Derivative Chromosome Research Articles

Low-level mosaicism of a de novo derivative chromosome 9 from a t(5;9)(q35.1;q34.3) has a major phenotypic impact

Microdeletion and microduplication syndromes are well-known causes of developmental delay and/or malformations of differing severity. Although homogeneous abnormalities can now be detected relatively easily using microarray technologies, they are more difficult to detect and interpret in cases of mosaicism. Here, we report on a male infant with a mosaic de novo derivative chromosome 9, featuring a 10.2 Mb 5q35 duplication (including the NSD1 gene) and a 687 kb 9q34 deletion (including EHMT1). The infant presented developmental delay, short stature, brachy/plagiocephaly and hyperactivity. The proportion of abnormal cells was 50% in saliva (in a microarray analysis) and 25% in lymphocytes (in a FISH analysis). Despite the low-level mosaicism in lymphocytes, this imbalance appears to be responsible for a distinctive phenotype (suggesting the presence of variable clinical expression and/or major somatic mosaicism).

Chromothripsis in Healthy Individuals Affects Multiple Protein-Coding Genes and Can Result in Severe Congenital Abnormalities in Offspring

Chromothripsis represents an extreme class of complex chromosome rearrangements (CCRs) with major effects on chromosomal architecture. Although recent studies have associated chromothripsis with congenital abnormalities, the incidence and pathogenic effects of this phenomenon require further investigation. Here, we analyzed the genomes of three families in which chromothripsis rearrangements were transmitted from a mother to her child. The chromothripsis in the mothers resulted in completely balanced rearrangements involving 8-23 breakpoint junctions across three to five chromosomes. Two mothers did not show any phenotypic abnormalities, although 3-13 protein-coding genes were affected by breakpoints. Unbalanced but stable transmission of a subset of the derivative chromosomes caused apparently de novo complex copy-number changes in two children. This resulted in gene-dosage changes, which are probably responsible for the severe congenital phenotypes of these two children. In contrast, the third child, who has a severe congenital disease, harbored all three chromothripsis chromosomes from his healthy mother, but one of the chromosomes acquired de novo rearrangements leading to copy-number changes. These results show that the human genome can tolerate extreme reshuffling of chromosomal architecture, including breakage of multiple protein-coding genes, without noticeable phenotypic effects. The presence of chromothripsis in healthy individuals affects reproduction and is expected to substantially increase the risk of miscarriages, abortions, and severe congenital disease.

APL-The Age-Old Harlequin: Do We See it All?

Dear Sir, Acute promyelocytic leukemia (APL) is a unique type of acute myeloid leukemia characterized by distinct cytomorphological features, immunophenotype, molecular rearrangement t(15;17) involving the PML and RARa genes and a very good response to all-trans retinoic acid (ATRA) [1]. However in rarity of cases, no evidence for rearrangement of RARa is found indicating that alternative mechanisms could mediate the differentiation block that typifies this disease. Here we describe a case of APL, with classical morphological and immunophenotypic features, which was of interest because of presence of isochromosome(17) along with the absence of rearrangement of RARa. APL hence always proves to be a harlequin that takes various molecular and morphological masquerades. An 11-year-old male child presented at our centre with high grade fever and cervical lymphadenopathy for 2 months duration. Baseline laboratory investigations showed TLC of 14,800/lL with 85 % abnormal promyelocytes with hemoglobin of 7.7 gm% and platelet count of 20,000/lL (Fig. 1). On flow cytometry, abnormal promyelocytes were positive for cMPO, CD117, CD13, CD33, CD9, CD64 and CD65. These cells were negative for CD34, CD2, cCD79a, CD3, CD11b, CD18, CD14, CD38, CD56, CD4 and HLA-DR. A diagnosis of APL was made and genetic studies performed. RT-PCR was negative for PML–RARa fusion transcript. Metaphase karyotyping revealed 45–48,XY,i(17)(q10), -6, ?8, -14, -21, ?21[cp10] (Fig. 2). ATRA based combination chemotherapy with Daunomycin was given. Patient was in morphological remission at day 28 of induction and at the end of first consolidation cycle. After completion of consolidation phase, patient received maintenance therapy and is doing well at a follow up of 4 months. APL can have varied clinical and morphogenetic presentation. Besides the conventional reciprocal translocation t(15;17)(q22;q21) disrupting the PML and RARa genes, other chromosomal abnormalities such as t(11;17) (q23;q21), t(5;17)(q35;q12–21), t(11;17)(q13;q21) and der(17) are also observed in APL whereby RARa is fused to the PLZF, NPM, NuMA, and STAT5b genes, respectively. An isochromosome of the long arm of the derivative chromosome 17 is rarely observed in APL patients. However our case is further rare as no evidence for rearrangement of RARa was found. Only 3 such cases have been reported previously in literature [2–4]. Two of these three patients responded poorly to therapy and died within a year [2, 4]. The prognosis of third cases is not known [3]. Our case responded to standard doses of ATRA and BM was in morphological remission at day 45. Consolidation and maintenance was uneventful and is doing well on maintenance. The present case hence highlights the importance of immune-morphological and genetic basis of APL. These cases can still be managed by ATRA which shows that differentiation block by any molecular mechanism in APL can still be overcome by its therapeutic doses as in our case. Management and prognosis of such patients remains unknown as there is not much literature on management of such patients. The lack of independent prognostic value of additional chromosomal abnormalities in APL does not support the use of alternative therapeutic strategies when such abnormalities are found [5]. ATRA however remains A. Misra S. Soni A. Dutta R. Kumar A. Chopra (&) Laboratory Oncology, BRA IRCH, AIIMS, New Delhi, India e-mail: chopraanita2005@gmail.com

