Complex Chromosomal Rearrangements Research Articles

Combining cytogenetic and genomic technologies for deciphering challenging complex chromosomal rearrangements.

Complex chromosomal rearrangements (CCRs), a class of structural variants (SVs) involving more than two chromosome breaks, were classically thought to be extremely rare. As advanced technologies become more available, it has become apparent that CCRs are more common than formerly thought, and are a substantial cause of genetic disorders. We attempted a novel approach for solving the mechanism of challenging CCRs, which involve repetitive sequences, by precisely identifying sequence-level changes and their order. Chromosomal microarray (CMA) and FISH analyses were used for interpretation of SVs detected by whole exome sequencing (WES). Breakpoint junctions were analyzed by Nanopore sequencing, a novel long-read whole genome sequencing tool. A large deletion identified by WES, encompassing the FOXF1 enhancer, was the cause of alveolar capillary dysplasia and respiratory insufficiency, resulting in perinatal death. CMA analysis of the newborn's mother revealed two duplications encompassing the deleted region in theproband, raising our hypothesis that the deletion resulted from themother's CCR. Breakpoint junctions of complex SVs were determined at thenucleotide level using Nanopore long-read sequencing. According to sequencing results of breakpoint junctions, the CCR in the newborn was considered the consequence of at least one double-strand break during meiosis, and reassembly of DNA fragments by intra-chromosomal homologous recombination. Our comprehensive approach, combining cytogenetics and long-read sequencing, enabled delineation of the exact breakpoints in a challenging CCR, and proposal of a mechanism in which it arises. We suggest applying our integrative approach combining technologies for deciphering future challenging CCRs, enabling risk assessment in families.

16p13.11p11.2 triplication syndrome: a new recognizable genomic disorder characterized by optical genome mapping and whole genome sequencing.

Highly identical segmental duplications (SDs) account for over 5% of the human genome and are enriched in the short arm of the chromosome 16. These SDs are susceptibility factors for recurrent chromosomal rearrangements mediated by non-allelic homologous recombination (NAHR). Chromosomal microarray analysis (CMA) has been widely used as the first-tier test for individuals with developmental disabilities and/or congenital anomalies and several genomic disorders involving the 16p-arm have been identified with this technique. However, the resolution of CMA and the limitations of short-reads whole genome sequencing (WGS) technology do not allow the full characterization of the most complex chromosomal rearrangements. Herein, we report on two unrelated patients with a de novo 16p13.11p11.2 triplication associated with a 16p11.2 duplication, detected by CMA. These patients share a similar phenotype including hypotonia, severe neurodevelopmental delay with profound speech impairment, hyperkinetic behavior, conductive hearing loss, and distinctive facial features. Short-reads WGS could not map precisely any of the rearrangement's breakpoints that lie within SDs. We used optical genome mapping (OGM) to determine the relative orientation of the triplicated and duplicated segments as well as the genomic positions of the breakpoints, allowing us to propose a mechanism involving recombination between allelic SDs and a NAHR event. In conclusion, we report a new clinically recognizable genomic disorder. In addition, the mechanism of these complex chromosomal rearrangements involving SDs could be unraveled by OGM.

EP314: Rare unbalanced complex chromosomal rearrangement in a case of infertility: Insight into the 8p23.1 duplication syndrome

Complex chromosomal rearrangements (CCR) are defined as structural abnormalities involving more than 2 chromosomal breaks and subsequent exchange of chromosomal material. These events are rare, with less than 255 cases reported to date involving 3 chromosomes. The majority of CCRs are detected in phenotypically normal individuals, presenting with abnormal sperm parameters in males and reproductive failure in females. In these patients, the rearrangement is usually balanced and inherited from a mother carrying the same translocations.

EP402: Partial trisomy of chromosome 22 mediated by chromoanasynthesis in an 8-month-old male

Chromoanasynthesis is a subtype of chromoanagenesis, a term used to describe the single-step processes by which complex chromosomal rearrangements lead to localized structural genomic reorganization of one or a few chromosomes. During chromoanasynthesis, complex chromosomal rearrangements are mediated by replication-based mechanisms including fork stalling and template switching (FoSTeS) or by microhomology-mediated break-induced replication (MMBIR). As a result, highly reorganized chromosomes with multiple copy number changes including deletions, duplications, and triplications are generated.

