Intrachromosomal Translocation Research Articles

A D e Novo Supernumerary Ring Chromosome 1 Causes B-Cell Acute Lymphoblastic Leukemia in Monozygotic Twins Due to Independent and Partially Convergent Evolutionary Trajectories

Introduction B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer. In recent years, our understanding of B-ALL biology has benefited from genomic and transcriptomic analyses that identify driver alterations in 98% of patients. In addition, prior studies of monozygotic twins, who both developed B-ALL, have provided insight into the developmental timing and natural history of the disease, as well as the identification of novel mechanisms of leukemogenesis. Herein we study a case of monozygotic twins, who developed B-ALL (Table 1). We report the characterization of a de novo supernumerary ring chromosome 1 (SRC1) as an initiator event that followed independent but partially convergent clonal trajectories in each twin. Thus, SRC1 resulted in genomic rearrangements of MEF2D in both twins but targeted distinct fusion gene partners ( BCL9 versus ARNT). To our knowledge, this study represents the first description of MEF2D-ARNT as an event involved in B-ALL, and the association of SRC1 Syndrome with leukemia. Methods We performed integrative whole genome sequencing (WGS) and RNAseq analysis on B-ALL samples at diagnosis and compared this to WGS data from minimal residual disease negative bone marrow aspirates. Coverage levels for tumors and matched normals were: Twin A=104 & 59; Twin B=81 & 53. Tumor purity estimations were 0.80 in Twin A and 0.31 in Twin B. For RNAseq, expression profile efficiencies were 61% and 36% respectively. Comprehensive variant calling was performed using the Isabl platform (Medina et al, 2020) to map all putative somatic and germline events. ALLCatchR (Beder et. al, 2022) was used for molecular B-ALL subtype allocation in 26 samples from our pediatric cohort of patients including the twins (age range 1-23), and MutationTimeR (Gerstung et al, 2020) to calculate the relative timing of copy number gains. All research studies were approved by NYU and MSKCC Institutional Review Boards. Results The tumor mutational burdens for each leukemia sample were 0.86 for Twin A and 0.47 mutations/Mb for Twin B, and they showed independent alterations in oncogenic genes (Table 1). The only B-ALL driver that both twins had partially in common was an intrachromosomal translocation resulting in MEF2D fusions, but each with a distinct partner gene ( BCL9 versus ARNT) which was confirmed by RNAseq (supporting reads: Twin A=48, Twin B=107) and PCR assays (Fig. 1). Each sample demonstrated enhanced transcription levels of MEF2D compared to our reference B-ALL cohort (transcripts per million: Twin A=48.0, Twin B=77.4, median=27.7, sd=19.9), and were transcriptionally classified as MEF2D-subtype by the ALLCatchR classifier. These results functionally supported the role of MEF2D-ARNT as a new oncogenic event. Analysis of germline variants showed a tandem duplication flanking the pericentromeric region of chromosome 1 in both twins (1:120,163,074-157,714,717) where MEF2D BCL9and ARNT are localized, that had been previously identified as SRC1 in only one of them by constitutional genotyping (Table 1). Cell fractions in normal and tumor tissues were: Twin A=14.8% & 50%, Twin B=8.6% & 26.5%. PCR interrogation of parental germline DNA failed to identify SRC1, suggesting this to be a de novo event in the affected twins (Fig. 1). We compared the relative timepoint in which the SRC1 took place in each sample using MutationalTimeR, but we were only able to obtain results in Twin A due to a higher mutational burden (number of variants in SRC1: Twin A=44, Twin B=13). As expected, the tandem duplication that formed the SRC1 was identified as an early mutational event (confidence interval of relative time of appearance: 0-0.4). Conclusions Our results identify SRC1 as the common initiating genomic event leading to MEF2D fusion driven B-ALL in both twins. These findings support a model in which a tandem duplication around a pericentromeric area was an initiating event which caused the scission and circularization of DNA without the loss of genetic material. This led to a de novo acquisition of a SRC1, that after independent but partially convergent clonal trajectories gave rise to 2 distinct oncogenic MEF2D fusion driven leukemias in each twin . Moreover, ARNT is a novel partner gene to MEF2D which has not been previously described. This is the first report of B-ALL association with SRC1 Syndrome, which emphasises the utility of the study of monozygotic twins to identify novel alterations underlying B-ALL.

Abstract 2053: Structural variants detected by optical genome mapping in acute lymphoblastic leukemia patient-derived xenografts models

