Balanced Chromosomal Translocation Research Articles

Identification of epigenetic modifiers essential for growth and survival of AML1/ETO-positive leukemia.

Aberrant gene expression patterns in acute myeloid leukemia (AML) with balanced chromosomal translocations are often associated with dysregulation of epigenetic modifiers. The AML1/ETO (RUNX1/MTG8) fusion protein, caused by the translocation (8;21)(q22;q22), leads to the epigenetic repression of its target genes. We aimed in this work to identify critical epigenetic modifiers, on which AML1/ETO-positive AML cells depend on for proliferation and survival using shRNA library screens and global transcriptomics approaches. Using shRNA library screens, we identified 41 commonly depleted genes in two AML1/ETO-positive cell lines Kasumi-1 and SKNO-1. We validated, genetically and pharmacologically, DNMT1 and ATR using several AML1/ETO-positive and negative cell lines. We also demonstrated in vivo differentiation of myeloblasts after treatment with the DNMT1 inhibitor decitabine in a patient with an AML1/ETO-positive AML. Bioinformatic analysis of global transcriptomics after AML1/ETO induction in 9/14/18-U937 cells identified 973 differentially expressed genes (DEGs). Three genes (PARP2, PRKCD, and SMARCA4) were both downregulated after AML1/ETO induction, and identified in shRNA screens. In conclusion, using unbiased shRNA library screens and global transcriptomics, we have identified several driver epigenetic regulators for proliferation in AML1/ETO-positive AML. DNMT1 and ATR were validated and are susceptible to pharmacological inhibition by small molecules showing promising preclinical and clinical efficacy.

Altered chromatin topologies caused by balanced chromosomal translocation lead to central iris hypoplasia

Despite the advent of genomic sequencing, molecular diagnosis remains unsolved in approximately half of patients with Mendelian disorders, largely due to unclarified functions of noncoding regions and the difficulty in identifying complex structural variations. In this study, we map a unique form of central iris hypoplasia in a large family to 6q15-q23.3 and 18p11.31-q12.1 using a genome-wide linkage scan. Long-read sequencing reveals a balanced translocation t(6;18)(q22.31;p11.22) with intergenic breakpoints. By performing Hi-C on induced pluripotent stem cells from a patient, we identify two chromatin topologically associating domains spanning across the breakpoints. These alterations lead the ectopic chromatin interactions between APCDD1 on chromosome 18 and enhancers on chromosome 6, resulting in upregulation of APCDD1. Notably, APCDD1 is specifically localized in the iris of human eyes. Our findings demonstrate that noncoding structural variations can lead to Mendelian diseases by disrupting the 3D genome structure and resulting in altered gene expression.

Which factors affect the live birth outcome of the first single euploid frozen-thawed blastocyst transfer in couples with balanced chromosomal translocations?

The objective of this study is to investigate the factors that influence the live birth rate (LBR) of the first single euploid frozen-thawed blastocyst transfer (FBT) cycles after preimplantation genetic testing for structural rearrangements (PGT-SR) in couples with balanced chromosomal translocations (BCT). Single center, retrospective and observational study. A total of 336 PGT-SR and the first single euploid FBT cycles between July 2016 and December 2022 were included in this study. The patients were divided into two groups according to the live birth outcomes. The parameters of the study population, controlled ovarian stimulation cycles, and FBT cycles were analyzed. Multivariable binary logistic regression was performed to find the factors that affected the LBR. The percentage of blastocysts at developmental stage Day 5 compared to Day 6 (51.8% vs. 30.8%; P<0.001) and with morphology ≥BB compared to <BB (49.7% vs. 32.2%; P=0.001) was significantly different between the group that resulted in live births (n=193) and the group that did not (n=143). The results of the multivariable binary logistic regression analysis indicated that the developmental stage (adjusted OR: 2.068, 95%CI 1.291-3.313; P=0.003) and morphology (adjusted OR: 1.697, 95%CI 1.039-2.773; P=0.035) of the blastocyst were significantly correlated with live birth. Patients with blastocysts that reached the developmental stage at Day 5 and had a morphology of ≥BB had a higher likelihood of having a live birth. The developmental stage and morphology of blastocyst affect the live birth outcome of the first single euploid FBT in BCT carriers undergoing PGT-SR.

