7q35 Microdeletion and 15q13.3 and Xp22.33 Microduplications in a Patient with Severe Myoclonic Epilepsy, Microcephaly, Dysmorphisms, Severe Psychomotor Delay and Intellectual Disability.

Copy number variations (CNVs) play a key role in the pathogenesis of several diseases, including a wide range of neurodevelopmental disorders. Here, we describe the detection of three CNVs simultaneously in a female patient with evidence of severe myoclonic epilepsy, microcephaly, hypertelorism, dimorphisms as well as severe psychomotor delay and intellectual disability. Array-CGH analysis revealed a ∼240 kb microdeletion at the 7q35 inherited from her father, a ∼538 kb microduplication at the 15q13.3 region and a ∼178 kb microduplication at Xp22.33 region, both transmitted from her mother. The microdeletion in 7q35 was included within an intragenic region of the contactin associated protein-like 2 (CNTNAP2) gene, whereas the microduplications at 15q13.3 and Xp22.33 involved the cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) and the cytokine receptor-like factor 2 (CRLF2) genes, respectively. Here, we describe a female patient harbouring three CNVs whose additive contribution could be responsible for her clinical phenotypes.

A novel flow cytometric method for enhancing acute promyelocytic leukemia screening by multidimensional dot-plots.

Acute promyelocytic leukemia (APL) is generally characterized by t(15;17)(q24;q21). In some cases, the classic translocation cannot be identified by conventional methods, since the PML-RARA fusion protein results from complex, variant, or cryptic translocation. The diagnostic algorithm of APL starts with screening methods, such as flow cytometry (FC), followed by fluorescence in situ hybridization or polymerase chain reaction to confirm the diagnosis. Our aim was to develop a novel protocol for analyzing APL samples based on multidimensional dot-plots that can provide comprehensive information about several markers at the same time. The protocol included four optimized multidimensional dot-plots, which were tested by retrospective reanalysis of FC results in APL (n = 8) and non-APL (n = 12) acute myeloid leukemia (AML) cases. After predicting the potential position of hypergranular- and microgranular-type aberrant promyelocytes, the percentages of blast populations were examined within the gates in all AML cases. The percentage of blasts in each predefined gate was well above the cut-off value (95%) in APL cases in all tubes. In non-APL AML cases, the percentage of blasts in the same gates never reached the cut-off value in all investigated tubes, and even when it did in a single tube, the pattern was markedly different from that observed in APL cases. In conclusion, multidimensional dot-plots can be used for screening APL even in cryptic APL cases, although reproducibility across several laboratories would require standardization of antibodies and fluorochromes. This easy-to-use and quick method can support the diagnosis of APL and the prompt initiation of the appropriate treatment.

Applications of imaging flow cytometry in the diagnostic assessment of acute leukaemia.

Automated imaging flow cytometry integrates flow cytometry with digital microscopy to produce high-resolution digital imaging with quantitative analysis. This enables cell identification based on morphology (cell size, shape), antigen expression, quantification of fluorescence signal intensity and localisation of detected signals (i.e. surface, cytoplasm, nuclear). We describe applications of imaging flow cytometry for the diagnostic assessment of acute leukaemia. These bone marrow malignancies are traditionally diagnosed and classified by cell morphology, phenotype and cytogenetic abnormalities. Traditionally morphology is assessed by light microscopy, phenotyping by conventional flow cytometry and genetics by karyotype and fluorescence in situ hybridisation (FISH) on interphase nuclei/metaphase spreads of cells on slides. Imaging flow cytometry adds a new dimension to the diagnostic assessment of these neoplasms. We describe three specific applications: From this we conclude that imaging flow cytometry offers benefits over conventional diagnostic methods. Specifically the ability to visualise the cells of interest, the pattern and localisation of expressed antigens and assess cytogenetic abnormalities in one integrated automated high-throughput test. Imaging flow cytometry presents a new paradigm for the diagnostic assessment of leukaemia.

Understanding the molecular pathogenesis of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a distinct subset of acute myeloid leukemia (AML) associated with peculiar biologic and clinical features and requiring specific management. At the genetic level, APL is featured by a unique chromosome translocation t(15;17) which results in the PML-RARα gene fusion and chimeric protein. APL is the first example of differentiation therapy targeted to a defined genetic target i.e. PML-RARα. PML-RARα behaves as an altered retinoic acid receptor with an ability of transmitting oncogenic signaling leading to accumulation of undifferentiated promyelocytes. All-trans-retinoic acid (ATRA) induces disease remission in APL patients by triggering terminal differentiation of leukemic promyelocytes. More recently, arsenic trioxide (ATO) has been shown to contribute degradation of the PML-RARα oncoprotein through bonding the PML moiety and has shown excellent synergism with ATRA in clinical trials. Elucidating the oncogenic signaling of PML-RARα through various transcription factors and the study of APL mouse models have greatly helped to understand the molecular pathogenesis of APL. However, the precise molecular mechanism by which t(15;17) is formed and initiates leukemia remains unknown. While transforming oncogenic potential of PML-RARα has been described extensively, the mechanistic events important for the formation of t(15;17) have been taken from the model of Therapy-related APL (t-APL).