EP060: Characterizing the complex chromosomal rearrangements in a Philadelphia negative BCR/ABL1 positive case

The Philadelphia chromosome (Ph), produced by the reciprocal translocation t(9;22)(q34;q11.2), is a derivative chromosome 22 with a chimeric BCR/ABL1 fusion gene encoding constitutively active tyrosine kinase protein. The Ph occurs in 95% of chronic myeloid leukemia (CML) cases and the BCR/ABL1 fusion is a diagnostic hallmark for CML. A small subset of patients (around 5-10%), displaying typical features of CML, lack a Ph by chromosome analysis, but are shown to carry BCR/ABL1 fusion either by fluorescence in situ hybridization (FISH) or molecular analysis. Two major mechanisms have been proposed to explain how BCR/ABL1 rearrangements arise in these Ph-negative BCR/ABL1 positive cases: a cryptic insertion between chromosomes 9 and 22; or via two sequential translocations where a standard t(9;22) translocation is followed by an additional reverse translocation resulting in the reconstitution of the normal morphology of the partner chromosomes. The bone marrow specimen of a 66 year old female patient, presented with persistent leukocytosis with myelocytes and metamyelocytes, along with thrombocytosis, was sent to our lab for both BCR/ABL1 RT-PCR analysis and chromosome analysis, due to suspicious myeloproliferative neoplasm. The RT-PCR analysis showed the presence of the BCR/ABL1 major (p210) breakpoint fusion transcript b2a2 (e13a2). Initial chromosome analysis did not identify a typical t(9;22)(q34;q11.2) reciprocal translocation; instead, structural abnormalities were observed on the short arm of chromosome 12 and the long arm of chromosome 22. Overall, the abnormal chromosome 22 resembles the morphology of a “Philadelphia chromosome” while both homologs of chromosome 9 show normal morphology. This leads us to hypothesize that a complex rearrangement involving chromosomes 9, 12, and 22 might be contributing to this case. Metaphase fluorescence in situ hybridization (FISH) analysis was performed to test our hypothesis using BCR/ABL1 fusion probes, where the only cryptic BCR/ABL1 fusion signal was mapped to the long arm of chromosome 9 that has normal morphology at band q34, while the long arm of the derivative chromosome 22 is absent for BCR/ABL1 fusion signals, suggesting that the Ph-like derivative chromosome 22 is not a real Philadelphia chromosome, making this case a true Ph negative case. The final ISCN for the case is designated as: 46,XX,der(9)(9pter->9q34::22q11.2->22q11.2::9q34->9qter),der(12)(22qter->22q11.2::12p13->12qter),der(22)(22pter->22q11.2::12p13->12pter), and this can be explained by an initial three way translocation between chromosomes 9, 12, and 22 followed by an additional sequential reverse translocation between the derivative chromosomes 9 and 22, leading the cryptic BCR/ABL1 fusion being inserted into the long arm of chromosome 9 and bringing chromosome 9 back to a normal morphology. Cryptic BCR/ABL1 fusion has been characterized as a variant rearrangement in CML patients lacking Ph, and up to 50% of these cases with variant BCR/ABL1 fusion exhibit a del(9q) of the derivative chromosome 9, which has been considered to be a marker for poor prognosis in CML patients. Thus, chromosomal microarray analysis (CMA) was also performed for this case, and no deletions in 9q were identified. Here, we presented a case where FISH mapping was utilized to further characterize the complex chromosomal rearrangements between chromosomes 9, 12, and 22 in a Ph negative BCR/ABL1 positive case, which reveals that the cryptic BCR/ABL1 is inserted into the long arm of chromosome 9 with normal morphology at band q34, while the Ph-like derivative chromosome 22 is not a true Ph chromosome. Our study illustrates the challenge of recognizing the complex pattern of cytogenetic aberrations in these Ph negative BCR/ABL1 positive CML cases with variant BCR/ABL1 rearrangements, and highlights the importance of utilizing comprehensive cytogenomic and molecular techniques (including metaphase FISH, RT-PCR and CMA) in characterizing these complex chromosomal abnormalities for proper diagnosis and prognosis prediction.