Abstract 1081: A role for the oncogenic driver fusion protein EWS-FLI1 in the targeting of DR5 by INBRX-109 in Ewing sarcoma

Abstract Background: Tumor necrosis factor receptor superfamily member 10b (TNFRSF10b, also known as DR5) agonists are attractive targeted agents for cancer cells because these agents activate cellular apoptosis. Ewing sarcoma (ES) is a bone and soft-tissue sarcoma occurring in adolescence defined by a t(11;22)(q24;q12) balanced chromosomal translocation that results in chimeric transcription factor and oncogenic driver protein, EWS-FLI1. Independent studies and unbiased cell line screens have reported ES to be sensitive to DR5 targeting. INBRX-109 is a tetravalent, DR5-targeted antibody. Here, we analyzed the sensitivity of ES models to DR5 targeting by INBRX-109 and explored how the EWS-FLI1 transcriptional program impacts the continuum of sensitivity-resistance to this targeting. Methods: In a panel of 11 ES lines, in vitro sensitivity was assessed to both single agent and INBRX-109 combinations. A screen of 1,363 FDA approved compounds identified cooperative combinations that were prioritized mechanistically. Differential sensitivity for single agent and in combination with irinotecan was confirmed in vivo in xenograft models. Resistant clones were isolated from in vivo experiments and evaluated for DR5 expression and sensitivity. Cellular factors associated with sensitivity and resistance were determined by siRNA knockdown and Cleavage Under Targets and Tagmentation assay (CUT and Tag). Results: ES models showed variable sensitivity to INBRX-109, with a GI90 as low as 10 picomolar in some models. All models showed robust expression of DR5, but absolute expression of DR5 did not correlate with sensitivity. In vitro sensitivity was replicated in vivo, with all 7 mice bearing RD-ES xenografts demonstrating initial complete tumor regression and 3 of 7 mice demonstrating no recurrence beyond 90 days. Importantly, cells isolated from recurrent tumors showed a novel mechanism of resistance: direct repression of DR5 by EWS-FLI1. INBRX-109 was ineffective as a single agent at 10 nM in resistant ES models. However, there was striking synergy in vitro with SN38, the active metabolite of irinotecan, and this effect translated into tumor regressions of a resistant TC32 xenograft in vivo. The FDA compound screen captured novel synergy with a panel of agents. Conclusion: INBRX-109 is an exciting candidate for ES as a single agent in subsets of tumors or in combination with irinotecan, which is currently being evaluated in the clinic. Investigation of the exact relationship between EWS-FLI1 and sensitivity to DR5 targeting is ongoing. Initial findings suggest EWS-FLI1, combined with other disease modifying mutations, is a key mediator of this sensitivity. Further, EWS-FLI1 plays a direct role repressing DR5 in the setting of therapeutic challenge in one model in vivo. These findings highlight the potential for INBRX-109 in combination with irinotecan or novel candidates as therapies for ES patients. Citation Format: Curtis C. Parker, Lauren M. Gaetano, Elissa A. Boguslawski, Seneca Kinn-Gurzo, Rebecca Kaufman, Matthew C. Stout, Chase Deveraux, Patrick J. Grohar. A role for the oncogenic driver fusion protein EWS-FLI1 in the targeting of DR5 by INBRX-109 in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1081.

Analysis of Preimplantation and Clinical Outcomes of Two Cases by Oxford Nanopore Sequencing.

It is challenging to distinguish embryos with a balanced translocation karyotype from a normal karyotype by existing conventional genetic testing methods. However, in germ-cell gamete generation, chromosome exchange and separation through cell meiosis form a different proportion of unbalanced gametes. Adverse birth events may occur, such as repeated miscarriages and fetal birth defects. In this study, the exact breakpoints of structural variation (SV) from two balanced translocation carrier families by using Nanopore long reads sequencing technology were obtained, and haplotype analysis and Sanger verified the accuracy of the detection results, confirming the application value of the Nanopore sequencing technology in the detection of balanced translocation before embryo implantation. Nanopore long-read sequencing was performed to find the precise breakpoint of chromosome-balanced translocation carriers. The breakpoints were subsequently verified by designing primers across the breakpoints and Sanger sequencing. Haplotype linkage analysis of SNPs which can be linked by a read block of families around the breakpoint regions was followed. After frozen (-thawed) embryo transfer (FET), prenatal cytogenetic analysis of amniotic fluid cells confirmed the predicted karyotypes from the transferred embryos. The presence of breakpoints was detected in three embryos of patient 1. No breakpoints were detected in either embryo of patient 2. One balanced translocated embryo from patient 1 and one normal euploid embryo from patient 2 were transplanted back into the patients, and amniotic fluid cells were analyzed for the karyotype of fetuses. The results were entirely consistent with the fetal karyotype. And through late follow-up, both patients successfully had a live birth fetus. The breakpoint location of the balanced chromosome translocation can be accurately found by Nanopore sequencing. The haplotype of carriers can be successfully constructed by Nanopore and sanger sequencing confirmed that the results were accurate. This is very advantageous for preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) detection in the families without proband.