Complex karyotype in a polycythemia vera patient with a novel SETD1B/GTF2H3 fusion gene.

The patient had been diagnosed with polycythemia vera (PV) in 1999, at the age of 61, according to the criteria of the Polycythemia Vera Study Group (PVSG) on the basis of the increased red cell mass by isotope determination, normal oxygen saturation, low plasma erythropoietin level, presence of endogenous erythroid colonies (EEC), and splenomegaly. Histopathology of bone marrow biopsy was also consistent with polycythemia vera with no evidence of increased reticulin fibrosis. A karyotype analysis was not performed at that time. He had been treated initially with phlebotomies and then with hydroxyurea with the aim to obtain a better control of hematocrit; he was under low-dose aspirin. In 2009, 10 years after the diagnosis, while the patient was still being treated with hydroxyurea and phlebotomies, he noticed worsening of general conditions and fatigue, and the appearance of night sweats; he also reported that his spleen volume had increased rapidly in the past few months. He complained of severe pruritus especially after (but not limited to) a shower. He was referred to our center for further evaluation. At presentation, his blood counts were as follows: hemoglobin 157 g/L, hematocrit 54.7%, leukocytes 13.1 × 109 /L, platelets 238 × 109 /L, LDH 856 U/L (normal upper limit, 250 U/L). Blood film examination showed neutrophilia (8.9 × 109 /L) but immature myeloid cells and nucleated erythroblasts were absent. The spleen was 14 cm below the left costal margin, the liver was at 4 cm below the right costal margin. He was found to harbor the JAK2V617F mutation with an allele burden of 85% and the circulating CD34⁺ cell count was 14 × 106 /L. A bone marrow biopsy showed the presence of hyperplasia of myeloid and erythroid lineages, increased number of scattered megakarocytes without overt morphologic abnormalities; reticulin fibrosis was grade 1 according to the European classification. On these basis, we considered the patient as presenting the features of PV according to the 2008 WHO classification of myeloid neoplasms associated with grade 1 reticulin fibrosis.

Novel BRD4-NUT fusion isoforms increase the pathogenic complexity in NUT midline carcinoma.

Nuclear protein in testis (NUT)-midline carcinoma (NMC) is a rare, aggressive disease typically presenting with a single t(15;19) translocation that results in the generation of a bromodomain-containing protein 4 (BRD4)-NUT fusion. PER-624 is a cell line generated from an NMC patient with an unusually complex karyotype that gave no initial indication of the involvement of the NUT locus. Analysis of PER-624 next-generation transcriptome sequencing (RNA-Seq) using the algorithm FusionFinder identified a novel transcript in which Exon 15 of BRD4 was fused to Exon 2 of NUT, therefore differing from all published NMC fusion transcripts. The three additional exons contained in the PER-624 fusion encode a series of polyproline repeats, with one predicted to form a helix. In the NMC cell line PER-403, we identified the 'standard' NMC fusion and two novel isoforms. Knockdown by small interfering RNA in either cell line resulted in decreased proliferation, increased cell size and expression of cytokeratins consistent with epithelial differentiation. These data demonstrate that the novel BRD4-NUT fusion in PER-624 encodes a functional protein that is central to the oncogenic mechanism in these cells. Genomic PCR indicated that in both PER-624 and PER-403, the translocation fuses an intron of BRD4 to a region upstream of the NUT coding sequence. Thus, the generation of BRD4-NUT fusion transcripts through post-translocation RNA-splicing appears to be a common feature of these carcinomas that has not previously been appreciated, with the mechanism facilitating the expression of alternative isoforms of the fusion. Finally, ectopic expression of wild-type NUT, a protein normally restricted to the testis, could be demonstrated in PER-403, indicating additional pathways for aberrant cell signaling in NMC. This study contributes to our understanding of the genetic diversity of NMC, an important step towards finding therapeutic targets for a disease that is refractory to current treatments.

Visualization of the spatial positioning of the SNRPN, UBE3A, and GABRB3 genes in the normal human nucleus by three-color 3D fluorescence in situ hybridization.