Abstract Background: The established classification of acute lymphoblastic leukemia (ALL) does not cover all high-risk patients because of the difficulty in detecting novel or rare structural variants (SVs). Furthermore, many SVs and formed gene fusions have adverse prognostic effects on hematological malignancies. This study aims to investigate SVs in 22 patient-derived xenografts (PDX) of ALL and to assess rare SVs and chromosomal aberrations, particularly hematological malignancy genes and gene fusions in different individuals detected by optical genome mapping (OGM). Methods: Leukemia cells from 16 patients with B-cell precursor acute lymphoblastic leukemia (B-ALL) and 6 patients with T-cell acute lymphoblastic leukemia (T-ALL) were inoculated into immuno-deficient mice, growth curves were monitored, and leukemia cells were collected after human CD45 was more than 80%. OGM (Bionano Genomics), RNA sequencing (RNA-seq) and whole genome sequencing (WGS) techniques were used to detect SVs and gene fusions. In validation, polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) tests were performed to identify specific SVs. Results: On average, each model detected 42 rare SVs with known overlapping genes in different types by OGM: 12 insertions, 25 deletions, 1 inversion, 3 duplications, 2 inter-chromosomal translocations and 1 intra-chromosomal translocation. 5 rare SVs were found in multiple models (>=5), and two of them were never reported in B-ALL and T-ALL. Gene fusions were detected in all ALL models by OGM, and 17% were novel fusions. At the mRNA level, gene fusions were detected only in 19 models, and 24% of the predicted gene fusions by OGM were identical to the results of RNA-seq (high confidence results including pseudogenes). These two technologies could detect different fusion partners. For example, MLLT1 was seen at the mRNA level, and ACER1 was detected at the DNA level in the same KMT2A rearrangement model. Of the 16 B-ALL models, 6 were p190 BCR-ABL1 (a fusion of BCR exon 1 and ABL1 exon 2), 1 was p210 BCR-ABL1 (a fusion of BCR exon 13 and ABL1 exon 2) and 5 of 7 had IKZF1 gene deletions. 2 p190 BCR-ABL1 models were insensitive to imatinib and 2 p190 BCR-ABL1 models as well as the model with IKZF1 exons 4-7 deletion were sensitive to imatinib when treatment was initiated early in the disease. A novel ARL15-IKZF1 fusion was identified in one of the sensitive models. Deletion of the PRKAR2B gene was another rare SV in both models sensitive to imatinib besides the IKZF1 deletion. Conclusion: OGM assays were more comprehensive, and novel rare SVs were identified in ALL. Combining the results of gene fusions between different technologies will provide more accurate predictions for ALL classifications. The combination of aberrant SVs may synergize to influence the efficacy of imatinib, which warrants further investigations. Citation Format: Yueying Wang, Mengting Qin, JinPing Liu, Xiaobo Chen, Jia Xue, Sheng Guo. Structural variants detected by optical genome mapping in acute lymphoblastic leukemia patient-derived xenografts models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2053.

Mapping of the novel powdery mildew resistance gene Pm2Mb from Aegilops biuncialis based on ph1b-induced homoeologous recombination.

A novel powdery mildew resistance gene Pm2Mb from Aegilops biuncialis was transferred into common wheat and mapped to chromosome 2MbL bin FL 0.49-0.66 by molecular cytogenetic analysis of 2Mb recombinants. Aegilops biuncialis, a wild relative of common wheat, is highly resistant to powdery mildew. Previous studies identified that chromosome 2Mb in Chinese Spring (CS)-Ae. biuncialis 2Mb disomic addition line TA7733 conferred high resistance to powdery mildew, and the resistance gene was temporarily designated as Pm2Mb. In this study, a total of 65 CS-Ae. biuncialis 2Mb recombinants were developed by ph1b-induced homoeologous recombination and they were grouped into 12 different types based on the presence of different markers of 2Mb-specificity. Segment sizes and breakpoints of each 2Mb recombinant type were further characterized using in situ hybridization and molecular marker analyses. Powdery mildew responses of each type were assessed by inoculation of each 2Mb recombinant-derived F2 progenies using the isolate E05. Combined analyses of in situ hybridization, molecular markers and powdery mildew resistance data of the 2Mb recombinants, the gene Pm2Mb was cytologically located to an interval of FL 0.49-0.66 in the long arm of 2Mb, where 19 2Mb-specific markers were located. Among the 65 2Mb recombinants, T-11 (T2DS.2DL-2MbL) and T-12 (Ti2DS.2DL-2MbL-2DL) contained a small 2MbL segment harboring Pm2Mb. Besides, a physical map of chromosome 2Mb was constructed with 70 2Mb-specific markers in 10 chromosomal bins and the map showed that submetacentric chromosome 2Mb of Ae. biuncialis was rearranged by a terminal intrachromosomal translocation. The newly developed 2Mb recombinants with powdery mildew resistance, the 2Mb-specific molecular markers and the physical map of chromosome 2Mb will benefit wheat disease breeding as well as fine mapping and cloning of Pm2Mb.

Transcriptome and Gene Fusion Analysis of Synchronous Lesions Reveals lncMRPS31P5 as a Novel Transcript Involved in Colorectal Cancer

Fusion genes and epigenetic regulators (i.e., miRNAs and long non-coding RNAs) constitute essential pieces of the puzzle of the tumor genomic landscape, in particular in mechanisms behind the adenoma-to-carcinoma progression of colorectal cancer (CRC). In this work, we aimed to identify molecular signatures of the different steps of sporadic CRC development in eleven patients, of which synchronous samples of adenomas, tumors, and normal tissues were analyzed by RNA-Seq. At a functional level, tumors and adenomas were all characterized by increased activity of the cell cycle, cell development, cell growth, and biological proliferation functions. In contrast, organic survival and apoptosis-related functions were inhibited both in tumors and adenomas at different levels. At a molecular level, we found that three individuals shared a tumor-specific fusion named MRPS31-SUGT1, generated through an intra-chromosomal translocation on chromosome 13, whose sequence resulted in being 100% identical to the long non-coding RNA (lncRNA) MRPS31P5. Our analyses suggest that MRPS31P5 could take part to a competitive endogenous (ce)RNA network by acting as a miRNA sponge or/and as an interactor of other mRNAs, and thus it may be an important gene expression regulatory factor and could be used as a potential biomarker for the detection of early CRC events.