Abstract B008: GPR64-targeting single-domain CAR-T cells for immunotherapy in Ewing sarcoma

Abstract Ewing sarcoma (EwS) is the second most frequent bone and soft tissue cancer of childhood and adolescence. EwS is characterized by balanced chromosome translocation resulting in an aberrant tumor-specific transcriptional factor, EWSR1-FLI1, in 85% of cases. EwS is a highly aggressive cancer with a 5-years overall survival of only 30% for patients with metastases. These metastatic diseases are often resistant to intensive therapy and associated with acute and chronic adverse effects. Thus, there is a need for new treatment options to improve survival and to limit the long-term side effect. Immune-based approaches can offer alternative options to conventional treatments modalities. In this context, the aim of this study is to identify therapeutic target for immunotherapy. We carried-out an integrative mass spectrometry analysis of whole proteome and phosphoproteome of 42 cells lines including EwS and non-EwS cancer cell lines as well as mesenchymal stem cells (MSCs), the putative cell-of-origin for EwS. This multi-omics study revealed expected EWSR1-FLI1-associated signatures. Additional surfaceome data permitted to highlight 70 cell surface proteins up-regulated by EWSR1-FLI1. Among these selected proteins, potential candidates for immunotherapy were selected according to their healthy tissue expression profiles based on integrated transcriptomic analysis using public resources and RNA-seq data of EwS tumors. A particular interest was dedicated to the expression of proteins that may derive from the recently described neogenes (Vibert et al, Mol Cell 2022). We identified the G protein-coupled receptor 64 (GPR64), as a potential specific EwS target for immunotherapy. Immunochemistry assay highlighted a high expression of GPR64 specifically in EwS PDXs and patients-derived tumors though the level of expression varies from one case to the other. GPR64 expression in normal tissues is only restricted to human epididymis. Alternative splicing occurs intensively in the N-terminal region, generating nine splice isoforms of the protein with slight differences in the extracellular domain. All splice isoforms are expressed in Ewing's sarcoma, with a majority over-representation of protein isoforms 2 and 4, compared to healthy tissues. GPR64-specific single-domain antibodies (sdAb) were selected by phage display libraries binding to the recombinant protein corresponding to an extracellular part of the receptor. The specificity of sdAb was verified by flow cytometry on GPR64-wild type and GPR64-knock down cells as well as MSCs. The activating ability of sdAb to activate T cells and their cytotoxic potential against EwS cells are currently in progress. Thus, our multi-omics study identified GPR64 as a potential target for immunotherapy due to its EwS-specificity and its limited expression in normal tissues. This work could highlight new therapeutic opportunities for EwS patients with refractory disease. Citation Format: Floriane Petit, Florent Dingli, Jacob Torrejon-Diaz, Bérangère Lombard, Stéphane Liva, Sakina Zaidi, Didier Surdez, Renaud Leclere, Thomas Grünewald, Sandrine Grossetete, Berengère Ouine, Véronique Marsaud, André Nicolas, Franck Perez, Ahmed El Marjou, Damarys Loew, Sandrine Moutel, Amaury Leruste, Olivier Ayrault, Olivier Delattre. GPR64-targeting single-domain CAR-T cells for immunotherapy in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B008.

Identification of Epigenetic Modifiers Essential for Growth and Survival of AML1/ETO-Positive Leukemia