The three-dimensional (3D) structure of the genome is organized non-randomly and plays a role in genomic function via epigenetic mechanisms in the eukaryotic nucleus. Here, we analyzed the spatial positioning of three target regions; the SNRPN, UBE3A, and GABRB3 genes on human chromosome 15q11.2-q12, a representative cluster of imprinted regions, in the interphase nuclei of B lymphoblastoid cell lines, peripheral blood cells, and skin fibroblasts derived from normal individuals to look for evidence of genomic organization and function. The positions of these genes were simultaneously visualized, and all inter-gene distances were calculated for each homologous chromosome in each nucleus after three-color 3D fluorescence in situ hybridization. None of the target genes were arranged linearly in most cells analyzed, and GABRB3 was positioned closer to SNRPN than UBE3A in a high proportion of cells in all cell types. This was in contrast to the genomic map in which GABRB3 was positioned closer to UBE3A than SNRPN. We compared the distances from SNRPN to UBE3A (SU) and from UBE3A to GABRB3 (UG) between alleles in each nucleus, 50 cells per subject. The results revealed that the gene-to-gene distance of one allele was longer than that of the other and that the SU ratio (longer/shorter SU distance between alleles) was larger than the UG ratio (longer/shorter UG distance between alleles). The UG distance was relatively stable between alleles; in contrast, the SU distance of one allele was obviously longer than the distance indicated by the genome size. The results therefore indicate that SNRPN, UBE3A, and GABRB3 have non-linear and non-random curved spatial positioning in the normal nucleus, with differences in the SU distance between alleles possibly representing epigenetic evidence of nuclear organization and gene expression.

Síndrome de inversión duplicación del cromosoma 15 (INVDUP15) «de novo»: presentación de tres casos clínicos / Chromosome 15 inversion duplication syndrome (INVDUP15) “de novo”: presentation of three clinical cases

Introducción. El síndrome de inversión duplicación del cromosoma 15 se refiere a un conjunto de características clínicas entre las que se encuentran hipotonía central desde el nacimiento, retraso psicomotor, epilepsia o trastorno del espectro autista. Una invdup(15) resulta de la tetrasomía parcial de 15q y generalmente está implicada la región del Síndrome de Prader-Willi (SPW). Se evalúan tres casos remitidos a Genética por hipotonía y retraso psicomotor. Material y métodos. Cultivo de linfocitos de sangre periférica, cariotipo de alta resolución, FISH, extracción de ADN de linfocitos de sangre periférica, MS-MLPA de SPW y estudio de microsatélites. Resultados. El primer caso presentó un cariotipo 47,XY+der(15)(q13;p11.2)(pter->q13::p11.2->pter) y un cariotipo molecular arr 15q12.1q13(18,432,358-26,658,490)x3∼4 con ganancia de 8,23Mb implicando a genes sometidos a imprinting de la región causante de los síndromes de PWS y Angelman (SA). En el segundo caso se obtuvo una fórmula cromosómica 47, XX, + mar.ish idic (15)(q13)(Acro p-arm ++, D15Z1 ++, D15S10 ++, PML-) y cariotipo molecular arr 15q11.2q13.3(18,432,358-30,230,511)x3, con duplicación de aproximadamente 12Mb. En el tercer caso la paciente resultó ser portadora de una doble línea celular en mosaico 47,XX+ der (15) inv (15)(q11;p11.2) [40%] / 46,XX [60%]. En los tres casos se analizó mediante MLPA la región del SPW encontrándose un patrón de metilación alterado y la causa genética resultó ser un síndrome invdup(15) «de novo». Discusión. A pesar de la dificultad para establecer una correlación fenotipo-genotipo en los casos con invdup(15) las técnicas genéticas más recientes pueden aportar información para el diagnóstico clínico de estos pacientes (AU)