Next-Generation Optical Mapping Reveals Numerous Previously Unrecognizable Structural Variants in Chronic Lymphocytic Leukemia

Introduction: Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults in western countries. Although a number of genetic aberrations (complex karyotype, del(17p), del(11q), unmutated IGVH status, TP53 mutations) having predictive and prognostic significance in CLL was reported, there is still a lack of information about large structural variants, repetitive regions, copy number variations, and inversions in the CLL genome due to limitations of current methods (NGS, cytogenetics). Aim: Using novel next-generation optical mapping technology, we analyzed leukemic cells from peripheral blood of CLL patients at different stages of the disease. Methods and Patients: High-molecular weight DNA was isolated from peripheral blood (>90% CD5+/CD19+/CD20+/CD23+ B-cells) from two CLL patients (P1: 70-y-old male, diagnosis of CLL in 2018, Binet Stage B, unmutated IGVH, untreated; P2: 59-y-old male, diagnosis of CLL in 2005, Binet Stage A, unmutated IGVH, on ibrutinib (2nd line) since 4/2018). For optical mapping, DNA was labeled by DLS chemistry and visualized on Saphyr system (BioNano Genomics). The optical genome maps (480 Gbp, coverage 124X) were aligned to a human reference genome (GRCh38) and the detected structural variants (SVs) were compared against the database of healthy controls; only rare de novo variants were analyzed. In both patients, cytogenetic analysis (karyotype, FISH, and arrayCGH) and mutational analysis by NGS (TP53, ATM, BIRC3, NOTCH1, SF3B1, POT1 genes) were available. Results: For both CLL patients, the optical mapping method identified 97% of chromosomal abnormalities previously reported by standard cytogenetic methods such as deletions on 6q, 13q, 11q, 17p and trisomy 12. In both patients, same large deletions of 17.5 kbp in 9q21 and 7.1 kbp in 14q21 chromosome region were detected, undetectable by standard methods. Both patients also carried intra-chromosomal translocation t(13;13) located in 13q14 region. Additionally, P1 genome carried 2 kbp deletion in oncogene EGFR and 500 bp insertion affecting tumor suppressor TCF4, both genes previously associated with CLL. Moreover, numerous additional copy number variations and large somatic SVs, including on average tens of deletions, insertions, inversions, duplications, and translocations were detected in both patients. Conclusion: Our study revealed numerous novel genomic rearrangements in CLL genome, unrecognizable by standard methods. These data highlights a high potential of optical maps for refinement of genomic variability in CLL, mainly for detection of large structural variations and copy number variants. The study on larger patient cohort and longer follow-up of patients may identify genetic aberrations associated with the clinical course and therapy response. Support: MZ ČR VES16-32339A, MZ ČR - RVO (FNOl, 00098892), IGA UP_2019_014, IGA UP_2019_01 Disclosures Minarik: Celgene: Consultancy, Honoraria, Research Funding; Amgen, BMS, Janssen-Cilag, Takeda: Consultancy, Honoraria.

Molecular mechanisms and pathobiology of oncogenic fusion transcripts in epithelial tumors.

Recurrent fusion transcripts, which are one of the characteristic hallmarks of cancer, arise either from chromosomal rearrangements or from transcriptional errors in splicing. DNA rearrangements include intrachromosomal or interchromosomal translocation, tandem duplication, deletion, inversion, or result from chromothripsis, which causes complex rearrangements. In addition, fusion proteins can be created through transcriptional read-through. Fusion genes can be transcribed to fusion transcripts and translated to chimeric proteins, with many having demonstrated transforming activities through multiple mechanisms in cells. Fusion proteins represent novel therapeutic targets and diagnostic biomarkers of diagnosis, disease status, or progression. This review focuses on the mechanisms underlying the formation of oncogenic fusion genes and transcripts and their impact on the pathobiology of epithelial tumors.

Difference of molecular alterations in HER2-positive and HER2-negative gastric cancers by whole-genome sequencing analysis.

