Aberrant GRIA3 transcripts with multi-exon duplications in a family with X-linked mental retardation.

Investigation of chromosomal rearrangements in patients with mental retardation (MR) is particularly informative in the search for novel genes involved in MR. We report on a family with a genomic duplication at Xq25 identified by oligo array-CGH. Further characterization showed a partial tandem duplication of GRIA3 extending from exon 1 to exon 12. This duplication is present in two brothers with MR and on one allele in their sister with normal phenotype and completely skewed X-chromosome inactivation. The duplication is inherited from the mother, whose cognitive level is low and X-chromosome inactivation is random. This is the second family with partial duplication of GRIA3 associated with MR. GRIA3 expression studies in our case demonstrated a new mechanism for GRIA3 dysfunction with the presence of aberrant GRIA3 transcripts carrying multi-exon duplications leading to a frameshift. Our study gives additional support to the implication of GRIA3 in X-linked MR.

Heterogeneous patterns of amplification of the NUP214-ABL1 fusion gene in T-cell acute lymphoblastic leukemia.

Episomes with the NUP214-ABL1 fusion gene have been observed in 6% of T-ALL. In this multicentric study we collected 27 cases of NUP214-ABL1-positive T-ALL. Median age was 15 years with male predominance. Outcome was poor in 12 patients. An associated abnormality involving TLX1 or TLX3 was found in all investigated cases. Fluorescent in situ hybridization revealed a heterogeneous pattern of NUP214-ABL1 amplification. Multiple episomes carrying the fusion were detected in 24 patients. Episomes were observed in a significant number of nuclei in 18 cases, but in only 1-5% of nuclei in 6. In addition, intrachromosomal amplification (small hsr) was identified either as the only change or in association with episomes in four cases and two T-ALL cell lines (PEER and ALL-SIL). One case showed insertion of apparently non-amplified NUP214-ABL1 sequences at 14q12. The amplified sequences were analyzed using array-based CGH.These findings confirm that the NUP214-ABL1 gene requires amplification for oncogenicity; it is part of a multistep process of leukemogenesis; and it can be a late event present only in subpopulations. Data also provide in vivo evidence for a model of episome formation, amplification and optional reintegration into the genome. Implications for the use of kinase inhibitors are discussed.

Cryptic 7q21 and 9p23 deletions in a patient with apparently balanced de novo reciprocal translocation t(7;9)(q21;p23) associated with a dystonia-plus syndrome: paternal deletion of the epsilon-sarcoglycan (SGCE) gene.

We report on a boy with myoclonus-dystonia (M-D), language delay, and malformative anomalies. Genetic investigations allowed the identification of an apparently balanced de novo reciprocal translocation, t(7;9)(q21;p23). Breakpoint-region mapping using fluorescent in situ hybridization (FISH) analysis of bacterial artificial chromosome (BAC) clone probes identified microdeletions of 3.7 and 5.2 Mb within 7q21 and 9p23 breakpoint regions, respectively. Genotyping with microsatellite markers showed that deletions originated from paternal alleles. The deleted region on chromosome 7q21 includes a large imprinted gene cluster. SGCE and PEG10 are two maternally imprinted genes. SGCE mutations are implicated in M-D. In our case, M-D is due to deletion of the paternal allele of the SGCE gene. PEG10 is strongly expressed in the placenta and is essential for embryo development. Prenatal growth retardation identified in the patient may be due to deletion of the paternal allele of the PEG10 gene. Other genes in the deleted region on chromosome 7 are not imprinted. Nevertheless, a phenotype can be due to haploinsufficiency of these genes. KRIT1 is implicated in familial forms of cerebral cavernous malformations, and COL1A2 may be implicated in very mild forms of osteogenesis imperfecta. The deleted region on chromosome 9 overlaps with the candidate region for monosomy 9p syndrome. The proband shows dysmorphic features compatible with monosomy 9p syndrome, without mental impairment. These results emphasize that the phenotypic abnormalities of apparently balanced de novo translocations can be due to cryptic deletions and that the precise mapping of these aneusomies may improve clinical management.

Clinical, cytogenetic and molecular characteristics of 14 T-ALL patients carrying the TCRbeta-HOXA rearrangement: a study of the Groupe Francophone de Cytogénétique Hématologique.