Introduction. The chromosome 15 inversion duplication syndrome refers to distinctive clinical findings, such as, early central hypotonia, developmental delay, epilepsy and autistic behaviour. Invdup(15) results from partial 15q tetrasomy and the Prader-Willi syndrome(PWS) region is generally involved. We have analyzed three clinical cases in a Genetics Unit diagnosed with hypotonia and developmental delay. Material and methods. Lymphocyte cultures from peripheral blood samples, high resolution karyotype, FISH, DNA isolation from peripheral blood leukocytes, PWS MS-MLPA and microsatellites study. Results. The first case showed a karyotype 47,XY+der(15)(q13;p11.2)(pter->q13::p11.2->pter) and a molecular karyotype arr 15q12.1q13(18,432,358-26,658,490)x3 ∼ 4 with an extra 8.23Mb genetic material involving imprinted genes from SPW and Angelman (SA) syndromes region. In the second case there was a karyotype 47, XX, + mar.ish idic (15)(q13)(Acro p-arm ++, D15Z1 ++, D15S10 ++, PML-) and a molecular karyotype arr 15q11.2q13.3(18,432,358-30,230,511)x3 with an approximately 12Mb duplication. The third patient was a carrier of a mosaic double line cell with karyotype 47,XX+ der (15) inv (15)(q11;p11.2) [40%] / 46,XX [60%]. In the three cases the SPW region was analysed using a modified methylation pattern and all resulted from a invdup(15) «de novo» genetic defect. Discussion. Although it is difficult to establish a phenotype-genotype correlation in invdup (15) cases, most recent genetic techniques should provide information for the clinical diagnosis in these patients (AU)

Centromere activity in dicentric small supernumerary marker chromosomes.

Twenty-five dicentric small supernumerary marker chromosomes (sSMC) derived from #13/21, #14, #15, #18, and #22 were studied by immunohistochemistry for their centromeric activity. Centromere protein (CENP)-B was applied as marker for all centromeres and CENP-C to label the active ones. Three different 'predominant' activation patterns could be observed, i.e., centric fusion or either only one or all two centromeres were active. In one inherited case, the same activation pattern was found in mother and son. In acrocentric-derived sSMC, all three activation patterns could be present. In contrary, in chromosome 18-derived sSMC, only the fusion type was observed. In concordance with previous studies a certain centromeric plasticity was observed in up to 13% of the cells of an individual case. Surprisingly, the obtained data suggests a possible influence of the sSMC carrier's gender on the implementation of the predominant activation pattern; especially, only one active centromere was found more frequently in female than in male carriers. Also, it might be suggested that dicentric sSMC with one active centromere could be less stable than such with two active ones-centromeric plasticity might have an influence here, as well. Also, centromere activity in acrocentric-derived dicentrics could be influenced by heteromorphisms of the corresponding short arms. Finally, evidence is provided that the closer the centromeres of a dicentric are and if they are not fused, the more likely it was that both of them became active. In concordance and refinement with previous studies, a distance of 1.4 Mb up to about 13 Mb the two active centromere state was favored, while centromeric distance of over approximately 15 Mb lead to inactivation of one centromere. Overall, here, the first and largest ever undertaken study in dicentric sSMC is presented, providing evidence that the centromeric activation pattern is, and parental origin may be of interest for their biology. Influence of mechanisms similar or identical to meiotic imprinting in the centromeric regions of human chromosomes might be present. Furthermore, centromeric activation pattern could be at least in parts meaningful for the clinical outcome of dicentric sSMC, as sSMC stability and mosaicism can make the difference between clinically normal and abnormal phenotypes.

Acquired acute myelogenous leukemia after therapy for acute promyelocytic leukemia with t(15;17): a case report and review of the literature.

Therapy-related myelodysplastic syndrome (t-MDS) and therapy-related acute myelogenous leukemia (t-AML) in patients with acute promyelocytic leukemia (APL) are rare events. The cumulative exposure to chemotherapy with alkylating agents and topoisomerase II inhibitors is associated with t-AML that may develop any time after the completion of the treatment. We report the case of an acquired AML who previously received therapy for APL, after two years of being diagnosed. The diagnosis was established by morphologic findings, membrane markers, cytogenetic studies, and fluorescence in situ hybridization (FISH). To our knowledge this is the first documented case in Puerto Rico of a patient with APL that developed a t-AML without the characteristic chromosomal and morphologic findings of APL.

Immunohistochemical and biogenetic features of diffuse-type tenosynovial giant cell tumors: the potential roles of cyclin A, P53, and deletion of 15q in sarcomatous transformation.