Introduction Aberrant gene expression patterns in acute myeloid leukemia (AML) with balanced chromosomal translocations are often associated with direct or indirect dysregulation of epigenetic modifiers. Previously, we and others have shown that the AML1/ETO (RUNX1/MTG8) fusion protein, encoded by the translocation (8;21)(q22;q22), leads to the epigenetic repression of its target genes (Rejeski et al., Oncogene 2021) Aim We aimed in this work to identify critical epigenetic modifiers, on which AML1/ETO-positive AML cells depend on for proliferation and survival using shRNA library screens and global transcriptomics approaches. Materials and methods In the customized shRNA library screen, 2009 shRNAs developed to target 671 genes were pool-cloned into a retroviral vector allowing doxycycline-inducible expression to generate a plasmid shRNA library (Antonova et al., Cell Reports 2019). We transduced the AML1/ETO-positive Kasumi-1 and SKNO-1 cell lines, selected with blasticidine, and the expression vectors were induced with doxycycline. After 12 days of doxycycline treatment, double-positive dsRed/GFP cells were FACS-sorted, sequenced and compared with FACS-sorted single GFP-positive cells from day 0. Single shRNAs against two selected genes of interest (DNMT1, ATR), were cloned and validated following the same protocol used for shRNA library screen. The 9/10/17-LacZ and 9/14/18-AML1/ETO clones were described previously (Duque-Afonso et al., 2011). Briefly, individual U937 clones were stably transduced with an ecdysone-inducible system for the expression of AML1/ETO (9/14/18-AML1/ETO) or LacZ gene (9/10/7-LacZ as control cells). Gene expression was induced by 5 uM ponasterone A (PonA). RNA was isolated, RNA libraries were prepared and sequenced. Bioinformatic analysis were performed on the European Galaxy instance platform. Results Using shRNA library screens, we identified 41 commonly depleted genes in two AML1/ETO-positive cell lines Kasumi-1 and SKNO-1. Among them, several epigenetic regulators as DNMT1, ATR, DMAP1, SMYD1, SMYD2, BRD4, SETD8, SETD1A and HDAC8 were depleted. We validated, genetically and pharmacologically, DNMT1 and ATR using several AML1/ETO-positive (SKNO-1, Kasumi-1) and AML1/ETO-negative cell lines (OCI-AML3, HL60) with median IC50 for decitabine 74 nM (range 44-219), azacytidine 146 nM (range 14-267), berzosertib 219 nM (range 16-460) and ceralasertib 85 nM (range 42-328). We also demonstrated in vivo differentiation of myeloblasts after treatment with the DNMT1-inhibitor decitabine in a patient with an AML1/ETO-positive AML. To address the regulation of epigenetic modifiyer genes by AML1/ETO in an unbiased fashion, bioinformatic analysis of global transcriptomics after AML1/ETO induction in 9/14/18-U937 cells identified 1319 differentially expressed genes (DEGs) (822 up- and 497 downregulated). Four genes (DNMT1, PARP2, SMARCA4, and PRKCD) were both downregulated after AML1/ETO induction, and also detected in the survival shRNA screens of both cell lines as decreasing survival and proliferation. Conclusions To our knowledge we show here the first shRNA knock-down library screen for epigenetic modifiyer genes in AML1/ETO-positive cells. Using unbiased shRNA library screens and global transcriptomics, we have identified several driver epigenetic regulators for proliferation in AML1/ETO-positive AML. Some of these epigenetic regulators, such as DNMT1 and ATR, are susceptible to pharmacological inhibition by small molecules showing promising preclinical and clinical efficacy, and confirmation studies (Baeten et al., ASH meeting 2022) appear warranted.

Preimplantation genetic testing using comprehensive genomic copy number analysis is beneficial for balanced translocation carriers.

Balanced chromosomal translocation is one of chromosomal variations. Carriers of balanced chromosomal translocations have an increased risk of spontaneous miscarriage. To avoid the risk, preimplantation genetic testing (PGT) using comprehensive genomic copy number analysis has been developed. This study aimed to verify whether and how embryos from couples in which one partner is a balanced translocation carrier have a higher ratio of chromosomal abnormalities. A total of 894 biopsied trophectoderms (TEs) were obtained from 130 couples in which one partner was a balanced translocation carrier (Robertsonian translocation, reciprocal translocation, or intrachromosomal inversion) and grouped as PGT-SR. Conversely, 3269 TEs from 697 couples who experienced recurrent implantation failure or recurrent pregnancy loss were included in the PGT-A group. The transferable blastocyst ratio was significantly lower in the PGT-SR group, even when bias related to the sample number and patient age was corrected. Subgroup analysis of the PGT-SR group revealed that the transferable blastocyst ratio was higher in the Robertsonian translocation group. Because the PGT-SR group had a higher proportion of untransferable embryos than the PGT-A group, PGT using comprehensive genomic copy number analysis was more beneficial for balanced translocation carriers than for infertility patients without chromosomal translocations. The frequencies of de novo aneuploidies were further analyzed, and the frequency in the PGT-SR group was lower than that in the PGT-A group. Therefore, we could not confirm the existence of interchromosomal effects in this study.