Exceptional complex chromosomal rearrangements in three generations.

We report an exceptional complex chromosomal rearrangement (CCR) found in three individuals in a family that involves 4 chromosomes with 5 breakpoints. The CCR was ascertained in a phenotypically abnormal newborn with additional chromosomal material on the short arm of chromosome 4. Maternal karyotyping indicated that the mother carried an apparently balanced CCR involving chromosomes 4, 6, 11, and 18. Maternal transmission of the derivative chromosome 4 resulted in partial trisomy for chromosomes 6q and 18q and a partial monosomy of chromosome 4p in the proband. Further family studies found that the maternal grandmother carried the same apparently balanced CCR as the proband's mother, which was confirmed using the whole chromosome painting (WCP) FISH. High resolution whole genome microarray analysis of DNA from the proband's mother found no evidence for copy number imbalance in the vicinity of the CCR translocation breakpoints, or elsewhere in the genome, providing evidence that the mother's and grandmother's CCRs were balanced at a molecular level. This structural rearrangement can be categorized as an exceptional CCR due to its complexity and is a rare example of an exceptional CCR being transmitted in balanced and/or unbalanced form across three generations.

Transformation of an Unclassified Myeloproliferative Neoplasm with a Rare BCR-JAK2 Fusion Transcript Resulting from the Translocation (9;22)(p24;q11).

BCR-ABL1 negative myeloproliferative neoplasms (MPNs) are known to contain alterations of the tyrosine kinase JAK2 (located on 9p24) that result in constitutive activation of the encoded protein. JAK2 fusions are reported in acute and chronic leukemias of myeloid and lymphoid phenotypes. Here, we report an unclassified case of MPN (MPN-U) showing a t(9;22)(p24;q11), which generates a BCR-JAK2 fusion gene by fusing the BCR at intron 13 to JAK2 at intron 17 on the derivative chromosome 22. Most reported JAK2 fusions cases reveal an aggressive clinical course and long-term remissions have only been achieved after allogeneic stem cell transplantation (ASCT). To the best of our knowledge, this is the thirteenth case reported worldwide to describe a BCR-JAK2 fusion transcript in MPN-U. The present report revealed a sustained complete clinical, hematologic, and cytogenetic remission 35 months after diagnosis and ~24 months after ASCT. Regarding BCR-ABL1 negative MPN patients this case report provides strong support for a role of JAK2 activation in the oncogenesis and suggests a possible diagnostic and therapeutic target that should be investigated.

Genomic Alterations in Ethanol-Exposed Trophoblast Cell Lines Induced by Chronic Ethanol Treatment

Background: Ethanol consumption during pregnancy results in a broad spectrum of damage, but the knowledge of its mechanism is lacking. Objective: The aim of this study is to determine ethanol-caused genomic alterations in placental cell lines after a repeated ethanol treatment in order to describe new genomic targets of cell damage. Methods: A model of sustained exposure to standard doses of ethanol on two in vitro human choriocarcinoma cell lines, JEG-3 and BeWo, was used. Chromosomic abnormalities and copy number alterations (CNAs) were assessed by G-Banding cytogenetics and oligonucleotide Single Nucleotide Polymorphism-Array analysis (CytoScan, Affymetrix). Results: Chromosomal abnormalities did not change despite ethanol exposure except for the presence of a derivative chromosome 4 [add(4)(p14)] in exposed BeWo cells. Regarding SNP-Array analysis, a total of 21 CNAs were found to be caused by ethanol exposure, 16 in JEG-3 cell line and 5 in BeWo cell line, which were not found in controls. There was no coincidence between JEG-3 and BeWo regions affected by ethanol. Conclusion: Trophoblast cell lines exposed repetitively to ethanol presented genomic instability resulting in CNAs. However, no region has been equally altered in both models to consider it an ethanol exposure target area. So, further studies involving different models and approaches that target gene regulation are required.