Clustering and graph mining techniques for classification of complex structural variations in cancer genomes

For many years, a major question in cancer genomics has been the identification of those variations that can have a functional role in cancer, and distinguish from the majority of genomic changes that have no functional consequences. This is particularly challenging when considering complex chromosomal rearrangements, often composed of multiple DNA breaks, resulting in difficulties in classifying and interpreting them functionally. Despite recent efforts towards classifying structural variants (SVs), more robust statistical frames are needed to better classify these variants and isolate those that derive from specific molecular mechanisms. We present a new statistical approach to analyze SVs patterns from 2392 tumor samples from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium and identify significant recurrence, which can inform relevant mechanisms involved in the biology of tumors. The method is based on recursive KDE clustering of 152,926 SVs, randomization methods, graph mining techniques and statistical measures. The proposed methodology was able not only to identify complex patterns across different cancer types but also to prove them as not random occurrences. Furthermore, a new class of pattern that was not previously described has been identified.

Complex Chromosomal Rearrangement Causes Male Azoospermia: A Case Report and Literature Review

Background: Male carriers of complex chromosomal rearrangements (CCRs) may have decreased fertility and usually present with azoospermia, oligospermia or teratospermia. Methods: High-resolution karyotype analysis using G-banding on peripheral blood lymphocytes was performed in an azoospermic male. Copy number variations (CNVs) were detected by chromosomal microarray analysis, and genetic variations were determined by long-read nanopore sequencing with Sanger sequencing for breakpoint confirmation. Results: The karyotype of the patient was 46,XY,t(4;21)(p11;p11),t(5;6;14)(p13q22;p22q22;q22), which did not involve CNVs with clinical significance. Twelve breakpoints in chromosomes 5, 6, and 14 were found by long-read nanopore sequencing. Reports on 17 males carrying CCRs with azoospermia were also reviewed. Conclusion: The extent of asynaptic regions in synaptonemal complexes during pachytene and the disruption of genes involved in male gametogenesis may cause azoospermia in CCR carriers.

Chromosomal abnormalities of 19,000 couples with recurrent spontaneous abortions: a multicenter study

To investigate the demographic data and karyotypes of 19,000 couples who experienced recurrent spontaneous abortion (RSA). A cross-sectional study of 19,000 couples. Five hospitals. A total of 19,000 couples experiencing RSA. Not applicable. Cytogenetic analysis of blood lymphocytes. A total of 844 couples (4.44%) showed chromosomal aberrations in either partner. Females were more likely to have chromosomal aberrations. The mean age of females and males with chromosomal aberrations was younger than that of females and males without chromosomal aberrations. Interestingly, sex and age distribution varied significantly depending on the subtypes of chromosomal aberrations. We detected 324 balanced translocations, including 223 novel ones. They were distributed across all chromosomes; the frequency of balanced translocations decreased according to the numerical order of autosomes (strong negative correlation; r = -0.84). Individuals with balanced translocations were younger than other groups. All 58 inversions, including 25 novel ones, were detected in autosomes; the negative correlation also existed. Thirteen Robertsonian translocations, 5 deletions, and 3 duplications were detected. Six types of Turner variants, triple X mosaicism, and mosaic Down syndrome were detected in females; Klinefelter variants and mosaic XYY syndrome were detected in males. Marker chromosomes at various mosaic levels and 7 different complex chromosomal rearrangements were also observed. Patients who experienced RSA induced by chromosomal aberrations experienced miscarriages at a younger age. Significant correlations existed between the patients' age or sex and the subtypes of chromosomal aberrations. This study detected several chromosomal abnormalities associated with RSA, including various novel aberrations.

Prenatal diagnosis and molecular cytogenetic characterization of hereditary complex chromosomal rearrangements in a Chinese family.

To report a family with an extremely rare and previously undescribed complexchromosomalrearrangement (CCR). To explore the molecular cytogenetic mechanism of 'octaradial chromosome'. G-banding karyotype analysis was performed on all the members of the family. Chromosomal microarray analysis(CMA) was performed on the five members of the family. This case presented with a karyotypically balanced CCR (46,XX,t(2;4;11;5)(p21;q34;q21;p15)). The familial CCR was stably transmitted across three generations. We report an extremely rare and previously undescribed complexchromosomal arrangement that is transmitted across three generations. The clinical outcome of this CCR is complex. Careful characterization of all the breakpoint regions is required for prenatal diagnosis and genetic counseling.

Target enrichment long-read sequencing with adaptive sampling can determine the structure of the small supernumerary marker chromosomes.