A Discussion of Chronic Myeloid Leukemia

Chronic Myeloid Leukemia (CML) is a triphasic hematological malignancy characterized by the presence of the abnormal Philadelphia chromosome, linked to causing uncontrolled myeloid cell proliferation in the bone marrow and marked changes in the peripheral blood and bone marrow microenvironments. The discovery of the BCR-ABL1 fusion oncogene from the balanced translocation of chromosomes 9 and 22, creating the Philadelphia chromosome, and its encoded tyrosine kinase protein has been linked to causing uncontrolled cell proliferation, leading to a subsequent development of tyrosine kinase inhibitors (TKI). TKIs, along with traditional chemotherapy drugs and interferon treatment, have revolutionized CML treatment, enabling deep remissions and prolonging of the disease’s chronic phase. This review will introduce CML and comprehensively explore the epidemiology, molecular basis, pathology, diagnostic approaches, and therapeutic strategies for CML, including their mechanisms of action and challenges, such as TKI resistance, with a focus on TKIs as the workhorses of CML therapy. By reviewing the current known literature, this paper aims to contribute to and guide the pursuit of new knowledge and research.

How chromosomal translocations arise to cause cancer: Gene proximity, trans-splicing, and DNA end joining

Chromosomal translocations (CTs) are a genetic hallmark of cancer. They could be identified as recurrent genetic aberrations in hemato-malignancies and solid tumors. More than 40% of all "cancer genes" were identified in recurrent CTs. Most of these CTs result in the production of oncofusion proteins of which many have been studied over the past decades. They influence signaling pathways and/or alter gene expression. However, a precise mechanism for how these CTs arise and occur in a nearly identical fashion in individuals remains to be elucidated. Here, we performed experiments that explain the onset of CTs: (1) proximity of genes able to produce prematurely terminated transcripts, which lead to the production of (2) trans-spliced fusion RNAs, and finally, the induction of (3) DNA double-strand breaks which are subsequently repaired via EJ repair pathways. Under these conditions, balanced chromosomal translocations could be specifically induced. The implications of these findings will be discussed.

Gene sequencing and result analysis of balanced translocation carriers by third-generation gene sequencing technology

Because the total gene copy number remains constant and all genes are normally expressed, carriers of balanced chromosomal translocations usually have a normal phenotype but are able to produce many different types of gametes during meiosis, and unbalanced gametes lead to increased risks of infertility, recurrent spontaneous abortion, stillbirth, neonatal death or malformations and intellectual abnormalities in offspring. The key to balanced translocations lies in finding the breakpoints, but current genetic testing techniques are all short-read sequencing, with the disadvantage of procedural complexity and imprecision for precisely identifying the breakpoints. The latest third-generation sequencing technology overcomes these drawbacks and uses robust long-read sequencing to accurately and rapidly detect genome-wide information and identify breakpoint locations. In this paper, we performed whole genome long-read sequencing using an Oxford Nanopore sequencer to detect the breakpoints of 4 balanced chromosomal translocation carriers. The results showed that employing about ~ 10× coverage confirmed 6 of the 8 breakpoints, of which, 2 had microdeletions/insertions identified near the breakpoints and 4 had breakpoints that disrupted the normal gene structure and were simultaneously tested for genome-wide structural variation (SV). The results show that whole genome long-read sequencing is an efficient method for pinpointing translocation breakpoints and providing genome-wide information, which is essential for medical genetics and preimplantation genetic testing.

Recurrent spontaneous abortion related to balanced translocation of chromosomes – A case report

INTRODUCTION: Recurrent spontaneous abortion (RSA) is defined as three or more consecutive pregnancy losses before the 20th week of gestation. RSA is often idiopathic, but structural chromosomal abnormality is an important cause. An unbalanced karyotype in the conceptus of a couple when one partner has a structural chromosomal abnormality may result in failure to implant, miscarriage, or pregnancy of a fetus with an unbalanced karyotype.CASE PRESENTATION: We report a rare case of RSA associated with balanced translocation of chromosomes. a woman who had four spontaneous abortions, all pregnancy loss happened before 12 weeks of gestation, no other known chronic diseases reported to the family nor medications taken during pregnancy. The karyotype was 46.XX, t(13p,21p) The abnormal karyotype was not found in any other chromosomes. Further spectral karyotyping was performed to rule out the involvement of any other chromosomal aberrations present in the genome.The cytogenetic analysis of the husband revealed a normal karyotype 46.XY.CONCLUSION: Couples with more than three miscarriages should be referred to the genetist for chromosomal analysis for possible hereditary etiology and chromosomal abnormalities responsible for miscarriages to plan prenatal diagnostics and genetic counseling for subsequent pregnancies.