Fluroscence in-situ hybridization negative PML/RARA: A cryptic puzzle

Acute promyelocytic leukemia (APL) has defined biology and clinical course that is, distinct from the other forms of acute myelogenous leukemia. It may present with potentially devastating coagulopathy and the sensitivity to retinoid differentiating agents, including all-trans retinoic acid and arsenic trioxide, hence a fast and definite diagnosis is imperative. Reciprocal 15, 17 translocation creates a PML/RARA fusion gene on the derivative chromosome 15, which can be detected by various molecular tests such as cytogenetics, fluroscence in-situ hybridization (FISH), reverse transcriptase-polymerase chain reaction. We present here a diagnostically challenging case, both morphologically and immunophenotypically proven to be APL, which was negative for the PML/RARA by FISH.

Molecular cytogenetic characterization of a patient diagnosed with dimorphic anemia carrying de novo rare ring chromosome 7 along with T(7;9)

A case of a derivative chromosome 7 formed by a ring chromosome 7 and t(7;9) was found who presented with dimorphic anaemia with no other anomaly. Ring chromosome 7 was characterized by conventional and molecular cytogenetic techniques.

Quantitative multigene FISH on breast carcinomas identifies der(1;16)(q10;p10) as an early event in luminal A tumors.

In situ detection of genomic alterations in cancer provides information at the single cell level, making it possible to investigate genomic changes in cells in a tissue context. Such topological information is important when studying intratumor heterogeneity as well as alterations related to different steps in tumor progression. We developed a quantitative multigene fluorescence in situ hybridization (QM FISH) method to detect multiple genomic regions in single cells in complex tissues. As a “proof of principle” we applied the method to breast cancer samples to identify partners in whole arm (WA) translocations. WA gain of chromosome arm 1q and loss of chromosome arm 16q are among the most frequent genomic events in breast cancer. By designing five specific FISH probes based on breakpoint information from comparative genomic hybridization array (aCGH) profiles, we visualized chromosomal translocations in clinical samples at the single cell level. By analyzing aCGH data from 295 patients with breast carcinoma with known molecular subtype, we found concurrent WA gain of 1q and loss of 16q to be more frequent in luminal A tumors compared to other molecular subtypes. QM FISH applied to a subset of samples (n = 26) identified a derivative chromosome der(1;16)(q10;p10), a result of a centromere-close translocation between chromosome arms 1q and 16p. In addition, we observed that the distribution of cells with the translocation varied from sample to sample, some had a homogenous cell population while others displayed intratumor heterogeneity with cell-to-cell variation. Finally, for one tumor with both preinvasive and invasive components, the fraction of cells with translocation was lower and more heterogeneous in the preinvasive tumor cells compared to the cells in the invasive component. © 2014 The Authors Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.

Evidence of an Epigenetic Origin for High-Risk 1q21 Copy Number Aberrations in Multiple Myeloma