Structural analysis of small supernumerary marker chromosomes (sSMCs) has revealed that many have complex structures. Structural analysis of sSMCs by whole genome sequencing using short-read sequencers is challenging however because most present with a low level of mosaicism and consist of a small region of the involved chromosome. In this present study, we applied adaptive sampling using nanopore long-read sequencing technology to enrich the target region and thereby attempted to determine the structure of two sSMCs with complex structural rearrangements previously revealed by cytogenetic microarray. In adaptive sampling, simple specification of the target region in the FASTA file enables to identify whether or not the sequencing DNA is included in the target, thus promoting efficient long-read sequencing. To evaluate the target enrichment efficiency, we performed conventional pair-end short-read sequencing in parallel. Sequencing with adaptive sampling achieved a target enrichment at about a 11.0- to 11.5-fold higher coverage rate than conventional pair-end sequencing. This enabled us to quickly identify all breakpoint junctions and determine the exact sSMC structure as a ring chromosome. In addition to the microhomology and microinsertion at the junctions, we identified inverted repeat structure in both sSMCs, suggesting the common generation mechanism involving replication impairment. Adaptive sampling is thus an easy and beneficial method of determining the structures of complex chromosomal rearrangements.

Canine Oral Melanoma Genomic and Transcriptomic Study Defines Two Molecular Subgroups with Different Therapeutical Targets.

Simple SummaryIn humans, mucosal melanoma (MM) is a rare and aggressive cancer. The canine model is frequently and spontaneously affected by MM, thus facilitating the collection of samples and the study of its genetic bases. Thanks to an integrative genomic and transcriptomic analysis of 32 canine MM samples, we identified two molecular subgroups of MM with a different microenvironment and structural variant (SV) content. We demonstrated that SVs are associated with recurrently amplified regions, and identified new candidate oncogenes (TRPM7, GABPB1, and SPPL2A) for MM. Our findings suggest the existence of two MM molecular subgroups that could benefit from dedicated therapies, such as immune checkpoint inhibitors or targeted therapies, for both human and veterinary medicine.Mucosal melanoma (MM) is a rare, aggressive clinical cancer. Despite recent advances in genetics and treatment, the prognosis of MM remains poor. Canine MM offers a relevant spontaneous and immunocompetent model to decipher the genetic bases and explore treatments for MM. We performed an integrative genomic and transcriptomic analysis of 32 canine MM samples, which identified two molecular subgroups with a different microenvironment and structural variant (SV) content. The overexpression of genes related to the microenvironment and T-cell response was associated with tumors harboring a lower content of SVs, whereas the overexpression of pigmentation-related pathways and oncogenes, such as TERT, was associated with a high SV burden. Using whole-genome sequencing, we showed that focal amplifications characterized complex chromosomal rearrangements targeting oncogenes, such as MDM2 or CDK4, and a recurrently amplified region on canine chromosome 30. We also demonstrated that the genes TRPM7, GABPB1, and SPPL2A, located in this CFA30 region, play a role in cell proliferation, and thus, may be considered as new candidate oncogenes for human MM. Our findings suggest the existence of two MM molecular subgroups that may benefit from dedicated therapies, such as immune checkpoint inhibitors or targeted therapies, for both human and veterinary medicine.

The Clinical Diagnostic Utility of Array CGH in Children with Syndromic Microcephaly.

A prospective study using array CGH in children with Syndromic microcephaly from a tertiary pediatric healthcare centre in India. To identify the copy number variations causative of microcephaly detected through chromosomal array CGH. Of the 60 patients, 33 (55%) males and 27 (45%) females who consulted the Rare Disease Clinic at Department of Pediatrics, SMS Medical College, Jaipur, with developmental delay/facial dysmorphism/congenital anomalies in combination with microcephaly were included. Children with acquired or non-genetic causes of microcephaly, craniosynostosis, metabolic diseases, known chromosomal aneuploidy such as trisomy 21, 13, and 18 and abnormal karyotype were excluded. The cohort was analyzed by array CGH in order to identify potentially pathogenic copy number variants (CNVs). Clinically relevant pathogenic or likely pathogenic copy number variations (CNVs) were identified in 20/60 (33.3%) patients, variant of uncertain significance (VOUS) in 4/60 (6.6%) cases and benign CNVs in 3/60 (5%) of total cases. Out of 20 cases with pathogenic CNVs, 12 (60%) patients detected with a deletion, five (25%) patients with duplication and three (15%) patients resulted with a complex chromosomal rearrangement. Twelve cases present CNVs containing genes known to be implicated in microcephaly etiology. This research highlights the contribution of submicroscopic chromosomal changes in the etiology of microcephaly in combination with developmental delay/facial dysmorphism/congenital anomalies (syndromic microcephaly). Our studies provide more insights into the benefits derived by using array CGH analysis in patients with syndromic microcephaly.

