Molecular cytogenetic map visualizes the heterozygotic genome and identifies translocation chromosomes in Citrus sinensis

Citrus sinensis is the most cultivated and economically valuable Citrus species in the world,whose genome has been assembled by three generation sequencings.However,chromosome recognition remains a problem due to the small size of chromosomes,and difficulty in differentiating between pseudo and real chromosomes because of a highly heterozygous genome.Here,we employ fluorescence in situ hybridization(FISH)with 9 chromosome painting probes,30 oligo pools,and 8 repetitive sequences to visualize 18 chromosomes.Then,we develop an approach to identify each chromosome in one cell through single experiment of oligo-FISH and Chromoycin A3(CMA)staining.By this approach,we construct a high-resolution molecular cytogenetic map containing the physical positions of CMA banding and 38 sequences of FISH including centromere regions,which enables us to visualize significant differences between homologous chromosomes.Based on the map,we locate several highly repetitive sequences on chromosomes and estimate sizes and copy numbers of each site.In particular,we discover the translocation regions of chromosomes 4 and 9 in C.sinensis“Valencia.”The high-resolution molecular cytogenetic map will help improve understanding of sweet orange genome assembly and also provide a fundamental reference for investigating chromosome evolution and chromosome engineering for genetic improvement in Citrus.

Genetic effects of Agropyron cristatum 2P chromosome translocation fragments in a wheat background

Agropyron cristatum(2n=4x=28,PPPP)is a wild relative of common wheat which contains a large number of desirable genes that can be exploited for wheat improvement.Wheat–A.cristatum 2P alien translocation lines exhibit many desirable traits,such as small flag leaves,a high spikelet number and density,and a compact plant type.An agronomic trait evaluation and a genetic analysis were carried out on translocation lines and backcross populations of these lines carrying different translocation fragments.The results showed that a translocation fragment from 2PT-3(2PL)reduced the length of the flag leaves,while translocation fragments from 2PT-3(2PL)and 2PT-5(2PL(0.60–1.00))reduced the width of the flag leaves.A translocation fragment from 2PT-13(2PS(0.18–0.36))increased the length and area of the flag leaves.Translocation fragments from 2PT-3(2PL)and 2PT-8(2PL(0.86–1.00))increased the density of spikelets.Translocation fragments from 2PT-7(2PL(0.00–0.09)),2PT-8(2PL(0.86–1.00)),2PT-10(2PS),and 2PT-13(2PS(0.18–0.36))reduced plant height.This study provides a scientific basis for the effective utilization of wheat–A.cristatum translocation lines.

Meiotic Behavior of 1BL/1RS Translocation Chromosome and Alien Chromosome in Two Tri-genera Hybrids

The behavior of wheat-rye translocation chromosome and alien chromosome including Thinopyrum and Haynaldia chromosome at meiosis was investigated in two hybrids by fluorescence in situ hybridization (FISH). Misdivision of translocation chromosome at anaphase I and rye chromatin micronucleus at tetrad stage were observed, A plant with one normal 1BL/1RS translocation chromosome and one 1BL/1RS translocation chromosome deleted about 1/3 of rye chromosome arm in length was identified. One plant with wheat-Thinopyrum non-Robertson translocation chromosome was also detected in the F-2 population of Yi4212 x Yi4095. That could be the results of unequal misdivision of wheat-rye 1BL/1RS translocation chromosome and Thinopyrum chromosome during meiosis. No interaction between translocation chromosome and alien chromosome at meiosis was supported by the data of the distribution frequencies of translocation chromosome and Thinopyrum or Haynaldia chromosome in the progeny of two hybrids. The results may be useful to cultivate new germplasms with different length of rye 1R short arm and wheat-alien non-Robertson translocation tines under wheat background.

Trisomy 21 with t(5; 11) Chromosomal Translocation as New Unfavorable Cytogenetic Abnormalities in Pediatric Acute Myeloid Leukemia Type M2: One Case Report of Nine-year Follow-up and Literature Review

We report one case of pediatric acute myeloid leukemia type 2（AML-M2） who presented with karyotypic aberration of trisomy 21 with the t（5;11） chromosomal translocation. The patient achieved complete remission after two cycles of chemotherapy of daunorubicin, cytarabine and etoposide. Then, follow-up cytogenetic analysis from bone marrow cell cultures demonstrated a normal karyotype of 46, XY. After 9 years, the patient relapsed and the karyotypic abnormalities of trisomy 21 with t（5;11） reappeared. It was concluded that trisomy 21 with t（5; 11） is a new unfavorable cytogenetic aberration in AML-M2.