Multiple myeloma (MM) is a B-cell malignancy stratified in part by cytogenetic aberrations including the high-risk copy number alterations (CNAs) of 17p- and +1q21. CNAs of 1q21 are known to occur as a result of jumping translocations of 1q (JT1q12) which increase the copy number (CN) of 1q21, while at the same time causing collateral genomic damage including deletions and amplifications in some receptor chromosomes (RC). We have previously reported the decondensation of the 1q12 region and triradial chromosomes in the bone marrow specimens of patients with aggressive disease, and have speculated that hypomethylation of the pericentromeric region of 1q12 may play a role in the origin of 1q21 CNAs. To test the hypothesis that 1q12 hypomethylation induces CN gains of 1q21, we used the hypomethylating agent 5-azacytidine to treat blood lymphocyte cultures of five myeloma patients with balanced constitutional 1q12 aberrations as well as five normal control subjects. The patients with constitutional aberrations of 1q12 were selected based on their potential to show either novel CN gains of regions translocated distal to 1q12 or CN gains of 1q21 when 1q12 was translocated to a non-homologous chromosome. The constitutional aberrations included two patients with pericentromeric inversions of chromosome 1 inv(1)(p11q12), and one patient each with a balanced translocation of t(1;2)(q12;p11), t(1;2)(q12;q34), or t(1;9)(q12;q11). These particular aberrations should provide a unique model for testing the association between the hypomethylation of 1q12 and the origin of CN gains of 1q21. In vitro blood lymphocyte cultures of patients and controls were treated for 72 hours with 5-azacytidine at a final concentration of 10uM. 5-azacytidine is known to cause site-specific hypomethylation and chromatin decondensation in the 1q12 region. Utilizing FISH probes for 1q12 (satII/III) and 1q21(CKS1B), we analyzed 100 metaphase cells from each patient and each control for CN gains for 1q21 and any novel region translocated distal to 1q12. To fully characterize the induced rearrangements of 1q12, we also applied inverse G-banding and spectral karyotyping in selected cells. Remarkably, all patients with the balanced constitutional 1q12 rearrangements showed CN gains of chromosome regions on the derivative chromosomes distal to the inverted or translocated 1q12 regions. Recurring aberrations of 1q12 included pericentromeric decondensation and triradials of chromosome arms 1q, 1p, and 2p, in addition to localized pulverizations distal to 1q12 of these same arms. Specifically, in both patients with inv(1), whole-arm CN gains of 1p were identified in 2-3% of cells, while in the patient with t(1;2)(q12;p11), whole-arm gains of 2p (MYCN) were identified in 3% of cells. In these same three patients, on the derivative chromosomes where the 1q12 region was separated from 1q21 by an inversion or translocation, no CN gains of the 1q21 occurred. In the patients with 1q12 translocated to a non-homologous chromosome, 1q21 was amplified on the RC in 4% of the cells in the patient with t(1;2)(q12;q34), and in 3% in the patient with t(1;9)(q12;q11). Control subjects showed CN gains of 1q21on both chromosomes 1 in the range of 3-13%. Whole-arm JT1q12s to RC16q were identified in three patients and three controls. This JT1q12 results in a CN gain of 1q21, and a whole-arm CN loss in RC16q, identical to those found in the bone marrow specimens of MM patients. Interestingly, one control showed JT1q12s to two different RCs, 9q and 12q, causing whole-arm deletions in the long arms of both. The key structural aberration responsible for the generation of CN gains of 1q21 and subsequent JT1q12s is the formation of a triradial involving the 1q12 region. The novel finding in this study is the induction of triradials and CN gains for chromosome arms of 1p and 2p, which demonstrates that epigenetic modifications to 1q12 can amplify non-homologous chromosome regions juxtaposed to it. Furthermore, the CN gains induced here occurred only distal to 1q12 on all normal and derivative chromosomes and mimic the triradials and JT1q12s reported in the bone marrow of patients with aggressive disease. These findings demonstrate for the first time that the hypomethylation of the 1q12 region can potentially amplify any genomic region distal to it, and provide evidence for an epigenetic origin of the high-risk 1q21 CNAs found in the progression of MM. DisclosuresNo relevant conflicts of interest to declare.

Four Genetic Lymphoma-Specific Events (MYC, BCL2, BCL6 and CCND1) in High Grade B-Cell Lymphoma: Aggressive Mantle Cell Lymphoma or Cyclin D1-Positive DLBCL?

The term « double » or « triple hit lymphoma » is commonly used to describe mature B cell neoplasms with either BCL2 and/or MYC and/or BCL6 gene rearrangements. These rare and aggressive lymphomas with high Ki67 expression are categorized as « B-cell lymphomas unclassified, with features intermediate between diffuse B-cell lymphoma and Burkitt lymphoma » category in the WHO 2008 classification. To our knowledge, only 2 cases of lymphoma have been described with four specific genetic events (quadruple hit) involving MYC, BCL2, BCL6 and CCND1 genes (Bacher U. et al., Genes Chromosomes Cancer 2011). We describe here the third observation.A patient, a 79 years old man, suffering from paraesthesias for 4 months, was admitted for polyneuritis in a context of poor general condition. Clinical examination showed the presence of numerous axillary, supraclavicular, mediastinal and inguinal lymphadenopathy, neuro-meningeal invasion and skin infiltration. The biopsy of a skin nodule of left arm revealed an infiltration consisting in large proliferating cells (Ki67 80%) stained by anti-CD20, BCL2 and BCL6 antibodies, but not with CD10 nor CD23, consistent with a diffuse large B-cell lymphoma (DLBCL), NOS diagnosis. Blood cell count showed 8.1 G/L leukocytes, 13.2 g/dL hemoglobin, 166 G/L platelets. LDH and β-2 microglobulin were elevated (989 U/I, and 9.14 mg/L respectively). Blood cell film examination showed the presence of 28% of pathological basophil lymphocytes (medium sized, with slightly clumped chromatin and frequent prominent nucleoli). Flow cytometry revealed that these cells expressed a monotypic lambda immunoglobulin light chain and were CD19, CD20, FMC7, CD22 positive, with a dim CD5 positivity and no CD10 staining. The negativity of CD23 associated to strong CD148 staining (Miguet et al, J Proteome Res2009) were in favour of the diagnosis of variant pleomorphic mantle cell lymphoma (MCL).Cytogenetic study performed in the WBC found a complex hyperdiploid karyotype (47 chromosomes) with a t(3;22) translocation involving BCL6 and IGL genes, structural abnormality of a chromosome 8 resulting in juxtaposition of 5’MYC and BCL2 in FISH (with break of the MYC probe in FISH), a derivative chromosome 18 from a t(14;18) translocation with fusion of 5’IGH and BCL2, and a t(11;14) complex translocation involving IGH and CCND1 (54% of the nuclei). Overexpression of cyclin D1 was detected in the WBC by RQ-PCR, as well as in the skin lesion using immunochemistry. Others numeral (trisomy 12) and structural abnormalities (involving the 1, 7, 14 and 21 chromosomes) were also detected.The patient was treated with a chemotherapy combining rituximab, ifosfamide, cytosine arabinoside and intrathecal methotrexate. He is still alive 5 months after the diagnosis.This third case of quadruple hit lymphoma confirms the complexity of the classification of such aggressive malignancies. Initial rearrangement of the CCND1 gene characterizes MCL that may harbour in very rare cases additional rearrangements of MYC or BCL6. Conversely, cyclin D1 overexpression is considered a rare feature in DLBCL. Recently, Ok CY et al. (Cancer 2014) proposed to reclassify DLBCL with expression of cyclin D1 and CCND1 chromosomal rearrangement and CD5 positivity as aggressive pleomorphic MCL variant. But no case with rearrangement of 2 (or more) genes (BCL2 and/or MYC and/or BCL6) was described in this study.Juskevicius D et al. (Am J Surg Pathol 2014) suggest the existence of a “gray zone” in which morphologic, clinical and genetic features are insufficient to segregate CD5- and SOX11-negative lymphomas with overexpression of cyclin D1/ translocation involving CCND1between blastoid MCL from cyclin D1-positive DLBCL.In our case, immunophenotyping of circulating cells (CD5+, CD148++) as well as genetic and molecular features (translocation involving CCND1 and IGH, and overexpression of cyclin D1) allow us to diagnose a probable genetically unstable aggressive pleomorphic MCL variant with rearrangement of several extra genetic hits. DisclosuresNo relevant conflicts of interest to declare.