Recently, we and others described a new chromosomal rearrangement, that is, inv(7)(p15q34) and t(7;7)(p15;q34) involving the T-cell receptor beta (TCRbeta) (7q34) and the HOXA gene locus (7p15) in 5% of T-cell acute lymphoblastic leukemia (T-ALL) patients leading to transcriptional activation of especially HOXA10. To further address the clinical, immunophenotypical and molecular genetic findings of this chromosomal aberration, we studied 330 additional T-ALLs. This revealed TCRbeta-HOXA rearrangements in five additional patients, which brings the total to 14 cases in 424 patients (3.3%). Real-time quantitative PCR analysis for HOXA10 gene expression was performed in 170 T-ALL patients and detected HOXA10 overexpression in 25.2% of cases including all the cases with a TCRbeta-HOXA rearrangement (8.2%). In contrast, expression of the short HOXA10 transcript, HOXA10b, was almost exclusively found in the TCRbeta-HOXA rearranged cases, suggesting a specific role for the HOXA10b short transcript in TCRbeta-HOXA-mediated oncogenesis. Other molecular and/or cytogenetic aberrations frequently found in subtypes of T-ALL (SIL-TAL1, CALM-AF10, HOX11, HOX11L2) were not detected in the TCRbeta-HOXA rearranged cases except for deletion 9p21 and NOTCH1 activating mutations, which were present in 64 and 67%, respectively. In conclusion, this study defines TCRbeta-HOXA rearranged T-ALLs as a distinct cytogenetic subgroup by clinical, immunophenotypical and molecular genetic characteristics.

[Microarray-based comparative genomic hybridization in the study of constitutional chromosomal abnormalities]. / L'hybridation génomique comparative sur microréseau d'ADN (puces à ADN) en pathologie chromosomique constitutionnelle.

Chromosomal aberrations are the first cause of mental impairment and dysmorphism. Rearrangements involving large chromosomal segments can be detected by standard chromosome analysis using GTG-banding, but this technique is not suited for the detection of small chromosome abnormalities. Array comparative genomic hybridisation (array-CGH) is a method used to detect segmental DNA copy number alterations. Recently, advances in this technology have enabled high-resolution examination for identifying genetic alterations and copy number variations on a genome-wide scale. This review describes the current genomic array platforms and CGH methodologies and highlights their applications for studying constitutional disease.

Pure de-novo 5 Mb duplication at Xp11.22-p11.23 in a male: phenotypic and molecular characterization.

Males with duplications within the short arm of the X chromosome are rare and most cases are inherited from a maternal heterozygote. Here we describe the first detailed characterization of a de-novo Xp duplication delineated to Xp11.22-->Xp11.23 in a 15-year-old male with moderate mental impairment, autistic-like behaviour, short stature, and mild dysmorphic features. Chromosome analysis (550 band resolution) was normal and comparative genomic hybridization (CGH) analysis on metaphase spreads detected duplication on Xp11. Further characterization of the duplication by array CGH, FISH experiments with specific BAC probes, and genotyping with microsatellite markers helped to determine proximal and distal breakpoints giving a size of the duplication of approximately 5 Mb. As far as we are aware this is the first described male with isolated microduplication on Xp11.22-Xp11.23. Among the genes included within the duplicated region, and particularly those which are outside copy number polymorphisms, we discuss the relationship of FTSJ1, PQBP1 and HDAC6 with the clinical symptoms of our patient.

[A case of de novo acute basophilic leukaemia: diagnostic criteria and review of the literature]. / A propos d'un cas de leucémie à basophiles de novo: critères diagnostiques et revue de la littérature.