PURPOSE: Diffuse-type tenosynovial giant cell tumor (D-TSGCT) is an aggressive proliferation of synovial-like mononuclear cells with inflammatory infiltrates. Despite the COL6A3-CSF1 gene fusion discovered in benign lesions, molecular aberrations of malignant D-TSGCTs remain unidentified. EXPERIMENTAL DESIGN: We used fluorescent in situ hybridization and in situ hybridization to evaluate CSF1 translocation and mRNA expression in six malignant D-TSGCTs, which were further immunohistochemically compared with 24 benign cases for cell cycle regulators involving G(1) phase and G(1)-S transition. Comparative genomic hybridization, real-time reverse transcription-PCR, and a combination of laser microdissection and sequencing were adopted to assess chromosomal imbalances, cyclin A expression, and TP53 gene, respectively. RESULTS: Five of six malignant D-TSGCTs displayed CSF1 mRNA expression by in situ hybridization, despite only one having CSF1 translocation. Cyclin A (P = 0.008) and P53 (P < 0.001) could distinguish malignant from benign lesions without overlaps in labeling indices. Cyclin A transcripts were more abundant in malignant D-TSGCTs (P < 0.001). All malignant cases revealed a wild-type TP53 gene, which was validated by an antibody specifically against wild-type P53 protein. Chromosomal imbalances were only detected in malignant D-TSGCTs, with DNA losses predominating over gains. Notably, -15q was recurrently identified in five malignant D-TSGCTs, four of which showed a minimal overlapping deletion at 15q22-24. CONCLUSIONS: Deregulated CFS1 overexpression is frequent in malignant D-TSGCTs. The sarcomatous transformation involves aberrations of cyclin A, P53, and chromosome arm 15q. Cyclin A mRNA is up-regulated in malignant D-TSGCTs. Non-random losses at 15q22-24 suggest candidate tumor suppressor gene(s) in this region. However, P53 overexpression is likely caused by alternative mechanisms rather than mutations in hotspot exons.

[New chromosomal syndromes]. / Syndromes chromosomiques émergents.

Mental retardation occurs in 2-3% of the general population either in isolation or in combination with facial dysmorphism and/or malformations. Chromosomal abnormalities are a most common etiology. Karyotype displays chromosome aberrations in about 10% of patients but it has a limited resolution (5 Mb). Recently, the development of new molecular cytogenetic tools, especially array CGH, allowed to detect smaller abnormalities and increased the diagnosis capability of 15-20%. Among these newly detected rearrangements, some of them are recurrent and define new recognized syndromes. We will first briefly explain the non-allelic homologous recombination (NAHR) mechanism that underlines the origin of recurrent microdeletions and microduplications. Then we will describe eight new syndromes, four microdeletions (del 17q21.31, del 3q29, del 15q24, del 2q32.3q33) and four microduplications (dup 22q11.2, dup 7q11.23, dup 17p11.2, duplication of MECP2). A better knowledge of these new recurrent chromosomal syndromes will allow to improve care for patients, to develop targeted chromosomal diagnosis and to identify genes involved in neurocognitive functions.

Nuclear organisation in totipotent human nuclei and its relationship to chromosomal abnormality.

Studies of nuclear organisation, most commonly determining the nuclear location of chromosome territories and individual loci, have furthered our understanding of nuclear function, differentiation and disease. In this study, by examining eight loci on different chromosomes, we tested hypotheses that: (1) totipotent human blastomeres adopt a nuclear organisation akin to that of committed cells; (2) nuclear organisation is different in chromosomally abnormal blastomeres; and (3) human blastomeres adopt a ;chromocentre' pattern. Analysis of in vitro fertilisation (IVF) conceptuses permits valuable insight into the cell biology of totipotent human nuclei. Here, extrapolations from images of preimplantation genetic screening (PGS) cases were used to make comparisons between totipotent blastomeres and several committed cells, showing some differences and similarities. Comparisons between chromosomally abnormal nuclei and those with no detected abnormality (NDA) suggest that the former display a significant non-random pattern for all autosomal loci, but there is a less distinct, possibly random, pattern in 'NDA' nuclei. No evidence was found that the presence of an extra chromosome is accompanied by an altered nuclear location for that chromosome. Centromeric loci on chromosomes 15 and 16 normally seen at the nuclear periphery were mostly centrally located in aneuploid cells, providing some evidence of a 'chromocentre'; however, the chromosome-18 centromere was more peripheral, similar to committed cells. Our results provide clues to the nature of totipotency in human cells and might have future applications for preimplantation diagnosis and nuclear transfer.

Distribution of the D15Z1 copy number polymorphism.

Using fluorescent in situ hybridization (FISH) with the probe p15 (D15Z1), we investigated the distribution of the polymorphic 15p signal which has been reported to occur on acrocentric chromosomes in addition to chromosome 15. The short arm of chromosome 15 has a characteristic signal pattern when hybridized with the FISH probe D15Z1. However, the D15Z1 signal can occasionally be seen on the short arm of other acrocentric chromosomes. We studied the distribution of the D15Z1 probe in 1657 patients consisting both of individuals with a normal karyotype and those with a variety of chromosome abnormalities involving the acrocentric chromosomes. Our results show that one in six individuals, regardless of their patient ascertainment category or karyotypic status, had one or more additional D15Z1 signals, and that there were no significant differences in the distribution of extra signals among the patient groups.