Pharmaceutical/Clinical Strategies in the Treatment of Acute Promyelocytic Leukemia: All-Trans Retinoic Acid Encapsulation by Spray-Drying Technology as an Innovative Approach-Comprehensive Overview.

Acute promyelocytic leukemia (APL) is phenotypically characterized by the accumulation of dysplastic promyelocytes, resulting from a cytogenetic condition due to the balanced chromosomal translocation t(15;17)(q22;q21). Current first-line treatment of APL includes all-trans retinoic acid (all-trans RA), with or without arsenic trioxide, combined with chemotherapy, and a chemotherapy-free approach wherein arsenic trioxide is used alone or in combination with all-trans RA. The usage of all-trans RA revolutionized the treatment of APL, with survival rates of 80 to 90% being achieved. The mechanism of action of all-trans RA is based on regulation of gene transcription, promoting the differentiation of leukemic promyelocytes. Encapsulation technology has been explored as an innovative strategy to overcome the major drawbacks related to the all-trans RA oral administration in the APL treatment. The most recently published works on this subject highlight the development and optimization of carrier-based delivery systems based in microparticle formulations obtained by spray-drying to be used in the treatment of APL. The ultimate goal is to obtain a controlled delivery system for RA oral administration capable of providing a slow release of this bioactive compound in the intestinal lumen.

First trimester recurrent pregnancy loss due to balanced chromosomal translocations in husband: A case report

Early pregnancy loss in a wanted pregnancy is stressful and frustrating to any couple. It becomes even more emotionally traumatic when it occurs repeatedly. Two or more of early pregnancy loss is known as recurrent pregnancy loss (RPL). RPL is a clinical challenge for physicians to unriddle. In this case report, we illustrate a case of a 40-year-old woman who experienced six recurrent first-trimester pregnancy losses. After the third pregnancy loss, initial maternal blood investigations were done, and the results were normal. After the fifth miscarriage, both partners’ blood was tested for the cytogenetic study, and her cytogenetic analysis revealed a normal karyotype. However, the paternal showed balanced translocation 46, XY, t(6;13) (q13;14.1) with a balanced translocation between the long arm of chromosome 6 at position 6q13 and the long arm of chromosome 13 at position 13q14.1. When the initial blood investigation is considered normal, a chromosomal study is one of important etiological investigation in couples with RPL.

Newly defined Unbalanced distributions of paternal balanced chromosomal translocation and review of the literature

Newly defined Unbalanced distributions of paternal balanced chromosomal translocation and review of the literature

Prenatal diagnosis of 21 fetuses with balanced chromosomal abnormalities (BCAs) using whole-genome sequencing.

Balanced chromosomal abnormalities (BCAs) are the most common chromosomal abnormalities and the frequency of congenital abnormalities is approximately twice as high in newborns with a de novo BCA, but a prenatal diagnosis based on BCAs is subject to evaluation. To detect translocation breakpoints and conduct a prenatal diagnosis, we performed whole-genome sequencing (WGS) in 21 subjects who were found BCAs, 19 balanced chromosome translocations and two inversions, in prenatal screening. In 16 BCAs on non-N-masked regions (non-NMRs), WGS detected 13 (81.2%, 13/16) BCAs, including all the inversions. All the breakpoints of 12 (12/14) cases of sufficient DNA were confirmed by Sanger sequencing. In 13 interrupted genes, CACNA1E (in case 12) and STARD7 (in case 17) are known causative and PDCL was found in subject (case 11) with situs inversus for the first time. Case 12 with abnormal ultrasound reached a definitive genetic diagnosis of CACNA1E-disease, while STARD7 exon deletion has never been found causative in patients. WGS provides the possibility of prenatal diagnosis in fetuses with BCAs, and its clinical significance also lies in providing data for postnatal diagnosis.