Research Article Characterization of a complex chromosomal rearrangement in a girl with PURA syndrome

Research Article Characterization of a complex chromosomal rearrangement in a girl with PURA syndrome

A complex KMT2A::AFF3 fusion resulting from a three-way chromosomal rearrangement in pediatric B lymphoblastic leukemia

The KMT2A::AFF3 fusion, t(2;11)(q11.2;q23.2), is a very rare fusion occurring in pediatric B-cell acute lymphoblastic leukemia (B-ALL). Our patient is a 2-year-old male who presented with three weeks of intermittent fever. Bone marrow biopsy showed 82% blasts and cytogenetic analysis demonstrated a complex 3-way chromosomal rearrangement involving KMT2A and an unknown fusion partner. Molecular testing identified the fusion partner as AFF3, a FLT3-TKD non-D835 mutation, and an NF1 mutation. This case demonstrates a highly complex three-way variant translocation resulting in the rare KMT2A::AFF3 fusion with only a few cases previously described in the literature.

Neurodevelopmental phenotype in 36 new patients with 8p inverted duplication-deletion: Genotype-phenotype correlation for anomalies of the corpus callosum.

Inverted duplication deletion 8p [invdupdel(8p)] is a complex and rare chromosomal rearrangement that combines a distal deletion and an inverted interstitial duplication of the short arm of chromosome 8. Carrier patients usually have developmental delay and intellectual disability (ID), associated with various cerebral and extra-cerebral malformations. Invdupdel(8p) is the most common recurrent chromosomal rearrangement in ID patients with anomalies of the corpus callosum (AnCC). Only a minority of invdupdel(8p) cases reported in the literature to date had both brain cerebral imaging and chromosomal microarray (CMA) with precise breakpoints of the rearrangements, making genotype-phenotype correlation studies for AnCC difficult. In this study, we report the clinical, radiological, and molecular data from 36 new invdupdel(8p) cases including three fetuses and five individuals from the same family, with breakpoints characterized by CMA. Among those, 97% (n=32/33) of patients presented with mild to severe developmental delay/ID and 34% had seizures with mean age of onset of 3.9 years (2 months-9 years). Moreover, out of the 24 patients with brain MRI and 3 fetuses with neuropathology analysis, 63% (n=17/27) had AnCC. We review additional data from 99 previously published patients with invdupdel(8p) and compare data of 17 patients from the literature with both CMA analysis and brain imaging to refine genotype-phenotype correlations for AnCC. This led us to refine a region of 5.1 Mb common to duplications of patients with AnCC and discuss potential candidate genes within this region.

Molecular characterization of Novel ATM fusions in chronic lymphocytic leukemia and T-cell prolymphocytic leukemia

ATM deletions and/or mutations are recurrent in lymphoid neoplasms while rearrangements are rare. In this study, we used mate pair sequencing (MPseq) technology to characterize two novel ATM rearrangements in one patient with chronic lymphocytic leukemia (CLL) and one patient with T-prolymphocytic leukemia (T-PLL). Both patients showed chromosome 11q22 aberrations encompassing ATM by conventional karyotype and fluorescence in situ hybridization: isolated t(11;13)(q22;q14) in CLL and a complex karyotype with apparent 11q deletion and unbalanced der(14)t(11;14)(q22;p11.2) in T-PLL. MPseq identified ATM-LINC00371 fusion in CLL and ATM-USP28 in T-PLL, both of which led to ATM inactivation, confirmed by loss of immunohistochemical protein expression. Next-generation sequencing mutation analysis detected concurrent ATM mutation(s) CLL patient, while T-PLL lacked ATM mutation. ATM rearrangements, not apparently detectable using standard laboratory technologies, represent another mechanism of loss-of-function. Recent high-throughput technologies such as MPseq can uncover novel pathogenic gene fusions and resolve complex chromosomal rearrangements in hematologic malignancies.