We report a case of a de novo acute basophilic leukaemia, revealed by an infectious pneumopathy in a 73 year old man. The full blood count revealed an hyperleucocytosis associated with an unregenerative normocytic normochrom anaemia and a thrombocytopenia. The blood and bone marrow smears showed a mixture of undifferentiated blast cells and basophiloblasts (high nucleo-cytoplasmic ratio, coarse basophilic cytoplasmic granules), along with basophilic precursors and basophilic polymorphonuclears. All the blasts were MPO negative but positive for the toluidine blue metachromatic coloration, which is considered as consistent with basophilic lineage. Immunophenotypic studies showed myeloid blasts, without maturity marker, CD 117 negative and CD203 cytoplasmic positive, the latter known to be highly representative of the basophilic lineage. This very clear-cut phenotype, associated with the morphology of cells, were arguments to ascertain the basophilic lineage of the blasts without the need of electron microscopic study. Cytogenetic and RNA analysis revealed the presence of a Philadelphia chromosome and of a BCR-ABL transcript with the unusual junction e6a2. Thus, imatinib was added to the conventional chimiotherapy and the patient is currently in complete remission. This clinical prompted allows us to review the literature on acute basophilic leukaemia and to state on the different diagnostic criteria of this rare disorder.

Terminal deletion of the long arm of chromosome 10.

The case of two female patients with de novo terminal deletion of the long arm of chromosome 10, one with del(10)(q26.2) and the other with del(10)(q26.3), is reported. Both presented with megabladder associated with urinary tract abnormalities. The case of four similar patients has been previously reported with bladder dilatation secondary to urinary obstruction. These new cases highlight the possible involvement of the bladder and the urethra in the syndrome of chromosome 10q terminal deletion, suggesting a careful renal and urinary tract evaluation in such situations. Moreover, the possibility of monosomy 10qter syndrome should be borne in mind in the case of prenatal diagnosis of apparently isolated bladder obstruction, especially in females.

t(5;14)/HOX11L2-positive T-cell acute lymphoblastic leukemia. A collaborative study of the Groupe Français de Cytogénétique Hématologique (GFCH).

To accurately estimate the incidence of HOX11L2 expression, and determine the associated cytogenetic features, in T-cell acute lymphoblastic leukemia (T-ALL), the Groupe Français de Cytogénétique Hématologique (GFCH) carried out a retrospective study of both childhood and adult patients. In total, 364 patients were included (211 children

[Acute myeloblastic leukemia without maturation (AML-M1) with basophilic elements and associated with translocation t(6;9)]. / Leucémie aiguë myéloblastique sans maturation (LAM1) avec éléments basophiles associée à la translocation t(6;9).

The clinical, hematological, and cytogenetic data from a 4 year-old child with acute myeloid (AML-M1) and basophilia is reported. Interestingly, cytogenetic investigations revealed the presence of the translocation t(6;9) (p23;q34). This abnormality is rare and associated with myelodysplastic syndromes or with subtypes of acute myeloid leukemia (M1, M2, M4, M7), usually with preceding or underlying myelodysplasia. The prognosis is poor, without response to chemotherapy regimen alone. Allogeneic bone marrow transplantation appears likely to be a more appropriate treatment.

Leukaemic small cell variant anaplastic large cell lymphoma during pregnancy.

The history of a 28-year-old woman with anaplastic large cell lymphoma (ALCL) in the first trimester of her pregnancy is reported. Investigations allowed to diagnose a T-cell CD30 positive ALCL, which appearance is rare during pregnancy. Moreover, the atypical lymphoid cells were found in the peripheral blood and were predominantly small to medium sized with nuclear irregularities and cytoplasmic azurophilic granules, which allowed the hypothesis of leukaemic presentation of a small cell variant ALCL. A variant of the t(2:5)(p23:q35) was found [del(2)(p22)]. The patient died shortly after diagnosis.

Increased usage of TCR V-beta8 in kidney transplant recipients with aberrant immune reconstitution and clinical complications.

BACKGROUND: The efficiency of immunosuppressive drugs prescribed after organ transplantation is mostly monitored through clinical and biological signs of organ rejection or infection. However, it may be expected that some patients develop subtle alterations of their reconstituting immune system, not immediately associated with clinical events. Identification of such anomalies could be useful to alert clinicians for possible future complications. METHODS: A systematic follow-up of peripheral lymphocyte subsets, performed in a cohort of 89 kidney transplant recipients, identified severely skewed CD4/CD8 ratios in 32 patients. For 19 patients, the expression of specific T cell receptor fragments was examined using a panel of 10 monoclonal antibodies. Abnormal control of spontaneously Epstein Barr virus-infected B cells was tested by investigating for the generation of spontaneous lymphoblastoid cell lines in 17 cases. The incidence of rejection and infectious episodes was monitored. RESULTS: A bias in T cell receptor fragments usage was detected in 14/19 cases, involving Vbeta8 in all cases. Spontaneous lymphoblastoid cell lines of Epstein Barr positive B blasts developed in 9 of 17 cases. Eleven patients had early rejection episodes and 16 presented with viral primo-infection or reactivation. The incidence of rejection and infectious episodes was higher in the group of 32 patients who developed such abnormal patterns than in the 57 who did not. CONCLUSION: Transient bias in the T cell receptor repertoire may be observed during immune reconstitution after kidney transplantation, perhaps related to abnormal lymphocyte functions and associated to an impaired control of rejection and/or infectious agents.