A case of complex balanced chromosomal translocations associated with adverse pregnancy outcomes

Complex chromosomal rearrangements (CCR) are rare chromosomal structural abnormalities. The chromosomal structural variants in CCR carriers are one of the factors contributing to a history of adverse pregnancy and childbirth. In this study, we report a patient with a history of adverse pregnancy and childbirth who exhibited complex balanced chromosomal translocations. The female patient was phenotypically and intellectually normal; in her first pregnancy, the embryo was damaged, and a histological examination of the chromosomes of the embryos revealed a deletion of approximately 4.66 Mb at 1p32.3p32.2, a duplication of approximately 1.02 Mb at 1p22.2p22.1, a duplication of approximately 1.46 Mb at 6q27 and a deletion of approximately 7.78 Mb at 9p24.3p24.1. Chromosomal examinations of the patient revealed the karyotype to be 46,XX,(1;9)(p32; p34). In the second pregnancy, the foetus was diagnosed prenatally with three or more positive ultrasound soft indicators. The patient's karyotype was re-examined and further confirmed by fluorescence in situ hybridisation as 46,XX,t(1;9;6)(p31;p22;q27), revealing this patient was a carrier of complex balanced chromosomal translocations. Carriers of CCR have a higher risk of spontaneous abortion, and genetic counselling clinicians should consider the karyotype analyses of such patients in clinical practice and recheck their chromosomes if necessary.

P-549 Paraclinical and spermogram evaluation of infertile males with balanced chromosomal rearrangements referred to Royan institute from 2009-2021

Abstract Study question Whether balanced chromosomal rearrangements are associated with altered semen parameters/increased DNA Fragmentation Index (DFI), are the common breakpoints related to male infertility in patients ? Summary answer Balanced chromosomal rearrangements is associated with abnormal semen parameters and increased DFI in carriers. Chromosomes 9,1,3 showed the highest incidence of chromosomal rearrangement What is known already Among many factors, balanced chromosomal rearrangements are thought to be one of the major genetic factors influencing male infertility which can cause disorders related to spermogram, DNA fragmentation, increased risk of miscarriage and ART failure. Finding common chromosomal breakpoints in infertile men carrying balanced chromosomal translocations is significant since these patients usually possess normal clinical features. Numerous studies have been performed on the various chromosome translocations. However, there has not been any report on the common breakpoints in balanced chromosomal abnormalities related to male infertility in Iran. Study design, size, duration This cross-sectional study performed on 25000 patients’ files admitted to the Royan institute during the period 2009- 2021.Infertile men carrying balanced chromosomal rearrangements were included as case group. Sperm parameters, DFI and common chromosomal break points were evaluated Participants/materials, setting, methods 25000 files of patients were reviewed. 252 infertile men carrying balanced chromosomal rearrangements and 252 men with normal karyotype were included (case and control group, respectively). Sperm concentration, motility (CASA), sperm morphology (Papanicolaou Staining),DFI (SCSA) and cytogenetic analysis of peripheral blood lymphocytes (G-banding) were evaluated. Numerical chromosomal abnormalities, deletions, marker chromosomes, alcohol or drug addiction, smoking, chemotherapy and radiation, varicocele and exposure to environmental pollution were excluded. The common chromosomal breakpoints in translocations were investigated. Main results and the role of chance Sperm concentration, total motility and normal morphology were significantly lower in balanced chromosomal rearrangements carriers compared to control group(p &amp;lt; 0.05). DFI in patient was significantly higher in comparison to control group(p &amp;lt; 0.05). Among different kind of chromosomal rearrangement carriers’, sperm concentration and total motility were lower in Robertsonian translocation carriers comparing to the Reciprocal translocation and inversion ones. The reciprocal translocation and inversion carriers did not differ regarding to sperm concentration and total motility. Meanwhile, normal morphology was lower in the translocation compared to inversion carriers. No difference was observed in DFI among carriers of balanced chromosomal rearrangements. Among case group, inversion accounted for the largest share, followed by Reciprocal, Robertsonian translocation and insertion. Group C had the highest frequency in reciprocal translocation, inversion and insertion among the chromosomal groups. The highest balanced translocation prevalence was related to three chromosomes 9,3, and1. 9q12,1p36,3p11 were the most frequent breakpoints among them. Chromosome 9 had the largest contribution to inversions,22 to reciprocal translocations and14 to Robertsonian translocations. In the Robertsonian translocation, group D showed the highest frequency. Interestingly, patients carrying normal variations such as pericentric inversion of chromosome 9showed a significant alteration in sperm parameters, therefore normal variations showed a possible relationship to infertility. Limitations, reasons for caution In order to achieve stronger statistical results on DFI, a larger sample size would be more helpful. Wider implications of the findings The results may be useful to identify the most common breakpoints and consequently genes related to male infertility and can suggest appropriate molecular mechanism for their infertility. Trial registration number not applicable

Prenatal detection of pure proximal 6q14.1 microduplication encompassing LCA5 gene: A variant of likely benign.

