Raouf Fetni

Whole-genome array CGH identifies pathogenic copy number variations in fetuses with major malformations and a normal karyotype

D’Amours G, Kibar Z, Mathonnet G, Fetni R, Tihy F, Désilets V, Nizard S, Michaud JL, Lemyre E. Whole‐genome array CGH identifies pathogenic copy number variations in fetuses with major malformations and a normal karyotype.

CD133 expression is associated with poor outcome in neuroblastoma via chemoresistance mediated by the AKT pathway

Sartelet H, Imbriglio T, Nyalendo C, Haddad E, Annabi B, Duval M, Fetni R, Victor K, Alexendrov L, Sinnett D, Fabre M & Vassal G 
(2012) Histopathology 60, 1144–1155

SNP genotyping of a sclerosing rhabdomyosarcoma: reveals highly aneuploid profile and a specific MDM2/HMGA2 amplification.

Since the first description of sclerosing rhabdomyosarcoma in 2000, 19 pediatric cases have been reported in the literature. However, it is debated whether sclerosing rhabdomyosarcoma represents a specific rhabdomyosarcoma entity or a variant of embryonal or alveolar rhabdomyosarcoma. To date, 6 sclerosing rhabdomyosarcoma karyotypes and 1 sclerosing rhabdomyosarcoma comparative genomic hybridization profile have been reported. We present the first whole-genome tumoral genotyping of a sclerosing rhabdomyosarcoma by high-density single nucleotide polymorphism array. The single nucleotide polymorphism genotyping revealed a complex pattern including gains and losses of whole chromosomes and an amplification of the 12q13-15 region. Amplification of the 12q13-q15 region containing SAS, GLI, CDK4, and MDM2 has been observed in rhabdomyosarcoma. In the present case, the 2 amplified target genes were MDM2 and HMGA2, excluding CDK4. The identification of a specific MDM2-HGMA2 amplicon excluding CDK4 has only been described so far in well-differentiated and dedifferentiated liposarcoma. Further studies are needed to assess if this anomaly is a specific marker of sclerosing rhabdomyosarcoma.

Fragile site and interstitial telomere repeat sequences at the fusion point of a de novo (Y;13) translocation

Abstract We describe a novel fragile site in a rearranged chromosome, associated with the presence of telomeric repeat sequences at the fusion point of a translocation between chromosomes 13 and Y. The case reported in this study shows a de novo (Y;13) translocation, which appears to represent fusion of an apparently intact chromosome Y with a chromosome 13 that has lost only part of its short arm. Ten percent of the cells show a normal karyotype without the (Y;13) translocation. Molecular cytogenetic studies of the derived Y;13 chromosome revealed three hybridization sites of the telomeric probes – one at each end and one at the breakpoint junction. A fragile site is also observed in the intrachromosomic telomeric region. This coincidence suggests that the telomere repeat sequences (TTAGGG)n, when present at an interstitial chromosomal location, can promote the formation of a novel fragile site.

Frequency of chromosome healing and interstitial telomeres in 40 cases of constitutional abnormalities.

Human telomeres play a major role in stabilizing chromosome ends and preventing fusions. Chromosomes bearing a broken end are rescued by the acquisition of a new telomeric cap without any subtelomeric sequences being present at the breakpoint, a process referred to as chromosome healing. Conversely, a loss of telomeric function or integrity can lead to the presence of interstitial telomeres at the junction site in translocations or ring chromosomes. In order to determine the frequency at which interstitial telomeres or chromosome healing events are observed in target chromosome abnormalities, we conducted a retrospective FISH study using pan-telomeric and chromosome-specific subtelomeric probes on archival material from 40 cases of terminal deletions, translocations or ring chromosomes. Of the 19 terminal deletions investigated, 17 were negative for the subtelomeric probe specific to the deleted arm despite being positive for the pan-telomeric probe. These 17 cases were thus considered as having been rescued through chromosome healing, suggesting that this process is frequent in terminal deletions. In addition, as 2 of these cases were inherited from a parent bearing the same deletion, chromosomes healed by this process are thus stable through mitosis and meiosis. Regarding the 13 cases of translocations and 8 ring chromosomes, 4 and 2 cases respectively demonstrated pan-telomeric sequences at the interstitial junction point. Furthermore, 2 cases of translocations and 1 ring chromosome had both interstitial pan-telomeres and subtelomeres, whereas 2 other cases of ring chromosomes and 1 case of translocation only showed interstitial subtelomeres. Therefore, interstitial (sub)telomeric sequences in translocations and ring chromosomes are more common than previously thought, as we found a frequency of 43% in this study. Moreover, our results illustrate the necessity of performing FISH with both subtelomeric and pan-telomeric probes when investigating these rearrangements, as the breakpoints can be either in the distal part of the pan-telomeres, or in between the 2 types of sequences.