Chromosomal disorders and male infertility

Infertility in humans is surprisingly common occurring in approximately 15% of the population wishing to start a family. Despite this, the molecular and genetic factors underlying the cause of infertility remain largely undiscovered. Nevertheless, more and more genetic factors associated with infertility are being identified. This review will focus on our current understanding of the chromosomal basis of male infertility specifically： chromosomal aneuploidy, structural and numerical karyotype abnormalities and Y chromosomal microdeletions. Chromosomal aneuploidy is the leading cause of pregnancy loss and developmental disabilities in humans. Aneuploidy is predominantly maternal in origin, but concerns have been raised regarding the safety of intracytoplasmic sperm injection as infertile men have significantly higher levels of sperm aneuploidy compared to their fertile counterparts. Males with numerical or structural karyotype abnormalities are also at an increased risk of producing aneuploid sperm. Our current understanding of how sperm aneuploidy translates to embryo aneuploidy will be reviewed, as well as the application of preimplantation genetic diagnosis （PGD） in such cases. Clinical recommendations where possible will be made, as well as discussion of the use of emerging array technology in PGD and its potential applications in male infertility.

Clinical Impact of t(14;18) in Diffuse Large B-cell Lymphoma

Objective： Recent studies have suggested that t（14;18） is present in a significant proportion of diffuse large B-cell lymphomas （DLBCLs）. However, the prognostic significance of this translocation and its relationship with BCL-2 protein expression remains controversial. Our study aimed to investigate the predictive power of t（14;18） and BCL-2 protein expression in the prognosis of DLBCLs. Methods： Biopsy specimens from 106 DLBCLs were analyzed using interphase fluorescence in situ hybridization （FISH）. Immunophenotypic analysis of CD20, CD3, CD10, BCL-6, MUM1 and BCL-2 was performed by immunohistochemistry. SPSS 13.0 software was used for statistical analysis. Results： The t（14;18） was identified in 27 of 106 cases （25.5%）. The percentages of tumor cells expressing CD10, BCL-6, MUM1 and BCL-2 were 21.7%, 26.4%, 56.6% and 73.6%, respectively. The presence of this translocation was significantly correlated with the expression of CD10 and immunophenotypic subtype （p0.001）. No association was observed between BCL-2 protein expression and the presence of t（14;18）. Multivariate analysis confirmed that both t（14;18） and BCL-2 expression were significantly associated with survival. Moreover, patients with t（14;18） had worse prognosis, compared with those with BCL-2 expression （for overall survival： hazard ratio, 4.235; 95%CI, 2.153-8.329, p0.001 vs. hazard ration, 2.743; 95%CI, 1.262-5.962, p=0.011）. Conclusions： The t（14;18） is a useful prognostic tool for the evaluation of DLBCL immunophenotype and prognosis. The prognosis of GCB （germinal centre-like B cell） DLBCL patients should be made with the consideration of the presence of this translocation, and the detection of t（14;18） should be included as a routine diagnostic test in these cases.

API2-MALT1 oncoprotein promotes lymphomagenesis via unique program of substrate ubiquitination and proteolysis

Lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is the most common extranodal B cell tumor and accounts for 8% of non-Hodgkin’s lymphomas. Gastric MALT lymphoma is the best-studied example and is a prototypical neoplasm that occurs in the setting of chronic inflammation brought on by persistent infection or autoimmune disease. Cytogenetic abnormalities are commonly acquired during the course of disease and the most common is chromosomal translocation t(11;18)(q21;q21), which creates the API2-MALT1 fusion oncoprotein. t(11;18)-positive lymphomas can be clinically aggressive and have a higher rate of dissemination than t(11;18)-negative tumors. Many cancers, including MALT lymphomas, characteristically exhibit deregulated over-activation of cellular survival pathways, such as the nuclear factor-κB (NF-κB) pathway. Molecular characterization of API2-MALT1 has revealed it to be a potent activator of NF-κB, which is required for API2-MALT1-induced cellular transformation, however the mechanisms by which API2-MALT1 exerts these effects are only recently becoming apparent. The API2 moiety of the fusion binds tumor necrosis factor (TNF) receptor associated factor (TRAF) 2 and receptor interacting protein 1 (RIP1), two proteins essential for TNF receptor-induced NF-κB activation. By effectively mimicking ligand-bound TNF receptor, API2-MALT1 promotes TRAF2-dependent ubiquitination of RIP1, which then acts as a scaffold for nucleating and activating the canonical NF-κB machinery. Activation occurs, in part, through MALT1 moiety-dependent recruitment of TRAF6, which can directly modify NF-κB essential modulator, the principal downstream regulator of NF-κB. While the intrinsic MALT1 protease catalytic activity is dispensable for this canonical NF-κB signaling, it is critical for non-canonical NF-κB activation. In this regard, API2-MALT1 recognizes NF-κB inducing kinase (NIK), the essential upstream regulator of non-canonical NF-κB, and cleaves it to generate a stable, constitutively active fragment. Thus, API2-MALT1 harnesses multiple unique pathways to achieve deregulated NF-κB activation. Emerging data from our group and others have also detailed additional gain-of-function activities of API2-MALT1 that extend beyond NF-κB activation. Specifically, API2-MALT1 recruits and subverts multiple other signaling factors, including LIM domain and actin-binding protein 1 (LIMA1) and Smac/DIABLO. Like NIK, LIMA1 represents a unique substrate for API2-MALT1 protease activity, but unlike NIK, its cleavage sets in motion a major NF-κB-independent pathway for promoting oncogenesis. In this review, we highlight the most recent results characterizing these unique and diverse gain-of-function activities of API2-MALT1 and how they contribute to lymphomagenesis.

C-type lectins and human epithelial membrane protein1:Are they new proteins in keratin disorders?

Here we report a family with a clinical spectrum of Pachyonychia Congenita Tarda (PCT) encompassing two generations via a balanced chromosomal translocation between 4q26 and 12p12.3. We discuss the effects of chromosomal translocations on gene expression through involved breakpoints and structural gene abnormalities detected by array CGH. We believe that the family we present gives further insight to the better understanding of molecular and structural basis of keratin disorders, and to the late onset and genetic basis of PCT through the possible role of C-type lectins and human epithelial membrane protein1 (EMP1). Better understanding of the molecular basis of keratin disorders is the foundation for improved diagnosis, genetic counseling and novel therapeutic approaches to overcome the current treatment limitations related to this disease.

Mass Production of Intergeneric Chromosomal Translocations through Pollen Irradiation of Triticum durum-Haynaldia villosa Amphiploid

Haynaldia villosa possesses a lot of important agronomic traits and has been a powerful gene resource for wheat improvement. However, only several wheat-H, villosa translocation lines have been reported so far. In this study, we attempted to develop an efficient method for inducing wheat-H, villosa chromosomal translocations. Triticum durum- Haynaldia villosa amphiploid pollen treated with 1 200 rad ^60Co-y-rays was pollinated to Triticum aestivum cv. ＇Chinese Spring＇. Ninety-eight intergeneric translocated chromosomes between T. durum and H. villosa were detected by genomic in situ hybridization in 44 of 61 M1 plants, indicating a translocation occurrence frequency of 72.1%; much higher than ever reported. There were 26, 62 and 10 translocated chromosomes involving whole arm translocations, terminal translocations, and intercarlary translocations, respectively. Of the total 108 breakage-fusion events, 79 involved interstitial regions and 29 involved centric regions. The ratio of small segment terminal translocations （W.W-V） was much higher than that of large segment terminal translocations （W-V.V）. All of the M1 plants were self-sterile, and their backcross progeny was all obtained with ＇Chinese Spring＇ as pollen donors. Transmission analysis showed that most of the translocations were transmittable. This study provides a new strategy for rapid mass production of wheat-alien chromosomal translocations, especially terminal translocations that will be more significant for wheat improvement.

Induction and transmission of wheat-Haynaldia villosa chromosomal translocations

In order to develop more wheat-Haynaldia villosa translocations involving different chromosomes and chromosome segments of H. villosa, T. durum-H, villosa amphiploid was irradiated with ^60Co γ-rays at doses of 800, 1,200, and 1,600 rad. Pollen collected from the spikes 1, 2, and 3 days after irradiation were transferred to emasculated spikes of the common wheat cv. ‘Chinese Spring＇. Genomic in situ hybridization was used to identify wheat-H, villosa chromosome translocations in the M1 generation. Transmission of the identified translocation chromosomes was analyzed in the BC1, BC2, and BC3 generations. The results indicated that all three irradiation doses were highly efficient for inducing wheat-alien translocations without affecting the viability of the M1 seeds. Within the range of 800-1,600 rad, both the efficiency of translocation induction and the frequency of interstitial chromosome breakage-fusion increased as the irradiation dosage increased. A higher translocation induction frequency was observed using pollen collected from the spikes 1 day after irradiation over that of 2 or 3 days after irradiation. More than 70% of the translocations detected in the M1 generation were transmitted to the BC1 through the female gametes. All translocations recovered in the BC1 generation were recovered in the following BC2, and BC3 generations. The transmission ability of different translocation types in different genetic backgrounds showed an order of ‘whole-arm translocation 〉 small alien segment translocation 〉 large alien segment translocation＇, through either male or female gametes, In general, the transmission ability through the female gametes was higher than that through the male gametes. By this approach, 14 translocation lines that involved different H. villosa chromosomes have been identified in the BC3 using EST-STS markers, and eight of them were homozygous.