In Multiple Myeloma, High-Risk Features Are Modulated By Other Chromosomal changes : A Large Snparray IFM Study

▪In multiple myeloma, cytogenetic changes display important value for patients’ outcome. In this setting, the most important changes are the del(17p), and the t(4;14), conferring a poor outcome. However, a certain heterogeneity is observed in survival of these high-risk patients. We hypothesized that other chromosomal changes may impact the outcome. We retrospectively analyzed a large series of 242 patients displaying either the t(4;14) (157 patients), or the del(17p) (110 patients), 25 patients presenting both abnormalities, using SNP-array. The main advantage of SNParray analyses is that all the unbalanced chromosomal changes are analyzed, in contrast to FISH which informs only for the chosed probes. For t(4;14), 144 patients have relapsed, and 103 have died. The median PFS was 1.4 year, and the median OS was 3.5 years. The median beta2-microglobulin (b2m) level was 4.0 mg/L (range, 1.2-38.3). The distribution of ISS stages 1, 2, and 3 was 35.4%, 26.4%, and 38.2%, respectively. Based on FISH results, 31.2% of the patients presented the abnormal configuration with only one fused signal. In all these patients, SNP-array analysis showed a loss of the telomeric part of one chromosome 4. In contrast to previous reports on this particularity in which the explanation was a loss of the derivative chromosome 14,17,18 we clearly show here that this configuration results from an unbalanced translocation. In patients with t(4;14), del(1p32) (p<0.001), del(16q) (p=0.03), del22q (p=0.04) and more than 30 chromosomal structural changes (p=0.02) negatively impacted PFS. For OS, del(13q14) (p=0.01), del(1p32) (p<0.001), and the number of chromosomal structural changes (p<0.05 and p=0.01 for [10;30[and more than 30 structural changes, respectively) worsened the prognosis of patients.In patients with del(17p) (110 patients), 99 patients relapsed, and 83 died. The median PFS was 1.3 year and the median OS was 2.7 years. The median beta2-microglobulin (b2m) level was 4.4 mg/L (range, 1.4-32.1). The distribution of ISS stages 1, 2, and 3 was 28.4%, 26.9%, and 44.8%, respectively. For patients with del(17p), del(1p32) (p<0.001), del(16q)] (p=0.03), del22q (p=0.04) and more than 30 chromosomal structural changes (p=0.02) negatively impacted PFS. For OS, del(1p32) (p=0.004) worsened the prognosis of patients, whereas more than 8 trisomies (p=0.008) was found to have a protective effect on survival.This study, which is the largest series of high-risk patients, analyzed with the most modern genomic technique, identified two main factors negatively impacting survival: del(1p32) and genomic complexity. Furthermore, we did show that 1/3 of patients with t(4;14) did not display upregulation of FGFR3 because of loss of the telomeric part of the chromosome 4. These patients do not present a better prognosis than those upregulating FGFR3, reinforcing the idea that FGFR3 activation is not important in the biology of the t(4;14). DisclosuresNo relevant conflicts of interest to declare.