Overexpression of PRDM16 in the presence and absence of the RUNX1/PRDM16 fusion gene in myeloid leukemias.

In a recent study published in Genes, Chromosomes & Cancer, Sakai et al. (2005) reported the identification of a novel RUNX1 partner gene, MDS1/EVI1-like gene 1 (PRDM16), in a patient with acute myeloid leukemia (AML) M4 with the t(1;21) translocation. This fusion gene has also been described recently in a case of t(1;21) positive therapy-related AML (Stevens-Kroef et al., 2006). The t(1;21)(p36;q22) is a rare but recurrent translocation associated with de novo and therapy-related AML and with myelodysplastic syndromes (MDS). We, here, describe the first case of chronic myeloid leukemia (CML) blast crisis cells in which the RUNX1/PRDM16 fusion gene likely contributed to clonal evolution from CML to AML. A 59-year-old woman was referred in July 2000 for a CML. Cytogenetic analysis showed 46,XX,t(9;22)(q34.1;q11.2) in 20 metaphases (UPN 00-H59). She was treated initially with IFN for 20 months with a minor cytogenetic response and then with imatinib mesylate. After 6 months of imatinib, she developed a blastic transformation with 68% blast cells in the peripheral blood. Cells were obtained after an informed consent. Conventional G-banding karyotype showed a possible deletion at band 21q22 in addition to the t(9;22) in all metaphases. Spectral karyotyping of these cells revealed a t(1;21) translocation in addition to the t(9;22) translocation (UPN 02-H056)(Fig. 1A and 1B). The t(1;21) translocation was not present in chronic phase cells isolated from this patient. Fluorescence in situ hybridization (FISH) experiments were performed on peripheral blood metaphases of the blast phase cells using bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) clones obtained from the BACPAC Resource Center (Children’s Hospital Oakland Research Institute in Oakland, California, http://bacpac.chori.org/ home.htm). Clone physical positions and covered genes are available at the UCSC Human Genome Browser (http://genome.ucsc.edu/). The RUNX1 gene located at band 21q22.12 was first studied using two selected BACs, RP11-771C10 (containing intron 6 to exon 8 and the 30 end of RUNX1) and RP11-299D9 (containing intron 1 to exon 8 and the 30 end of RUNX1). Split signals were observed with BAC RP11-299D9 (Fig. 1C). The candidate gene, PRDM16, located at band 1p36.32 was studied using two PACs, RP1-163G9 (containing the 50 end of PRDM16, exon 1 and intron 1) and RP4-785P20 (containing intron 3 to exon 17 of PRDM16) and the BAC RP11-659J6 (containing part of intron 1, exons 2 and 3, part of intron 3 of PRDM16). A split signal was detected using PAC RP1-163G9, indicating that the breakpoint was in intron 1 of PRDM16 (Fig. 1D). On the basis of the results of the FISH studies, we designed PCR primers covering most exons of RUNX1 in both directions (forward and reverse), and for PRDM16 in exon 1 (forward), exons 2–4 (reverse) to assess the expression of PRDM16/RUNX1 and RUNX1/ PRDM16 fusion products. RT-PCR analysis and sequencing of the amplicons revealed eight RUNX1/PRDM16 transcripts joining RUNX1 exons 5 or 6 and PRDM16 exons 2 or 3. Two RUNX1/ PRDM16 cDNAs represented an open reading frame encoding exons 1–5 or exons 1–6 of the RUNX1 gene containing the RUNT domain fused to exon 2 of PRDM16 that contain the PR domain (Fig. 2). However, the reciprocal PRDM16/RUNX1 fusion transcript was not detected. We analyzed the relative expression levels of PRDM16 using real-time PCR (ABI Prism 7900HT Sequence Detection System) in our CML case at the time of chronic phase (UPN 00-H59) and blast crisis (UPN 02-H056), and we compared PRDM16 transcript levels in this case to a series of 88 other myeloid malignancies without evident karyotypic abnormalities of chromosomal band 1p36.3 (including 4 CML-blast crisis derived cell lines, 6 CML blast crisis samples, 14 MDS and MDS transformed to AML samples, and 68 AML samples of different morphological and cytogenetic groups). The Ct (threshold cycle) values of PRDM16 were normalized to an endogenous control gene (GAPDH)

Genotype analysis of tumor-initiating cells expressing CD133 in neuroblastoma.