Identifying normal embryos from reciprocal translocation carriers by whole chromosome haplotyping

Reciprocal translocation is a chromosomal structural abnormal- ity that arises when two non-homologous chromosomes rearrange and attach with each other, an incidence that occurs in about 1/500 to 1/625 newborns （Mackie and Scriven, 2002）. This event typically does not lead to any significant loss of genetic material, thus recip- rocal translocation carriers do not exhibit any severe abnormal phenotypes （Scriven et al., 1998; Zhang et al., 2016）.

Mechanisms and impacts of chromosomal translocations in cancers

Chromosomal aberrations have been associated with cancer development since their discovery more than a hundred years ago.Chromosomal translocations,a type of particular structural changes involving heterologous chromosomes,have made a critical impact on diagnosis,prognosis and treatment of cancers.For example,the discovery of translocation between chromosomes 9 and 22 and the subsequent success of targeting the fusion product BCR-ABL transformed the therapy for chronic myelogenous leukemia.In the past few decades,tremendous progress has been achieved towards elucidating the mechanism causing chromosomal translocations.This review focuses on the basic mechanisms underlying the generation of chromosomal translocations.In particular,the contribution of frequency of DNA double strand breaks and spatial proximity of translocating loci is discussed.

Site-specific transfer of chromosomal segments and genes in wheat engineered chromosomes

Recently, engineered minichromosomes have been produced using a telomere-mediated truncation technique in some plants. However, the study on transferring genes to minichromosomes is very limited.Here, telomere-mediated truncation was successfully performed in common wheat（Triticum aestivum）to generate stable truncated chromosomes accompanied by a relatively high frequency of chromosomal rearrangements. After the cross between transgenic parents, a promoter-less DsRed gene in a chromosome from one parent was transferred to another chromosome from the other parent at the site behind a maize ubiquitin promoter via the Cre/lox system. DsRed transcripts and red fluorescent proteins were detected in the recombinant plants. In one such seedling, transgenic signals were detected at the centric terminus of chromosome 4D and the distal terminus of chromosome 3A. Clear translocations could be detected at the transgenic loci of these two chromosomes. Intriguingly, signals of centric-specific sequences were co-localized with the translocated D-group chromosomal segment in the terminal region of chromosome 3A. Our results indicate that the Cre/lox system induces the gene swapping to the target chromosome and non-homologous chromosomal recombination simultaneously. These approaches could offer a platform to transfer large DNA fragments or even terminal chromosomal segments to other chromosomes of the natural genome.

Global methylation status of sperm DNA in carriers of chromosome structural aberrations

Male infertility might be clearly associated with aberrant DNA methylation patterns in human spermatozoa. An association between oxidative stress and the global methylation status of the sperm genome has also been suggested. The aim of the present study was to determine whether the global sperm DNA methylation status was affected in the spermatozoa of carriers of chromosome structural aberrations. The relationships between the 5-methylcytosine （msC） levels in spermatozoa and chromatin integrity status were evaluated. The study patients comprised male carriers of chromosome structural aberrations with reproductive failure （n = 24）, and the controls comprised normozoospermic sperm volunteers （n = 23）. The global msC level was measured using thin-layer chromatography （TLC） and immunofluorescence （IF） techniques. The sperm chromatin integrity was assessed using aniline blue （AB） staining and TUNEL assay. The mean msC levels were similar between the investigated chromosome structural aberrations carriers （P） and controls （K）. However, sperm chromatin integrity tests revealed significantly higher values in chromosomal rearrangement carriers than in controls （P 〈 0.05）. Although the potential relationship between sperm chromatin integrity status and sperm DNA fragmentation and the msC level juxtaposed in both analyzed groups （P vs K） was represented in a clearly opposite manner, the low chromatin integrity might be associated with the high hypomethylation status of the sperm DNA observed in carriers of chromosome structural aberrations.