The t(6;8)(q27;p11) translocation in a stem cell myeloproliferative disorder fuses a novel gene, FOP, to fibroblast growth factor receptor 1.

In patients with an atypical stem-cell myeloproliferative disorder with lymphoma (B or T cell), myeloid hyperplasia, and eosinophilia, the chromosome 8p11-12 region is the site of a recurrent breakpoint that can be associated with three different partners, 6q27, 9q32-34, and 13q12. Rearrangements are supposed to affect a pluripotent stem cell capable of myeloid and lymphoid differentiation and to involve the same 8p11-12 gene. The t(8;13) translocation has recently been shown to result in a fusion between the FGFR1 gene that encodes a tyrosine kinase receptor for fibroblast growth factors and a novel gene, FIM (also called RAMP or ZNF198), belonging to a novel family of zinc finger genes. In the present study, we have cloned the t(6;8)(q27;p11) translocation in two patients and found a fusion between FGFR1 and a novel gene, FOP (FGFR1 Oncogene Partner), located on chromosome band 6q27. This gene is alternatively spliced and ubiquitously expressed. It encodes a protein containing two regions of putative leucine-rich repeats putatively folding in alpha-helices and separated by a hydrophobic spacer. The two reciprocal fusion transcripts were evidenced by reverse transcription-polymerase chain reaction in the tumoral cells of the patients. The predicted chimeric FOP-FGFR1 protein contains the FOP N-terminus leucine-rich region fused to the catalytic domain of FGFR1. It may promote hematopoietic stem cell proliferation and leukemogenesis through a constitutive phosphorylation and activation of the downstream pathway of FGFR1.

In-vitro growth patterns of bone marrow erythroid progenitors from patients with post-renal transplant erythrocytosis.

BACKGROUND: Erythrocytosis is relatively common after renal transplantation and is associated with a higher risk of thromboembolism. Its aetiology is unclear and there is still debate about the most frequently suggested causes. The culture in vitro of erythroid progenitors is regarded as a useful tool for the differential diagnosis of patients with unclear erythrocytosis. We studied the growth in vitro of bone marrow erythroid progenitors from renal transplant patients with erythrocytosis and controls without erythrocytosis. SUBJECTS AND METHODS: Thirteen renal transplant patients with erythrocytosis and 12 normocythaemic renal transplant controls were studied. The clinical characteristics of these patients were evaluated and serum erythropoietin (Epo) and ferritin levels were determined. Bone marrow erythroid progenitors were cultured both with and without the addition of Epo to the medium. RESULTS: Samples from six polycythaemic patients and seven controls did not grow spontaneously in the absence of exogenous Epo. Three cases of post-transplant erythrocytosis and five controls produced CFU-E, but not BFU-E. A few CFU-E and BFU-E grew spontaneously in samples from four polycythaemic patients but not in samples from the controls. Addition of 1 unit per millilitre Epo caused similar increases in the number of colonies in both polycythaemic patients and controls. Of the nine patients eligible for follow-up, all four with spontaneous growth of BFU-E had transient erythrocytosis and four of the five patients with no spontaneous growth or spontaneous growth of CFU-E only had persistent erythrocytosis requiring treatment with ACE inhibitors. CONCLUSIONS: Pathophysiology of post-transplant erythrocytosis is heterogeneous. In one-third of the patients, there was unexpected, spontaneous and transient growth of BFU-E which was not predictive of permanent erythrocytosis. The results of stem-cell studies suggest that in these cases erythrocytosis may be caused by defective regulation of erythroid progenitor proliferation, possibly due to particular cellular interactions or the effect of cyclosporin on erythropoiesis.

t(6;8), t(8;9) and t(8;13) translocations associated with stem cell myeloproliferative disorders have close or identical breakpoints in chromosome region 8p11-12.