ObjectiveThe aim of this study was to compare the molecular profiling, including somatic mutation and somatic copy number variation (SCNV), between human epidermal growth factor receptor 2 (HER2)-positive (HER2+) and HER2-negative (HER2−) gastric cancer patients.Patients and methodsTumor samples were collected from 15 gastric cancer patients, including 10 HER2+ samples and five HER2− samples, which were diagnosed by immunohistochemistry. Whole-genome sequencing was performed by Illumina HiSeq PE150 instrument, along with somatic single nucleotide variant (SNV), somatic structural variation (SV) and SCNV analyses.ResultsThe average number of somatic SNVs and mutation spectrum were similar between HER2+ and HER2− samples. Transition of C>T was the main type of mutation. For somatic SV, number of intrachromosomal translocation (2,850.3±1,260.4 vs 1,157±586.6, P=0.015) and insertion of large fragment (1,125.6±457.4 vs 500±138.9, P=0.002) in HER2+ samples were higher than those in HER2− samples. For all samples, lysine methyltransferase 2C (KMT2C), ZNF91, TAF1 and MAP4 genes were identified as new significant mutated driver genes. KMT2C gene mutations were mainly detected in HER2+ samples (7/10), which were correlated with the lysine degradation pathway. SERF2 gene mutations were more common in HER2− samples (3/5) than in HER2+ samples (1/10). Copy number gain was the major type of SCNV in both groups, and the average number of SCNVs was similar. In the HER2+ samples, by using the GISTIC algorithm, amplification of known driver genes cyclin-dependent kinase 12 (CDK12, 6/10) and RARA (5/10) was mainly observed, and other amplifications including JUP, GJD3, KRT39, CDC6, RAPGEFL1, WIPF2, FAM65C, KLF5, DACH1 and PIBF1 genes were also observed. Amplifications of solute carrier family 12 member 7 (SLC12A7, 5/5), TTC40 (4/5) and GALNT9 (4/5) genes were mainly detected in HER2− samples.ConclusionDifferences in genomic landscape between HER2+ and HER2− gastric cancer samples were revealed in this study. KMT2C mutation and CDK12 amplification were mainly detected in HER2+ gastric cancer, whereas SERF2 mutation and SLC12A7 amplification were detected in HER2− gastric cancer.

Conservation and lineage-specific rearrangements in the GOBP/PBP gene complex of distantly related ditrysian Lepidoptera

General odorant binding proteins (GOBPs) and pheromone binding proteins (PBPs) form a monophyletic subfamily of insect odorant binding proteins (OBPs) specific for Lepidoptera, butterflies and moths. The GOBP/PBP genes include six subgroups (GOBP1–2, PBP-A–D) previously reported to form a complex arrayed in a conserved order in representative moths (superfamily Bombycoidea) and butterflies (Nymphalidae). Although our knowledge of lepidopteran genomes has increased greatly recently, the structure of the GOBP/PBP complex has been studied only for species that represent limited lineages of the highly diverged Ditrysia. To understand the evolution of this functionally important gene complex, we determined 69–149 kb genomic sequences that include GOBP2 and five PBP genes in three Ostrinia moths (Pyraloidea), O. nubilalis, O. furnacalis, and O. latipennis, using bacterial artificial chromosome (BAC) and fosmid clones. The structure of the GOBP2/PBP gene cluster was well conserved despite the different sex pheromone composition utilized by the three moths. Five expressed PBP genes in Ostrinia moths were the result of two duplications of PBP-A genes. Surprisingly, an allele containing a fusion gene between tandemly arrayed PBP-A genes was observed in O. nubilalis. We also revealed duplication and intra-chromosomal translocation of the GOBP1 gene in P. xylostella by fluorescence in situ hybridization (FISH) analysis. Additionally, we compared the structure of the GOBP/PBP gene complex of seventeen species covering six superfamilies and twelve families of the lepidopteran clade, Ditrysia, and found the gene order was basically conserved despite the frequent occurrence of lineage-specific gains, losses, inversions and translocations of these genes, compared with their neighboring genes. Our findings support the hypothesis that the structure of the GOBP/PBP gene complex was already established in the common ancestor of Ditrysia.

A High-Density Genetic Map of Wild Emmer Wheat from the Karaca Dağ Region Provides New Evidence on the Structure and Evolution of Wheat Chromosomes.

Wild emmer (Triticum turgidum ssp. dicoccoides) is a progenitor of all cultivated wheat grown today. It has been hypothesized that emmer was domesticated in the Karaca Dağ region in southeastern Turkey. A total of 445 recombinant inbred lines of T. turgidum ssp. durum cv. ‘Langdon’ x wild emmer accession PI 428082 from this region was developed and genotyped with the Illumina 90K single nucleotide polymorphism Infinium assay. A genetic map comprising 2,650 segregating markers was constructed. The order of the segregating markers and an additional 8,264 co-segregating markers in the Aegilops tauschii reference genome sequence was used to compare synteny of the tetraploid wheat with the Brachypodium distachyon, rice, and sorghum. These comparisons revealed the presence of 15 structural chromosome rearrangements, in addition to the already known 4A-5A-7B rearrangements. The most common type was an intra-chromosomal translocation in which the translocated segment was short and was translocated only a short distance along the chromosome. A large reciprocal translocation, one small non-reciprocal translocation, and three large and one small paracentric inversions were also discovered. The use of inversions for a phylogeny reconstruction in the Triticum–Aegilops alliance was illustrated. The genetic map was inconsistent with the current model of evolution of the rearranged chromosomes 4A-5A-7B. Genetic diversity in the rearranged chromosome 4A showed that the rearrangements might have been contemporary with wild emmer speciation. A selective sweep was found in the centromeric region of chromosome 4A in Karaca Dağ wild emmer but not in 4A of T. aestivum. The absence of diversity from a large portion of chromosome 4A of wild emmer, believed to be ancestral to all domesticated wheat, is puzzling.