Limited Introgression between Rock-Wallabies with Extensive Chromosomal Rearrangements.

Chromosome rearrangements can result in the rapid evolution of hybrid incompatibilities. Robertsonian fusions, particularly those with monobrachial homology, can drive reproductive isolation amongst recently diverged taxa. The recent radiation of rock-wallabies (genus Petrogale) is an important model to explore the role of Robertsonian fusions in speciation. Here, we pursue that goal using an extensive sampling of populations and genomes of Petrogale from north-eastern Australia. In contrast to previous assessments using mitochondrial DNA or nuclear microsatellite loci, genomic data are able to separate the most closely related species and to resolve their divergence histories. Both phylogenetic and population genetic analyses indicate introgression between two species that differ by a single Robertsonian fusion. Based on the available data, there is also evidence for introgression between two species which share complex chromosomal rearrangements. However, the remaining results show no consistent signature of introgression amongst species pairs and where evident, indicate generally low introgression overall. X-linked loci have elevated divergence compared with autosomal loci indicating a potential role for genic evolution to produce reproductive isolation in concert with chromosome change. Our results highlight the value of genome scale data in evaluating the role of Robertsonian fusions and structural variation in divergence, speciation, and patterns of molecular evolution.

Whole-genome analysis of a putative rare and complex interchromosomal reciprocal insertion: thorough investigations for a straightforward interpretation

Research questionShould whole-genome investigations be considered systematically before a complex chromosomal abnormality preimplantation genetic testing for structural chromosomal rearrangements (PGT-SR) management is carried out using conventional cytogenetic techniques? DesignA male carrying a putative rare interchromosomal reciprocal insertion (IRI) 46,XY,ins(14;?)(q11;?).ish der(14)ins(14;22)(q11.2;q11.2q11.2)(xcp14+,xcp22+,N25+,3’TRA/D+),der(22)ins(22;14)(q11.2;q11.2q11.2)(xcp22+,xcp14+,N25-,5’TRA/D+), and his partner were referred to our centre for preimplantation genetic testing analysis after three spontaneous miscarriages. Whole-genome sequencing was used to distinguish between the proposed IRI and an alternative explanation of reciprocal translocation. Fluorescence in-situ hybridization was used to detect all chromosome segments involved in this chromosomal rearrangement, to identify transferable normal and balanced embryos. ResultsWhole-genome sequencing allowed the determination of the number of chromosomal breakpoints involved in chromosomal rearrangement between chromosomes 14 and 22. Finally, only two breakpoints were identified instead of four in IRI rearrangements, which suggests a reciprocal translocation rearrangement. A probe strategy was established to highlight all chromosomal imbalances, whether IRI or reciprocal translocation, and preimplantation genetic testing cycles were achieved. ConclusionConventional cytogenetic techniques are not capable of identifying all complex chromosomal rearrangements, especially those involving centromeric regions and short arms of acrocentric chromosomes. The advent of new sequencing technologies has allowed for a better appreciation of genome complexity. In this study, whole-genome analysis provided additional information to explain the occurrence of genomic events and confirmed that the initial diagnosis of IRI identified by conventional cytogenetic techniques was, in fact, a simple reciprocal translocation. A reliable PGT-SR strategy was proposed for this couple to achieve their parental project.

Diagnosis of Shashi-Pena Syndrome Caused by Chromosomal Rearrangement Using Nanopore Sequencing.

Background and ObjectivesThe aim of this study was to uncover the genetic cause of delayed psychomotor development and variable intellectual disability in a proband whose previous genetic analyses, including chromosome microarray and whole exome sequencing, had been negative.MethodsLong-read sequencing Oxford Nanopore Technology and RNA-seq analysis were performed on peripheral blood mononuclear cells. Genes with a fold change ≥ 1.5 and p ≤ 0.05 were identified as differentially expressed.ResultsClinical examinations showed that the proband's features were similar to a rare autosomal-dominant neurodevelopmental syndrome, Shashi-Pena syndrome (MIM #617190). Karyotyping showed that a chromosomal balanced translocation t(2; 11) (p23; q23) was detected in the proband, her father, and her grandmother. Meanwhile, long-read sequencing identified 102 balanced translocations and 145 inversions affecting ASXL2 at an average of 15×. Combined with the family's RNA-seq results, the average mRNA expression of ASXL2 decreased in the patients.DiscussionWe identified a complex chromosomal rearrangement affecting ASXL2 as a pathogenic mechanism of Shashi-Pena syndrome in a Chinese family. This case study suggests that nanopore sequencing is suitable for pathogenic analysis of complex rearrangements, providing new avenues for the diagnosis of genetic diseases.