A stem-cell myeloproliferative disorder involving T- and B-cell, and myeloid lineages, is associated with three different translocations with a breakpoint in region p11-12 of chromosome 8: t(6;8)(q27;p11), t(8;9)(p11;q33), and t(8;13)(p12;q12), respectively. Using fluorescence in situ hybridization (FISH), we have analysed blood cells from a series of five patients carrying these different translocations. We have identified cosmids from chromosome region 8p11-12 that span the breakpoint in all the cases. They are specific for the FCFR1 gene that encodes a receptor for members of the FGF family. The breakpoint was further detected by Southern and pulsed-field gel electrophoresis analyses with probes from the FGFR1 locus.

De novo methylation of tumour suppressor genes CDKN2A and CDKN2B is a rare finding in B-cell chronic lymphocytic leukaemia.

De novo methylation of the 5'CpG island has been recently reported as an alternative mechanism of inactivation for the tumour suppressor genes CDKN2A and CDKN2B. We examined CDKN2A methylation status at diagnosis in 42 B-cell chronic lymphocytic leukaemia (CLL) patients, in 19 cases the CDKN2B methylation status was also analysed. No homozygous CDKN2A/2B deletion was detected, but four patients (9%) displayed an aberrant CDKN2A methylation status and only one had hypermethylated CDKN2B. De novo methylation was associated with silencing of gene expression. These results confirm that CDKN2A/2B inactivation by deletion is a rare event in CLL and suggest that aberrant methylation could be an alternative way of inactivation very rarely involved in the development of some CLL.

Structure-photodynamic activity relationships of a series of 4-substituted zinc phthalocyanines.

Radioiodinated zinc phthalocyanine including [125I]ZnPcI4 and differently sulfonated [65Zn]ZnPcS (ZnPcS4, ZnPcS3, ZnPcS2 and ZnPcS1.75, a mixture of adjacent di and 25% mono) were prepared in order to study cell uptake and release kinetics in EMT-6 cells. The same compounds were evaluated for their in vitro phototoxicity and the biological parameters were compared to partition coefficients to arrive at quantitative structure-activity relationships (QSAR). At 1 microM in 1% serum, at 37 degrees C, all dyes showed rapid cell uptake during the first hour followed by a slow accumulation phase. After 24 h, the highest cellular concentration was observed with the lipophilic ZnPcI4, followed by the amphiphilic ZnPcS2 and ZnPcS1.75. The hydrophilic ZnPcS4 and ZnPcS3 showed lower uptake. Dye release from dye-loaded cells during incubation in dye-free medium could reach up to 60% and was shown to depend mainly on the amount of drug incorporated rather than the type of compound. These results suggest that care should be taken in interpreting dye toxicity data, which involve in vitro cell manipulations in dye-free medium, particularly during in vitro-in vivo protocols. The EMT-6 cell survival after 1 h or 24 h incubation with 1 microM dye in 1% serum followed by exposure to red light was assessed by means of the colorimetric 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) assay. Photocytotoxicities correlated inversely with the tendencies of the dyes to aggregate. Increased dye uptake by the cells also correlated with their activities, except for the lipophilic ZnPcI4, which showed the highest cell uptake but little phototoxicity. The QSAR between phototoxicity and the log of the partition coefficients (phosphate-buffered saline and n-octanol) gave a parabola with optimal partition values corresponding to the adjacent sulfonated ZnPcS2.

Epstein-Barr virus lymphoproliferative disease of donor origin after kidney transplantation: a case report.

A case of an Epstein-Barr virus (EBV)-associated B lymphoproliferative disorder presented as a renal transplant obstruction is reported. The diagnosis was made from histology, immunohistochemistry, and EBV expression studies. Cytogenetic analysis showed the tumor to be of donor origin and revealed chromosomal translocation 46, XY, inv (1)(p35; q41), involving the EBV insertion site 1(1p35) and transforming growth factor beta 2(1q41) loci.