A single amino acid mutation in Spo0A results in sporulation deficiency of Paenibacillus polymyxa SC2

Sporulating bacteria such as Bacillus subtilis and Paenibacillus polymyxa exhibit sporulation deficiencies during their lifetime in a laboratory environment. In this study, spontaneous mutants SC2-M1 and SC2-M2, of P. polymyxa SC2 lost the ability to form endospores. A global genetic and transcriptomic analysis of wild-type SC2 and spontaneous mutants was carried out. Genome resequencing analysis revealed 14 variants in the genome of SC2-M1, including three insertions and deletions (indels), 10 single nucleotide variations (SNVs) and one intrachromosomal translocation (ITX). There were nine variants in the genome of SC2-M2, including two indels and seven SNVs. Transcriptomic analysis revealed that 266 and 272 genes showed significant differences in expression in SC2-M1 and SC2-M2, respectively, compared with the wild-type SC2. Besides sporulation-related genes, genes related to exopolysaccharide biosynthesis (eps), antibiotic (fusaricidin) synthesis, motility (flgB) and other functions were also affected in these mutants. In SC2-M2, reversion of spo0A resulted in the complete recovery of sporulation. This is the first global analysis of mutations related to sporulation deficiency in P. polymyxa. Our results demonstrate that a SNV within spo0A caused the sporulation deficiency of SC2-M2 and provide strong evidence that an arginine residue at position 211 is essential for the function of Spo0A.

Identification of Driving ALK Fusion Genes and Genomic Landscape of Medullary Thyroid Cancer.

The genetic landscape of medullary thyroid cancer (MTC) is not yet fully understood, although some oncogenic mutations have been identified. To explore genetic profiles of MTCs, formalin-fixed, paraffin-embedded tumor tissues from MTC patients were assayed on the Ion AmpliSeq Cancer Panel v2. Eighty-four sporadic MTC samples and 36 paired normal thyroid tissues were successfully sequenced. We discovered 101 hotspot mutations in 18 genes in the 84 MTC tissue samples. The most common mutation was in the ret proto-oncogene, which occurred in 47 cases followed by mutations in genes encoding Harvey rat sarcoma viral oncogene homolog (N = 14), serine/threonine kinase 11 (N = 11), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (N = 6), mutL homolog 1 (N = 4), Kiesten rat sarcoma viral oncogene homolog (N = 3) and MET proto-oncogene (N = 3). We also evaluated anaplastic lymphoma kinase (ALK) rearrangement by immunohistochemistry and break-apart fluorescence in situ hybridization (FISH). Two of 98 screened cases were positive for ALK FISH. To identify the genomic breakpoint and 5’ fusion partner of ALK, customized targeted cancer panel sequencing was performed using DNA from tumor samples of the two patients. Glutamine:fructose-6-phosphate transaminase 1 (GFPT1)-ALK and echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusions were identified. Additional PCR analysis, followed by Sanger sequencing, confirmed the GFPT1-ALK fusion, indicating that the fusion is a result of intra-chromosomal translocation or deletion. Notably, a metastatic MTC case harboring the EML4-ALK fusion showed a dramatic response to an ALK inhibitor, crizotinib. In conclusion, we found several genetic mutations in MTC and are the first to identify ALK fusions in MTC. Our results suggest that the EML4-ALK fusion in MTC may be a potential driver mutation and a valid target of ALK inhibitors. Furthermore, the GFPT1-ALK fusion may be a potential candidate for molecular target therapy.

Abstract 5167: Identification of inactivating mutations in stepwise hepatocarcinogenesis using next generation sequencer

Abstract Early hepatocellular carcinoma (HCC) is histologically diagnosed as the existence of intratumoral portal tracts with stromal invasion. It consists of small hepatocytes with little cellular atypia but with structural atypia. This type of very well-differentiated cancerous tissue is usually characterized by indistinct margins and many portal tracts within the tumor, and develops into classical HCC. To identify sequential genomic changes in stepwise hepatocarcinogenesis, we analyzed fusion genes and mutations using next generation sequencer about 43 early and 105 advanced HCCs. First, exome sequence showed that base sequence aberrations (non-silent) were more frequent in advanced HCC than early HCC (70.9 ± 29.0 vs 53.7 ± 23.8 genes per tumor, P = 0.0002). Mutations in p53/RB pathway including TP53, ATM, RB1, CDKN2A, and CDKN1A were observed in 27.9% and 38.1% in early and advanced HCCs, respectively (P = 0.161) and those of WNT/b-catenin pathways consisting of CTNNB1, AXIN1, and APC were recurrently observed in both early and advanced HCCs (27.9% vs 41.0%, P = 0.095). On the other hand, mutations in SWI/SNF complex genes were more frequently observed in advanced HCC (35.2%) than early HCC (16.3%, P = 0.016). Especially, inactivating mutation of ARID was found in 16 advanced HCCs (15.2%) while only 1 case in early HCC (2.3%, P = 0.018). Consistent with our previous report homozygous deletions near CSMD1 and CDKN2A were also found in 8 and 5 cases, respectively, only in advanced HCC. Next, we detected 4 types of fusion gene, including interchromosomal (translocation), intrachromosomal (deletion), intrachromosomal (translocation), and inversion by RNA sequencing. We also found that every HCC harbored 2 to 5 fusion genes, which were more frequent in advanced than early HCC, although there were no recurrent rearrangements in neither advanced nor early HCCs. Especially chromathripsis, in which 10 to 100 rearrangements were localized in the specific genomic regions and genomic features imply chromosome breaks occur in one-off crisis, was the event observed only in advanced HCC. Taken together, genomic changes were more frequent in advanced HCC, and inactivating mutations of p53/RB and WNT/b-catenin signaling pathways are the driver mutation in the initiation of hepatocarcinogenesis and that of ARID2 may play pivotal roles in the progression from early to advanced HCC. Citation Format: Yutaka Midorikawa, Shogo Yamamoto, Hiroo Ueda, Kotaro Sonoda, Shingo Tsuji, Kenji Tatsuno, Tatsuhiro Shibata, Kyle Covington, David Wheeler, Tadatoshi Takayama, Hiroyuki Aburatani. Identification of inactivating mutations in stepwise hepatocarcinogenesis using next generation sequencer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5167. doi:10.1158/1538-7445.AM2014-5167