The ASXL1 Gene Alterations in Patients with Myeloid Diseases and Chromosomal 20q Deletion

Deletion of the long arm of chromosome 20 - del(20q) - is a recurrent abnormality observed in various myeloid disorders, including myelodysplastic syndromes (MDSs), myeloproliferative neoplasms (MPNs), or acute myeloid leukemia (AML). It is a primary cytogenetic aberration, occurring often as a sole abnormality or the first abnormality in complex karyotypes, therefore it is assumed to play a key role in pathogenesis of myeloid malignancies. The proximal breakpoints of the deletion are consistently located in the 20q11.21 band, and the distal breakpoints span from 20q13.13 to band 20q13.33. In our previous study we showed a fusion of the ASXL1 and TSHZ2 genes resulting from an isochromosome of a deleted 20q in a patient with MDS (Brezinova et al Br J Haematol 2014). The ASXL1 gene is one of the most frequently mutated genes in myeloid disorders, mutations are generally associated with more aggressive course of the disease and poor clinical outcome. The aim of this study was to determine the frequency of ASXL1 breakpoints and/or ASXL1/TSHZ2 fusion in del(20q) cases.Fluorescence in situ hybridizations (FISH) with locus specific probes for 20q11 and 20q12 regions (Abbott, Des Plaines, IL; Kreatech Diagnostics, Amsterdam, The Netherlands) confirmed the cytogenetically observed deletions of 20q in a cohort of 20 patients with myeloid malignancies (MDS 13x, MPN 3x, AML 2x, myelofibrosis 1x, thrombocytopenia 1x). In 15 patients deletion of 20q was a sole aberration, in 2 patients a variant of del(20q), an isochromosome of deleted 20q, was detected by FISH with a subtelomeric probe, Vysis ToTel 20p/20q (Abbott). Metaphase FISH mapping with a set of 7 bacterial artificial chromosome (BAC) probes (BlueGnome, Cambridge, UK) distributed in 20q11.21 and 20q13.2, with a chromosome-20-specific centromeric probe (Kreatech Diagnostics) as a control was used for determination of the breakpoints. Array comparative genomic hybridization (CytoChip Cancer 180K, BlueGnome) was performed on DNA samples of bone marrow cells of 11 patients with suspected ASXL1 gene deletion to find out the gene copy number variation.A weak signal of RP11-358N2 BAC probe (30.93-31.12 Mb from telomere on the short arm, 20q11.21) was observed in 6 patients(30%), suggesting the proximal breakpoint of the deletion in the ASXL1 gene (30.95-31.03 Mb). However, the distal breakpoint in the TSHZ2 gene (51.80-52.11 Mb; 20q13.2) was found in one patient only, as previously reported. In 6 patients (30%) the signal of RP11-358N2 BAC probe was not present on the derivative chromosome confirming the ASXL1 gene deletion. In the remaining 8 patients the proximal breakpoint of the deletion was determined distally to the ASXL1 gene, in 3 patients into 9.3 Mb region between the MAPRE and the PTPRT genes. None but 1 patient had the distal breakpoint localized in the TSHZ2 gene. Three patients died in a group of 12 patients with ASXL1 gene alteration (deletion or partial deletion).Using combination of molecular cytogenetic methods we proved that the extent of 20q deletions varied among the patients with a frequent proximal breakpoint in the ASXL1 gene. In our cohort, the ASXL1 gene was altered (deleted or partially deleted) in totally 60% of patients. The determination of the ASXL1 gene alteration in del(20q) cases may have a clinical and prognostic impact, but the relations with clinical data should be studied in a larger cohort of patients.Supported by MHCR project for conceptual development of research organization 00023736, RVO-VFN64165/2012, and GACR P302/12/G157/1. DisclosuresNo relevant conflicts of interest to declare.

Multiplex ligation-dependent probe amplification and array comparative genomic hybridization analyses for prenatal diagnosis of cytogenomic abnormalities.