Trisomy 6q is a recognizable syndrome which exhibits psychomotor/growth retardation, developmental/intellectual disabilities, feeding difficulties, facial dysmorphism, hearing loss, brain and heart malformations. The purpose of this study was to delineate the prenatal features of proximal 6q14.1 duplication in fetal period, which was rarely reported in clinic. Eight pregnant women who opted for amniocentesis due to the fetal ultrasound abnormalities, maternal serum screening or other indications for prenatal diagnosis between 2019 and 2020. Chromosomal microarray analysis and G-banding analysis were offered after informed consents were obtained. Cytogenetic prenatal investigation showed all fetuses presented normal karyotypes except case 4 exhibiting a balanced chromosomal translocation 46,XX,t (4;8)(p16;q24). The chromosomal microarray analysis detected 0.211–0.242 Mb duplications of 6q14.1 (chr6: 80109532–80351666, hg19) in all 8 cases, encompassing the morbid gene LCA5 in common. Seven pregnant women (P1-P7) continued their pregnancies and delivered healthy infants at term while the parents of case 8 opted for termination of pregnancy for severe abnormal ultrasound findings. Overall, all neonates were in a good healthy condition with no evident anomalies, ranging from 2 m to 16 m. It is proposed that 6q14.1 duplication involving LCA5 gene detected in our study might be variants of likely benign. However, further large-scale studies should be gathered to assess its pathogenicity. To our knowledge, our study is the first report focusing on prenatally detected proximal 6q14.1 duplication, accompanied by detailed clinic phenotypes. Diverse ultrasound findings were observed in these cases, ranging from normal to abnormal. More evidence should be gathered to interpret the prenatal genotype-phenotype correlation of 6q14.1 duplication. For these cases with 6q14.1 microduplication, long term follow up should be carried out in case abnormal clinical symptoms or developmental-behavioral disorders emerge.

Abstract 6193: TWIST2 promotes tumor progression and represses myogenic differentiation in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma of myoblast origin. RMS tumors are unable to terminally differentiate into syncytial muscle despite elevated expression of the muscle master regulators, MyoD and Myf5. Fusion-positive RMS (FP-RMS) and fusion-negative RMS (FN-RMS) have emerged as the two major RMS subtypes. The pathogenesis of FP-RMS is commonly defined by the balanced translocation of chromosomes 2 and 3 resulting in the misexpression of a chimeric PAX3/7-FOXO1 oncoprotein that derails normal differentiation pathways. However, the molecular basis of FN-RMS is poorly understood as single nucleotide variations in genes like KRAS, NRAS, and TP53 have limited recurrence (less than 10% of all patient tumors). In contrast, DNA copy number variations are highly recurrent and the genes they encode represent vulnerabilities in FN-RMS. Strikingly, we discovered that 72% of FN-RMS patient tumors harbor copy number amplifications at loci of TWIST1, TWIST2, or both, resulting in the upregulation of TWIST gene expression. We demonstrated that depletion of TWIST2 using a doxycycline inducible shRNA system caused cell cycle arrest and increased myogenic differentiation in xenograft models of FN-RMS. To interrogate whether TWIST2 can directly regulate the expression of these genes, we performed RNA-seq and ChIP-seq for TWIST2, H3K27Ac, and MYOD in TWIST2 knockdown and control FN-RMS cells. Utilizing this genome-wide data of chromatin binding, we comprehensively demonstrated that TWIST2 competes with MYOD preferentially at a subset of enhancers required for myogenic differentiation, causing repression of these loci. Interestingly, while MYOD binding is gained at myogenic loci in the absence of TWIST2, binding of MYOD is not lost at growth-promoting, oncogenic enhancers despite a decrease in their associated gene expression. This suggests that TWIST2 interacts with a different slew of transcription factors or chromatin regulators to activate gene expression. To interrogate this possibility, we are characterizing the TWIST2 protein interactome in FN-RMS cells using BioID as well as motifs enriched at TWIST2-regulated peaks. By studying TWIST2-associated pathways that affect RMS growth and differentiation, we see an opportunity to unlock novel therapeutic strategies for RMS. Citation Format: Akansha Shah, Lei Guo, Priscilla Jaichander, Kenian Chen, Karla Cano Hernandez, Lin Xu, Rhonda Bassel-Duby, Eric Olson, Ning Liu. TWIST2 promotes tumor progression and represses myogenic differentiation in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6193.