Neuroblastoma (NB) is the most common and lethal extracranial solid tumor of childhood. Despite aggressive therapy, more than half of the children with advanced NB will die of uncontrolled metastatic disease. After chemotherapy, tumor‐initiating cells (TICs) could persist, cause relapses and metastasis. The aim of this study is to demonstrate the tumor‐initiating properties of CD133high NB cells and to identify new specific genetic abnormalities. Isolation of the CD133high cell population from NB cell lines was followed by neurosphere formation, soft agar assays, and orthotopic injections in NOD/SCID/IL2Rγc‐null mice. A differential genotyping analysis was performed with Affymetrix SNP 6.0 arrays on CD133low and CD133high populations and the frequency of the abnormalities of 36 NB tumors was determined. Our results show that CD133high NB cells possess tumor‐initiating properties, as CD133high cells formed significantly more neurospheres and produced significantly more colonies in soft agar than CD133low. Injection of 500 CD133high cells was sufficient to generate primary tumors and frequent metastases in mice. Differential genotyping analysis demonstrated two common regions with gains (16p13.3 and 19p13.3) including the gene EFNA2 in the CD133high population, and two with loss of heterozygosity (16q12.1 and 21q21.3) in the CD133low population. The gain of EFNA2 correlated with increased expression of the corresponding protein. These abnormalities were found in NB samples and some were significantly correlated with CD133 expression. Our results show that CD133high NB cells have TICs properties and present different genotyping characteristics compared to CD133low cells. Our findings reveal insights into new therapeutic targets in NB TICs.

Detection of small, single-copy genes on protein-G-banded chromosomes by electron microscopy

A method for the detection by electron microscopy of chromosome banding after in situ hybridization of small, nonradioactive DNA sequences is described. Typical high-resolution G-banding is produced by adding 5-bromodeoxyuridine (BrdU) during the last part of the S-phase and by applying a monoclonal antibody against the BrdU-substituted chromosome segments, followed by the addition of protein G, but no further treatment. A protocol for in situ hybridization of small, single-copy biotinylated DNA sequences and their detection by immunogold tagging on banded chromosomes is also described. This combined approach permits high-resolution mapping of small DNA sequences and should be useful in discriminating between neighboring DNA fragments.

Diagnostic utility of molecular and cytogenetic analysis in lipoblastoma: a study of two cases and review of the literature

Lipoblastoma is a benign neoplasm of embryonic white fat tissue that results from the proliferation of primitive adipocytes, in which histological features can be ambiguous. In order to discriminate between lipoblastoma and other lipogenic and lipomatous tumours, we studied chromosomal alterations and protein expression in two cases of lipoblastoma in infants.

A B-cell lymphoma–associated chromosomal translocation in a progressive transformation of germinal center

Progressive transformation of germinal center (PTGC) is a pattern of lymph node reactive hyperplasia. It can also be the predominant pattern in a hyperplastic lymph node known as florid PTGC. It is characterized histologically by the expansion of the mantle zone lymphocytes into both the adjacent sinusoids and germinal centers. The lymphocytes destroying the germinal centers are predominantly B cells, with a minor population of T cells. Morphologically, it can be confused with nodular lymphocyte-predominant Hodgkin disease (NLPHD) because of its nodular pattern and because of the presence of large cells that can be incorrectly identified as lymphocytic and histiocytic cells. A relationship between PTGC and NLPHD remains unclear, and many authors have suggested that PTGC can represent a precursor lesion of NLPHD. Here we report the first karyotype obtained in PTGC, in a 12-year-old boy. It shows a t(3;22)(q27;q11) translocation, probably involving the BCL6 gene. This translocation has previously been described in diffuse large B-cell lymphomas and in NLPHD with BCL6 rearrangement. This finding offers an insight into a possible tumorigenic pathway from PTGC to NLPHD. Further studies will be required to confirm this hypothesis.