Localización cromosómica de duplicaciones submicroscópicas en pacientes con trastornos del neurodesarrollo para identificar casos con alto riesgo de recurrencia familiar

Background and objectiveAn important proportion of neurodevelopmental disorders (NDDs) results from unbalanced genomic alterations (duplication or deletion). These chromosomal rearrangements may be considered as de novo, despite they arise as a result of a balanced rearrangement not detected in a phenotypically normal parent. Therefore, if the rearrangements are inherited, the recurrence risk and the genetic counseling of these cases change radically. Fluorescence in situ hybridization (FISH) is a technique that allows detecting both balanced and unbalanced rearrangements, identifying also the location of duplicated segments. We tried to locate in the genome the duplicated segments detected in patients with NDDs in order to identify those cases due to inherited rearrangements. Patients and methodThe study was conducted in 13 patients with NDDs and genomic duplications detected by compared genomic hybridization-array (CGH-array). Two approaches of FISH technique were taken: hybridization with painting chromosome probes and with specific probes for each duplication. ResultsIn the studied series of 13 patients with duplication, 11 patients were found to carry tandem duplications, one with an intrachromosomal insertional translocation, and another with an interchromosomal insertional translocation. Therefore, 2 of the duplications considered de novo were actually an unbalanced rearrangement inherited from a parent who is a balanced carrier. ConclusionThe results illustrate the need to characterize by FISH technique the rearrangements that are detected by CGH-array to identify those cases with a high risk of recurrence.

Abstract 4020: Genome-wide analysis of stepwise hepatocarcinogenesis using next generation sequencer.

Abstract Early hepatocellular carcinoma (HCC) originates from the hepatocytes of chronic liver disease and develops into classical HCC. To identify sequential genomic changes in multistep hepatocarcinogenesis, we analyzed fusion genes and mutations using next generation sequencer about 40 early and 80 advanced HCCs. First, exome sequence showed that base sequence aberrations were more frequent in advanced HCC than early HCC, and mutations including TP53 (24%), CTNNB1 (24%), and ARID1A (16%) were recurrently observed in both early and advanced HCCs. Consistent with our previous report homozygous deletions near CSMD1 and CDKN2A were also found in 8 and 5 cases, respectively, only in advanced HCC. Next, we detected 4 types of fusion gene, including interchromosomal (translocation), intrachromosomal (deletion), intrachromosomal (translocation), and inversion by RNA sequencing. We also found that every HCC harbored 2 to 5 fusion genes, which were more frequent in advanced than early HCC, although there were no recurrent rearrangements in neither advanced nor early HCCs. Especially chromathripsis, in which 10 to 100 rearrangements were localized in the specific genomic regions and genomic features imply chromosome breaks occur in one-off crisis, was the event observed only in advanced HCC. Taken together, genomic changes were more frequent in advanced HCC, suggesting early HCC originates in the chronic liver disease and progresses into advanced HCC with genomic aberrations. Citation Format: Yutaka Midorikawa, Tadatoshi Takayama, Shogo Yamamoto, Hiroo Ueda, Kotaro Sonoda, Kiyoko Takane, Shingo Tsuji, Genta Nagae, Kenji Tatsuno, Margaret Morgan, David Wheeler, Hiroyuki Aburatani. Genome-wide analysis of stepwise hepatocarcinogenesis using next generation sequencer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4020. doi:10.1158/1538-7445.AM2013-4020

Abstract 812: Whole genome discovery of genetic alterations carried by pancreatic adenocarcinoma.