Molecular pathogenesis of lymphomas of mucosa-associated lymphoid tissue-from(auto)antigen driven selection to the activation of NF-_(k)B signaling

Lymphomas of mucosa-associated lymphoid tissue(MALT) are typically present at sites such as the stomach, lung or urinary tract, where lymphoid tissues scatter in mucosa lamina propria, intra- or sub-epithelial cells. The infection of certain pathogens, such as Helicobacter pylori, Chlamydophila psittaci, Borrelia burgdorferi, hepatitis C virus, or certain autoantigens cause these sites to generate a germinal center called the "acquired lymphoid tissue". The molecular pathogenesis of MALT lymphoma is a multi-step process. Receptor signaling, such as the contact stimulation of B cell receptors and CD4 positive T cells mediated by CD40/CD40-ligand and T helper cell type 2 cytokines like interleukin-4, contributes to tumor cell proliferation. A number of genetic alterations have been identified in MALT lymphoma, and among them are important translocations, such as t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21) and t(3;14)(p13;q32). Fusion proteins generated by these translocations share the same NF-?B signaling pathway, which is activated by the caspase activation and recruitment domain containing molecules of the membrane associated guanylate kinase family, B cell lymphoma-10 and MALT1(CBM) protein complex. They act downstream of cell surface receptors, such as B cell receptors, T cell receptors, B cell activating factors and Toll-like receptors, and participate in the biological process of MALT lymphoma. The discovery of therapeutic drugs that exclusively inhibit the antigen receptor signaling pathway will be beneficial for the treatment of B cell lymphomas in the future.

Meiotic Chromosome Behavior in a Human Male t(8;15)Carrier

Reciprocal translocation is one of the most common structural chromosomal rearrangements in human beings; it is widely recognized to be associated with male infertility. This association is mainly based on the abnormal chromosome behavior of the translocated chromosomes and sex chromosomes during meiosis prophase I in reciprocal translocation carriers. However, the underlying mechanisms are not completely known. Here we report a reciprocal translocation carrier of t（8;15）, who is oligozoospermic due to apoptosis of primary spermatocytes and to premature germ cell desquamation from seminiferous tubules. Further analysis showed abnormal synapsis and recombination frequency in this patient, indicating a connection between chromosome behavior and apoptosis of primary spermatocytes. We also compared these observations with recently reported findings on spermatogenesis defects in reciprocal translocation carriers, and discuss the possible mechanisms underlying both common and unique phenotypes of reciprocal translocations involving different chromosomes with the aim of further understanding the regulation of human spermatogenesis.

New insights into transcriptional and leukemogenic mechanisms of AML1-ETO and E2A fusion proteins

BACKGROUND： Nearly 15% of acute myeloid leukemia （AML） cases are caused by aberrant expression of AML 1-ETO, a fusion protein generated by the t（8;21） chromosomal translocation. Since its discovery, AML 1-ETO has served as a prototype to understand how leukemia fusion proteins deregulate transcription to promote leukemogenesis. Another leukemia fusion protein, E2A-Pbx 1, generated by the t（1; 19） translocation, is involved in acute lymphoblastic leukemias （ALLs）. While AML 1 - ETO and E2A-Pbxl are structurally unrelated fusion proteins, we have recently shown that a common axis, the ETO/E-protein interaction, is involved in the regulation of both fusion proteins, underscoring the importance of studying protein-protein interactions in elucidating the mechanisms of leukemia fusion proteins. OBJECTIVE： In this review, we aim to summarize these new developments while also providing a historic overview of the related early studies. METHODS： A total of 218 publications were reviewed in this article, a majority of which were published after 2004. We also downloaded 3D structures of AML1-ETO domains from Protein Data Bank and provided a systematic summary of their structures. RESULTS： By reviewing the literature, we summarized early and recent findings on AML 1-ETO, including its protein-protein interactions, transcriptional and lenkemogenic mechanisms, as well as the recently reported involvement of ETO family corepressors in regulating the function of E2A-Pbxl. CONCLUSION： While the recent development in genomic and structural studies has clearly demonstrated that the fusion proteins function by directly regulating transcription, a further understanding of the underlying mechanisms, including crosstalk with other transcription factors and cofactors, and the protein-protein interactions in the context of native proteins, may be necessary for the development of highly targeted drugs for leukemia therapy.