BackgroundThe aims of this study were to evaluate the clinical utility of multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (aCGH) analyses on prenatal cases and to review prenatal ultrasound findings of cytogenomic syndromes.ResultsOf the 54 prenatal cases analyzed, cytogenomic abnormalities were characterized in 14 cases. In four fetuses with abnormal ultrasound findings, a 40.701 Mb duplication of 8q22.3-q24.3 and a 23.839 Mb deletion of 7q33-q36.3 derived from a paternal balanced translocation, a de novo 13.062 Mb deletion of 11q24.1-q25 for Jacobsen syndrome, a de novo 19.971 Mb deletion of 7q11.23-q21.3 for type 1 split-hand/foot malformation (SHFM1), and a de novo 28.909 Mb duplication of 3q21.1-q25.1 were detected. A 699.8 Kb deletion at 5p15.33 for Cri du Chat syndrome was confirmed in a fetus with abnormal MLPA result. A fetus with abnormal maternal screening was detected with a de novo distal 1.747 Mb duplication at 2q37.1-q37.2 and a 6.664 Mb deletion at 2q37.2-q37.3. Of the eight cases referred by history of spontaneous abortions, derivative chromosomes 11 from paternal carriers of a balanced 8q/11q and a 10q/11q translocation were noted in two cases, simple aneuploids of trisomy 2 and trisomy 21 were seen in three cases, and compound aneuploids of two or three chromosomes were found in three cases. Post-test genetic counseling was performed with detailed genomic information and well characterized postnatal syndromic features.ConclusionsThese results demonstrated that coupling MLPA screening and aCGH analysis are a cost-effective approach to detect cytogenomic abnormalities in a prenatal setting. The aCGH analysis provided not only genomic maps of breakpoints and gene content of imbalanced regions but also better inference of related phenotypes for genetic counseling. Prenatal ultrasound findings reported in the literature for Jacobsen syndrome, SHFM and Cri du Chat syndrome were summarized for use as diagnostic references.

Intellectual disability secondary to a 16p13 duplication in a 1;16 translocation. Extended phenotype in a four-generation family.

We describe a large family from the Gaza Strip presented with multiple congenital anomalies. The proband was presented with intellectual disability and multiple congenital anomalies including cleft palate, low-set ears, everted upper lip, diaphragmatic hernia, and arthrogryposis. Pedigree analysis showed 19 affected patients over five generations, only 6 were alive and 11 individuals were obligate carriers. The proband had an apparently normal karyotype, although FISH studies showed a derivative chromosome 1 with duplication of 16p13.3 and deletion of the 1p subtelomere. Her father however had a balanced translocation. The seven affected patients had a similar phenotype, one of them died before genetic testing was carried out and the living six patients had the same unbalanced translocation. Array CGH revealed an 8.8 Mb duplication in 16p13 and 200,338 bp deletion in 1p36.3. Accordingly, intellectual disability, hypertelorism, cupped ears, everted upper lip, and limb anomalies were presenting clinical features of the 16p13 duplication syndrome while deep set eyes were perhaps related to the 1p terminal deletion. Prevention of recurrent intellectual disability in this family can be achieved through carrier detection and prenatal genetic diagnosis.

Noninvasive detection of a balanced fetal translocation from maternal plasma.

Massively parallel sequencing of circulating cell free (ccf) DNA from maternal plasma has been demonstrated to be a powerful method for the detection of fetal copy number variations (CNVs). Although the detection of CNVs has been described by multiple independent groups, genomic aberrations resulting in copy number-neutral events including balanced translocations have proven to be more challenging to detect noninvasively from ccf DNA. Data modeling was initially performed to evaluate multiple methods, ultimately leveraging the short length of ccf DNA and paired-end sequencing to construct read-specific mapping characteristics. After testing in a model system, we evaluated the methods on ccf DNA isolated from the plasma of a donor known to be carrying a fetus with a balanced translocation [t(8;11)]. Sequencing was performed with Illumina sequencing technology. Our methodology identified the known translocation (P = 1.21 × 10(-8)) and discounted the likelihood of others, enabling the base specific identification of the rearrangement positions. In total, 402 unique sequencing reads spanned the putative breakpoints, of which 76 contained the structural rearrangement. In addition, 38 of the chimeric reads were mapped to each of the resulting derivative chromosomes, supporting the presence of a reciprocal translocation. Finally, we identified a 6-bp deletion present within der(8) that was absent from the der(11) reciprocal rearrangement. We have developed an algorithm to detect balanced rearrangements and applied our methodology to demonstrate the first proof-of-principle study on the noninvasive detection of a fetal-specific balanced translocation by sequencing ccf DNA from maternal plasma.

New BAC probe set to narrow down chromosomal breakpoints in small and large derivative chromosomes, especially suited for mosaic conditions.

