Jingwei Yu

Introduction: MECOM rearrangements are frequently observed in myeloid neoplasms and associated with poor prognosis. Among the genomic alterations leading to MECOM rearrangements, t(2;3)(p13∼p25;q26.2) accounts for approximately 13% of reported cases. However, the precise DNA breakpoints of this translocation have not been previously reported, nor has the mechanism by which it alters MECOM expression been fully elucidated. In this report, we describe two cases with myelodysplastic syndromes (MDS) and t(2;3)(p23;q26.2). Our genomic characterization of these two t(2;3) translocations provided insights into the molecular mechanism of MECOM activation. Case Presentation: Case 1 is a 44-year-old female who presented with new anemia and thrombocytopenia. She was treated with azacitidine. After two allogeneic stem cell transplants, her disease relapsed with rapid progression to acute myeloid leukemia (AML). The patient passed away 1 year after progression to AML and 8 years after initial diagnosis. Case 2 is a 75-year-old female who was incidentally found to have macrocytic anemia with rare circulating blasts. She remained asymptomatic from anemia and did not require transfusions or treatment. Her disease progressed to MDS with excess blasts 3 years later. Patient was treated with azacitidine. Fifteen months later, her disease further progressed to AML. She passed away 5 months later and four and a half years after initial diagnosis. DNA sequencing analysis of these two cases revealed that the t(2;3)(p23;q26.2) breakpoints were within the regulatory regions of ZFP36L2 and THADA on chromosome 2 and the proximity of MECOM on chromosome 3, creating a novel regulatory configuration for MECOM. Notably, the translocation breakpoints differed by 270 kb on chromosome 2 and 93 kb on chromosome 3, resulting in distinct translocated regulatory elements with varying sizes and proximities to MECOM. These structural differences may influence the level of MECOM upregulation and contribute to variation in disease severity and progression. Conclusion: Our findings highlighted that, despite cytogenetic similarity, different t(2;3) translocations may exert distinct regulatory effects depending on the precise breakpoint locations. Thus, molecular characterization of MECOM rearrangements is critical for understanding disease pathogenesis and prognosis in myeloid neoplasms and may lead to the development of novel treatment.

Hematological neoplasms are heterogeneous diseases with various subtypes, each with its unique genomic features. Cell culture and slide preparation are essential steps to enrich and collect sufficient neoplastic cells for cytogenetic studies of the neoplasms. This chapter describes methods that are commonly used for culturing hematological neoplastic cells and preparing cytogenetic slides for clinical diagnosis and research of the neoplasms.

ETV6::ABL1 gene fusion is a rare recurrent genomic rearrangement associated with hematologic malignancies, and frequently occurs with additional anomalies. Due to the opposite chromosome orientations of the ETV6 and ABL1 genes, an oncogenic in-frame ETV6::ABL1 gene fusion cannot be formed by a simple translocation. The molecular mechanism of the ETV6::ABL1 fusion and the significance of co-occurring anomalies are not fully understood. We characterized genomic alterations in an individual with ETV6::ABL1 gene-fusion-positive myeloid neoplasm using various genomic technologies. Our findings uncovered a molecular mechanism of the ETV6::ABL1 fusion, in which a paracentric inversion within the short arm of chromosome 12 (12p) and a translocation between the long arm of a chromosome 9 and the 12p with the inversion were involved. In addition, we detected multiple additional anomalies in the individual, and our findings suggested that the ETV6::ABL1 fusion occurred as a secondary event in a subset of cells with the additional anomalies. We speculate that the additional anomalies may predispose to further pathogenic changes, including ETV6::ABL1 fusion, leading to neoplastic transformation.

Non-invasive prenatal screening (NIPS) is a DNA sequencing-based screening test for fetal aneuploidies and possibly other pathogenic genomic abnormalities, such as large deletions and duplications. Validation and quality assurance (QA) of this clinical test using plasmas with and without targeted chromosomal abnormalities from pregnant women as negative and positive controls are required. However, the positive plasma controls may not be available for many laboratories that are planning to establish NIPS. Limited synthetic positive plasmas are commercially available, but the types of abnormalities and the number/quantity of synthetic plasmas for each abnormality are insufficient to meet the minimal requirements for the initial validation. We report here a method of making synthetic positive plasmas by adding cell-free DNA (cfDNA) isolated from culture media of prenatal cells with chromosomal abnormalities to the plasmas from non-pregnant women. Thirty-eight positive plasmas with various chromosomal abnormalities, including autosomal and sex chromosomal aneuploidies, large deletions and duplications, were synthesized. The synthetic plasmas were characterized side-by-side with real positive plasmas from pregnant women and commercially available synthetic positive plasmas using the Illumina VeriSeq NIPT v2 system. All chromosomal abnormalities in the synthetic plasmas were correctly identified with the same testing sensitivity and specificity as in the real and commercial synthetic plasmas. The findings demonstrate that the synthetic positive plasmas are excellent alternatives of real positive plasmas for validation and QA of NIPS. The method described here is simple and straightforward, and can be readily used in clinical genetics laboratories with accessibility to prenatal cultures.

Interstitial deletion in the long arm of chromosome 9 [del(9q)] is a fairly common cytogenetic finding associated with acute myeloid leukemia (AML), seen in approximately 2–5% of AML patients. However, the genomic features of the deletion remain largely unknown. Using chromosome analysis, single nucleotide polymorphism microarray, and next-generation sequencing, we characterized del(9q)s and other genomic alterations in 9 AML patients. We found several distinct features of the del(9q)s. The proximal breakpoints of the deletions are clustered within a 2.5-Mb region (chr9: 68,513,625–70,984,372; GRCh37) enriched with segmental duplications, which may represent a “hotspot” for genomic rearrangements. However, the distal breakpoints of the deletions vary significantly. In addition, the overall deleted region could be divided into a 14.4-Mb proximal constitutional region (chr9: 70,950,015–85,397,699; 9q21.11q21.32) and a 24.0-Mb distal oncogenic region (chr9: 85,397,700–109,427,261; 9q21.32q31.1). We further identified a 6.8-Mb common overlapped deletion region (CODR) in the distal region (chr9: 90,590,650–97,366,400). This CODR carries multiple genes that are reportedly involved in cancer pathogenesis. The prognostic value of the del(9q) in AML apparently depends on additional genomic alterations in the patients.

Key Points. 1. We identified a new pediatric type of PCNS-LBCL that has distinct molecular features compared to PCNS-LBCL occurring in adults.2. Pediatric PCNS-

Next‐generation sequencing strategies have resulted in mutation detection rates of 21% to 61% in small cohorts of patients with microphthalmia, anophthalmia and coloboma (MAC), but despite progress in identifying novel causative genes, many patients remain without a genetic diagnosis. We studied a cohort of 19 patients with MAC who were ascertained from a population with high rates of consanguinity. Using single nucleotide polymorphism (SNP) arrays and whole exome sequencing (WES), we identified one pathogenic variant in TENM3 in a patient with cataracts in addition to MAC. We also detected novel variants of unknown significance in genes that have previously been associated with MAC, including KIF26B, MICU1 and CDON, and identified variants in candidate genes for MAC from the Wnt signaling pathway, comprising LRP6, WNT2B and IQGAP1, but our findings do not prove causality. Plausible variants were not found for many of the cases, indicating that our current understanding of the pathogenesis of MAC, a highly heterogeneous group of ocular defects, remains incomplete.