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a 5-year survival rate of 4%. Even if some genetic hits leading to PDAC development are known, a deeper knowledge of the genetic landscape of tumor is necessary to identify druggable targets. Methods: PDAC samples from 14 localized and advanced cases of pancreatic adenocarcinomas were collected by ultrasound-guided biopsy used for DNA and RNA extraction. High resolution copy number analysis was performed on Affymetrix SNP array 6.0 and analyzed with segmentation algorithm against a reference of 270 Ceu HapMap individuals (Partek Genomic Suite). Whole transcriptome massively parallel sequencing was performed at 75x2 bp on a HiScanSQ Illumina platform. An average of 7,3x107 reads per sample were generated, with a mean read depth of 50X. Single nucleotide variants (SNVs) were detected with SNVMix2 and compared with genetic variation databases (dbSNP, 1000genomes, Cosmic). Non-synonimous SNVs were analyzed with SNPs&GO and SIFT to predict disease association. Results: Whole transcriptome sequencing showed that PDAC samples exhibited a mean of 145 (range: 61-240) non-synonimous SNVs, of which 16 on average are potentially disease-related. 9/14 patients exhibited both macroscopic and cryptic cytogenetic alterations, with a mean of 10 copy number alterations per patient, while 5 patients did not show any copy number gain or loss. Most frequent gains were observed in 18q11.2 involving GATA6 (3/14) and 19q13 targeting AKT2 (3/14) while hotspot deleted regions were found on 18q21 (7/14), 17p13 (6/14), 9p21.3 (6/14), 15q (5/14) and 1q35 (4/14). Merging copy number and RNAseq data we highlighted the major oncogenic hits of PDAC, confirming the prevalence (9/14) of KRAS mutations, in one case also NRAS (G13D), and the central role of the three oncosuppressor CDKN2A (mutated in 3 cases and deleted in 6 cases, either in hetero- or homozygosity), SMAD4 (altered by either point mutation or gene deletion in 8/14 cases), and TP53 (lost in 7/14 samples). We identified novel pathogenic single nucleotide variants and frameshift mutations, and inter- and intra-chromosomal translocation events that give rise to chimeric fusion transcripts. Even if the mutation profile was heterogeneous in different patients the signaling pathways affected were shared, and included KRAS/MAPK, TGFbeta and integrin signaling, proliferation and apoptosis, DNA damage response, and epithelial to mesenchymal transition. New oncogenic alterations emerged, as ARID1A that was inactivated by somatic mutation (G91R and P1425fs) or copy number loss in 6 patients, NOTCH2 and HMGCR, that displayed pathogenic mutations in 15-20% of the analyzed patients. Conclusions: Next generation sequencing combined with high resolution cytogenetic analysis can improve the understanding of carcinogenesis and highlight new biomarkers for early diagnosis and potential therapeutic targets in PDAC. Citation Format: Marina Macchini, Annalisa Astolfi, Riccardo Casadei, Valentina Indio, Carla Serra, Silvia Vecchiarelli, Elisa Grassi, Claudio Ricci, Marielda D'Ambra, Giovanni Taffurelli, Donatella Santini, Raffaele Pezzilli, Francesco Minni, Guido Biasco, Mariacristina Di Marco. Whole genome discovery of genetic alterations carried by pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 812. doi:10.1158/1538-7445.AM2013-812

Maternal intrachromosomal insertional translocation leads to recurrent 1q21.3q23.3 deletion in two siblings

We identified a novel 6.33 Mb deletion of 1q21.3q23.3 (hg18; chr1: 153035245-159367106) in two siblings presenting with blepharophimosis, ptosis, microbrachycephaly, severe psychomotor, and intellectual disability. Additional common features include small corpus callosum, normal birth length and head circumference, postnatal growth restriction, low anterior hairline, upturned nose, bilateral preauricular pits, widely spaced teeth, gingival hypertrophy, left ventricular dilatation with decreased biventricular systolic function, delayed bone age, 5th finger clinodactyly, short 3rd digit, hyperconvex nails, obstructive and central sleep apnea, and bilateral heel contractures. Fluorescence in situ hybridization (FISH) performed in the mother of both children showed an apparently balanced, intrachromosomal insertional translocation of 1q21.3q23.3 to 1q42.12. The sibling recurrence likely arose by a maternal meiotic crossing over on the rearranged chromosome 1 between the deleted region and the insertion. We hypothesize that the decreased cardiac function and contractures may be related to LMNA haploinsufficiency. This case illustrates the importance of FISH when attempting to determine inheritance of a copy-number variation and emphasize the value of evaluating known haploinsufficiency phenotypes for genes in deleted regions.

The abnormalities of chromosome 8 in two hepatocellular carcinoma cell clones with the same genetic background and different metastatic potential.

Two hepatocellular carcinoma (HCC) cell clones named MHCC97-H and MHCC97-L with different metastatic potential have recently been established from the same parent cell line MHCC97 in our institute. The cytogenetic alterations of these two clones were investigated in this study to explore the possible clues to the mechanism involved in HCC metastasis. Their chromosomal aberrations were analyzed with comparative genomic hybridization (CGH), chromosome-specific painting, and two-color fluorescence in situ hybridization (FISH). The aberrations were found in a total of 17 chromosomes, and six kinds of the aberrations including gains of 1q, 7q, 8q, 20, and the losses of 8p23, 21q were found both in the two cell clones and their parent cell line MHCC97. Using modified CGH, with the DNA of MHCC97-L as control to test the MHCC97-H clone, the loss of 8p23 and the gain of 1q31-32, 8q21.3-22, 13q22, 17q22 were highlighted, and the most significant finding was on chromosome 8. Dual color FISH combining a pericentromeric probe and a BAC probe mapping at 8q23.1 was then performed to verify this result, and the signal ratios of the BAC to centromere were 1.43 in MHCC97-H and 1.45 in MHCC97-L, confirming the over-representations at 8q in both cells. Another interesting finding in the dual-color metaphase FISH was the intrachromosomal translocation of 8q to 8p (looked like an isochromosome 8) and non-reciprocal translocation of part of 8q to 4q, which was further clarified and proved by the FISH with whole chromosome 8 painting probe. The high copies amplification on 8q, the formation of isochromosome 8, non-reciprocal translocation of partial 8q to 4q, and loss of 8p occurred at the same time and are the characteristic chromosomal aberrations of the two cell clones. The chromosome 8p, especially 8p23, might harbor some novel genes related to the HCC metastasis.