Loss of Lamin B1 in Myeloid Neoplasms with 5q Deletion Causes Myeloid-Biased Hematopoiesis and Pelger-Huet Nuclear Anomaly

Hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations and cytogenetic abnormalities leading to myeloid neoplasms (MDS/AML). A subgroup of 10-20% MDS and AML cases undergo complex chromosomal rearrangements associated with TP53 mutations and poor prognosis. One of the most common chromosomal events in high-risk myeloid neoplasms is deletion of chromosome 5q (del(5q)). In contrast to 5q- syndrome, characterized by a distal commonly deleted region (CDR) and good prognosis, the critical CDR in high-risk MDS/AML centers on 5q31. However, the key genes in the high-risk 5q-deleted region that contribute to dysregulation of hematopoiesis, chromosomal instability, and disease progression remain poorly characterized.Progression to high-risk MDS/AML is often accompanied by morphologic dysplasia in the form of Pelger-Huët cells, abnormal neutrophils with bilobed or unilobed nuclei and coarse clumping of the nuclear chromatin. The inherited form of Pelger-Huët anomaly (PHA) is caused by mutations in LBR, which encodes lamin B receptor. Acquired PHA or pseudo-PHA is commonly seen in myeloid malignancies, often in high risk MDS and AML with TP53 mutations. While LBR is not mutated in MDS/AML, its main interaction partner lamin B1/LMNB1 is encoded in the high-risk 5q-deleted region. Lamins organize chromatin into perinuclear compartments and regulate diverse biological processes, including gene expression and DNA repair. The frequent deletion of LMNB1 in 5q-deleted MDS and AML led us to hypothesize that LMNB1 loss drives both nuclear dysmorphology and functional HSC defects in malignant transformation.LMNB1 expression was broadly decreased in MDS and AML, particularly in del(5q) cases, 83% of which had deletions of the LMNB1 locus. We show that in a clinical cohort of MDS patients, the Pelger-Huët nuclear phenotype is strongly associated with del5q. To understand the role of LMNB1, we performed lentiviral shRNA knockdown in normal umbilical cord blood (CB) CD34 + HSPCs followed by transplantation into immunodeficient NSG mice. To study the role of lamin B1 in 5q-deleted MDS, we established an in vitro patient-derived induced pluripotent stem cell (iPSC) model by reprograming an MDS patient with TP53 mutation and complex karyotype, and deriving MDS HSPCs with TP53+/R209fs alone or TP53+/R209fs with an isolated del5q spanning 5q22-5q31.1 encoding LMNB1 (TP53;del5q).Using these model systems we show that LMNB1 loss is both necessary and sufficient to cause Pelger-Huët nuclear morphology in normal and MDS neutrophils. Additionally, we find that LMNB1-deficient human HSCs display increased self-renewal and profound myeloid lineage bias in vitro and in vivo. Single cell RNA expression data from LMNB1-deficient cells in vivo shows mis-expression of lineage-specifying transcription factors in single multipotent progenitors. LMNB1 deficiency also results in poor marking and propagation of unrepaired DNA breaks, leading to genome instability. The role of LMNB1 in maintaining chromatin organization prompted us to use chromosome conformation capture (Hi-C) to visualize the 3D chromatin organization of LMNB1-deficient HSPCs. We show that lamin B1 regulates enhancer-promoter loops suggesting that lamin B1 loss dysregulates HSC function by altering enhancer-promoter interactions at lineage-specifying transcription factor loci.Together, we identify lamin B1 as a novel 5q gene that contributes to malignant transformation by driving enhanced self-renewal, myeloid-bias, and genome instability. Furthermore, lamin B1 deletion is the genetic cause of acquired PHA in myeloid malignancies, providing a potential biomarker for high risk neoplasms. DisclosuresBecker: Glycomimetics: Research Funding; CVS Caremark: Consultancy; Abbie: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Cardiff Oncology: Research Funding; SecuraBio: Research Funding.