Fluorescence in situ hybridization (FISH) and/or array-comparative genomic hybridization (aCGH) performed after initial banding cytogenetics is still the gold standard for detection of chromosomal rearrangements. Although aCGH provides a higher resolution, FISH has two main advantages over the array-based approaches: (1) it can be applied to characterize balanced as well as unbalanced rearrangements, whereas aCGH is restricted to unbalanced ones, and (2) chromosomal aberrations present in low level or complex mosaics can be characterized by FISH without any problems, while aCGH requires presence of over 50 % of aberrant cells in the sample for detection. Recently, a new FISH-based probe set was presented: the so-called pericentric-ladder-FISH (PCL-FISH) that enables characterization of chromosomal breakpoints especially in mosaic small supernumerary marker chromosomes (sSMC). It can also be applied on large inborn or acquired derivative chromosomes. The main feature of this set is that the probes are applied in a chromosome-specific manner and they align along the chromosome in average intervals of ten megabasepairs. Hence PCL-FISH provides denser coverage and a more precise anchorage on the human DNA-sequence than most other FISH-banding approaches.

Cytogenetic and molecular genetic study of fetal supernumerary marker chromosomes and derivative chromosomes

Objective To investigate the prenatal diagnosis and phenotypic assessment strategies for fetal supernumerary marker chromosomes and derivative chromosomes. Methods Five cases of fetal supernumerary marker chromosomes and one case of fetal derivative chromosomes were diagnosed in the First Affiliated Hospital of Sun Yat-Sen University from March 12, 2010 to November 9, 2012 by conventional chromosome banding, fluorescence in situ hybridization （FISH） and spectral karyotyping （SKY）. These cases were retrospectively reviewed. Combined with the results of ultrasonography, abnormal phenotypes and pregnancy outcomes were evaluated in these cases. Results All of the five supernumerary marker chromosomes were de novo, in which two were mosaic and the remaining three cases were non-mosaic. Of these five cases, two were type 47, XX＋mar and ultrasound indicated abnormal phenotypes. FISH and SKY confirmed that they were＆amp;nbsp;derived from chromosome 4 and 22, respectively. The other three cases were marker chromosome with Turner syndrome karyotype （abnormal phenotypes were not found by ultrasound）, in which two cases were derived from chromosome Y （by FISH） and one case was identified as ring chromosome X （by FISH and SKY）. One de novo derivative chromosome was verified as a product of reciprocal translocation between chromosome 2 and 6 （by FISH and SKY）. Induced abortion was performed in all cases between 25 and 32 gestational weeks. Conclusions By combining conventional chromosome banding, FISH and SKY, the origin and content of supernumerary marker chromosomes and derivative chromosomes can be identified. On this basis, clinical phenotype evaluation and genetic counseling may be offered with the ultrasonographic result. Key words: Chromosome aberrations; Prenatal diagnosis; Genetic markers; Chromosome banding; In situ hybridization,fluorescence; Spectral karyotyping

IGF2BP1: a novel IGH translocation partner in B acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common form of childhood malignancy. Detecting and characterizing recurrent translocations is critical for ALL diagnosis and treatment. IGH (immunoglobulin heavy chain) rearrangements are relatively common in lymphoproliferative disorders, including ALL. Here we report a 16-year-old boy who was diagnosed with B acute lymphoblastic leukemia. Chromosome analysis showed a t(14;17)(q32;q21) with an additional copy of the derivative chromosome 14. IGH rearrangement was confirmed by concurrent FISH analysis. Chromosomal microarray analysis (CMA) showed the breakpoint at the 5 prime end of the IGF2BP1 gene located at 17q21.32. To the best of our knowledge, this is the first report of ALL with a 14;17 translocation resulting in an IGH-IGF2BP1 fusion; however, previous studies have implicated a role for up-regulation of IGF2BP1 in ALL.

Identification of chromosomal aberration in esophageal cancer cells by mixed BAC DNA probes of chromosome arms and regions

Chromosomal aberration is an important genetic feature of malignant tumor cells. This study aimed to clarify whether BAC DNA could be used to identify chromosome region and arm alterations. For each chromosome region, five to ten 1 Mb BAC DNA clones were selected to construct mixed BAC DNA clones for the particular region. All of the mixed clones from regions which could cover the whole chromosome arm were then mixed to construct mixed BAC DNA clones for the arms. Mixed BAC DNA probes of arms and regions were labeled by degenerate oligonucleotide primed PCR (DOP-PCR) and Nick translation techniques, respectively. The specificities of these probes were validated by fluorescence in situ hybridization (FISH) on the metaphase chromosomes of normal human peripheral blood lymphocytes. FISH with arm-specific mixed BAC DNA probes showed that chromosomal rearrangements and involved chromosome arms were confirmed in several esophageal cancer cells. By using region-specific mixed probes, the breakpoint on 1q from the derivative chromosome t(1q;7q) was identified in 1q32-q41 in esophageal KYSE140 cells. In conclusion, we established an effective labeling method for 1 Mb BAC DNA mixed clone probes, and chromosome arm and region rearrangements could be identified in several esophageal cancer cells by using these probes. Our study provides a more precise method for identification of chromosomal aberration by M-FISH, and the established method may also be applied to the karyotype analysis of hematological malignancies and prenatal diagnosis.