Structural rearrangement in chromosome 2M of Aegilops comosa has prevented the utilization of the Compair and related wheat-Ae. comosa translocations in wheat improvement

The genetic constitutions of chromosome 2M of Aegilops comosa and the derived wheat-Ae. comosa translocations were analyzed by molecular cytogenetic techniques. Hybridization of 15 RFLP markers covering the entire length of the group-2 chromosomes revealed that chromosome 2M was structurally rearranged compared to the homoeologous chromosomes of wheat by either a pericentric inversion or a terminal intrachromosomal translocation. The breakpoint of the rearrangement was located in a region between the loci Xpsr131 and Xcdo405, resulting in the translocation of 47% of 2MS to 2ML. This aberrant structure of 2M allowed homoeologous recombination between 2M and its wheat counterpart only in the translocated segment on 2ML. C-banding and genomic in situ hybridization analyses confirmed that all translocation chromosomes consisted of the complete 2MS arm, a large part of 2ML, and very small distal segments derived from 2AS or 2DS, as expected from the aberrant structure of chromosome 2M. Thus, the translocation in the line 2A-2M?4/2 can be described as T2AS-2M?1L ⋅ 2M?1S and the translocations in the lines Compair and 2D-2M?3/8 as T2DS-2M?1L ⋅ 2M?1S. RFLP analysis determined the breakpoints in these translocation chromosomes to be within the telomeric 16% of the wheat chromosome arms. The breakpoint of the 2A/2M translocation was between Xbcd348 and Xcdo783, and that of the 2D/2M translocation was between Xcdo783 and Xpsr666. Because the translocation chromosomes retain the structural aberration found in chromosome 2M, further exploitation of the wheat-Ae. comosa translocations for cultivar improvement is questionable.

Introgression and DNA marker analysis of Lycopersicon peruvianum, a wild relative of the cultivated tomato, into Lycopersicon esculentum, followed through three successive backcross generations

Segregation of the Lycopersicon peruvianum genome was followed through three generations of backcrossing to the cultivated tomato L. esculentum cv ‘E6203’ using molecular markers. Thirteen BC1 plants were genotyped with 113 markers, 67 BC2 plants with 84 markers, and finally 241 BC3 plants were genotyped with 177 markers covering the entire genome and a BC3 map constructed. Several segments of the genome, including parts of chromosomes 3, 4, 6, and 10, quickly became fixed for esculentum alleles, possibly due to sterility problems encountered in the BC1. Observed overall heterozygosity and chromosome segment lengths at each generation were very near the expected theoretical values. Markers located near the top telomeric region of chromosome 9 showed segregation highly skewed towards the wild allele through all generations, suggesting the presence of a gamete promoter gene. One markers, TG9, mapped to a new position on chromosome 9, implying an intrachromosomal translocation event. Despite the great genetic distance between the two parents, overall recombination was only 25% less than that observed in a previous tomato cross, indicating that L. peruvianum genes may be more readily introgressed into cultivated germplasm than originally believed.

Establishing a physical map of chromosome No. 4 of Plasmodium falciparum

We isolated a panel of 20 DNA probes that hybridize specifically to chromosome No. 4 of Plasmodium falciparum and used them to construct a restriction map of chromosome No. 4 in the FCR3 strain. These probes were partially sequenced and had an insert size range of 70–310 bp (average 160 bp) and a GC content range of 19–53% (average 35%). Three probes were identical to previously described P. falciparum sequences. Two probes were homologous to an 18-bp repetitive sequence in a previously cloned P. falciparum gene but were not homologous to other parts of the gene. One probe sequence is a homologue of the heat shock protein, DnaJ. The location of these probes and four previously cloned probes on chromosome No. 4 were determined by using eight restriction enzymes that recognize 6-bp sites containing only G or C and 10 restriction enzymes that recognize 6-bp sites containing four G or C and two A or T. The locations of the probes were well distributed along the chromosome. Three probes were located at two sites and two probes were found at at least four sites on chromosome No. 4. Maps of chromosome No. 4 of the FCR3 strain, and three laboratory-selected, pyrimethamine-resistant derivative strains were constructed. Two of the strains, FCR3-D81 and FCR3-D85, each had two polymorphic forms of chromosome No. 4. Each of those polymorphic chromosomes had a duplicated part of the center of the chromosome making the cell diploid for the genetic material in that region. Those chromosomes also had an amplification and probable intrachromosomal translocation of a 50-kb fragment of chromosome No. 4. One strain derived from FCR3-D7, FCR3-D7-1 contained 20 copies of a tandemly amplified fragment carrying the dihydrofolate reductase-thymidylate synthase gene and an amplification of an unrelated part of the chromosome.