dependent kinase 6 (CDK6), tyrosine protein-kinase Met (MET), and smoothened (SMO). Elevated pERK staining in tumor cells provided evidence of activated mitogen-activated protein kinase (MAPK) signaling. This report raises the possibility that gene fusion events may be involved in meningioma pathogenesis and warrant further investigation.

Lipoblastomas (LPBs) are rare benign neoplasms derived from embryonal adipose that occur predominantly in childhood. LPBs typically present with numeric or structural rearrangements of chromosome 8, the majority of which involve the pleomorphic adenoma gene 1 (PLAG1) proto-oncogene on chromosome 8q12. Here, we report on a LPB case on which showed evidence of chromothripsis. This is the second reported case of chromothripsis in LPB.

Optical genome mapping (OGM), which allows analysis of ultra-high molecular weight (UHMW) DNA molecules, represents a response to the restriction created by short-read next-generation-sequencing, even in cases where the causative variant is a neutral copy-number-variant insensitive to quantitative investigations. This study aimed to provide a molecular diagnosis to a boy with Marfan syndrome (MFS) and intellectual disability (ID) carrying a de novo translocation involving chromosomes 3, 4, and 13 and a 1.7 Mb deletion at the breakpoint of chromosome 3. No FBN1 alteration explaining his Marfan phenotype was highlighted. UHMW gDNA was isolated from both the patient and his parents and processed using OGM. Genome assembly was followed by variant calling and annotation. Multiple strategies confirmed the results. The 3p deletion, which disrupted ROBO2, (MIM*602431) included three copy-neutral insertions. Two came from chromosome 13; the third contained 15q21.1, including the FBN1 from intron-45 onwards, thus explaining the MFS phenotype. We could not attribute the ID to a specific gene variant nor to the reshuffling of topologically associating domains (TADs). Our patient did not have vesicular reflux-2, as reported by missense alterations of ROBO2 (VUR2, MIM#610878), implying that reduced expression of all or some isoforms has a different effect than some of the point mutations. Indeed, the ROBO2 expression pattern and its role as an axon-guide suggests that its partial deletion is responsible for the patient\'s neurological phenotype. Conclusion: OGM testing 1) highlights copy-neutral variants that could remain invisible if no loss of heterozygosity is observed and 2) is mandatory before other molecular studies in the presence of any chromosomal rearrangement for an accurate genotype-phenotype relationship.

Metastatic adrenocortical carcinoma (ACC) often has a poor outcome, with a five-year survival of less than 25%. We report a rare case of metastatic ACC with a myxoid variant with chromothripsis. We review the histologic variants of ACC, including myxoid type, molecular drivers, and current and investigational therapies for adrenocortical carcinoma. We also discuss the mechanism of chromothripsis, chromothripsis in ACC tumorigenesis, and propose potential therapies targeting chromothripsis.

Acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) is a heterogeneous hematological disorder defined by morphological, genetic, and clinical features. Patients with AML-MRC often show cytogenetic changes, which are associated with poor prognosis. Straightforward criteria for AML-MRC diagnosis and a more rigorous characterization of the genetic abnormalities accompanying this disease are needed. Here we describe an informative AML-MRC case, showing two separate, but concurrent, chromothripsis events, occurred at the onset of the tumor, and originating an unbalanced t(5;7) translocation and a derivative chromosome 12 with a highly rearranged short arm. Conversely, despite chromothripsis has been often associated with genomic amplification in cancer, in this case a large marker chromosome harboring amplified sequences from chromosomes 19 and 22 arose from a stepwise mechanism. Notably, the patient also showed a TP53 mutated status, known to be associated with an increased susceptibility towards chromothripsis and a poor prognosis. Our results indicate that multiple chromothripsis events may occur early in neoplastic transformation and act in a synergistic way with progressive chromosomal alterations to determine a dramatic impact on disease outcome, as suggested by the gene expression profile analysis.

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