A mesenchymal glioma stem cell profile is related to clinical outcome.

Recent studies have demonstrated a relationship between the expression of stem cell-associated genes and relapses in glioblastoma (GBM), suggesting a key role for tumor stem cells in this process. Although there is increasing interest in this field, glioma stem cells (GSCs) are still poorly characterized, their 'stemness' state and factors maintaining these properties remain largely unknown. We performed an expression profiling analysis of pluripotency in gliomaspheres derived from 11 patients. Comparative analysis between GSCs and H1 and H9 human embryonic stem cells as well as H9-derived neural stem cells indicates major variations in gene expression of pluripotency factors Nanog and OCT4, but a stable pattern for SOX2 suggesting its important function in maintaining pluripotency in GSCs. Our results also showed that all GSC lines have the capacity to commit to neural differentiation and express mesenchymal or endothelial differentiation markers. In addition, hierarchical clustering analysis revealed two groups of GSCs reflecting their heterogeneity and identified COL1A1 and IFITM1 as the most discriminating genes. Similar patterns have been observed in tumors from which gliomaspheres have been established. To determine whether this heterogeneity could be clinically relevant, the expression of both genes was further analyzed in an independent cohort of 30 patients with GBM and revealed strong correlation with overall survival. In vitro silencing of COL1A1 and IFTM1 confirmed the effect of these mesenchymal-associated genes on cell invasion and gliomasphere initiation. Our results indicate that COL1A1 and IFITM1 genes could be considered for use in stratifying patients with GBM into subgroups for risk of recurrence at diagnosis, as well as for prognostic and therapeutic evolution.

Stroke and aspirin non-responder patients: relation with hypertension and platelet response to adenosine diphosphate.

Despite its widespread use, there are many concerns about the efficacy of aspirin in the secondary prevention of cardiovascular events after stroke, leading to the concept of aspirin non-response (ANR). Although the mechanisms of ANR remain uncertain, it is expected to be due to a combination of clinical, biological and genetic characteristics affecting platelet function. In this study, we investigated whether clinical and/or biological factors such as hypertension and platelet response to ADP could contribute to the ANR. As a secondary objective, we determine whether ANR and collagen/ADP closure time (CADP-CT) could be related to platelet glycoprotein single nucleotide polymorphisms (SNPs). One hundred patients on aspirin (160 mg/day) were enrolled. ANR was measured with a platelet function analyzer (PFA-100); genotyping of four SNPs (GP IIIa, GP Ia, P2Y12 and GP VI) was performed using a tetra-primer amplification refractory mutation system. Using a collagen/epinephrine-coated cartridge on the PFA-100, the prevalence of ANR was 15% (n = 15). In the ANR group, (i) CADP-CT was significantly shorter and (ii) hypertension was an independent clinical predictive factor of ANR (OR = 4.25; 95%CI: 1.06-17.11). No clear relation was found between CADT-CT and platelet gene polymorphism as well as ANR status and SNPs. In conclusion our study confirms the independent relationship between hypertension, platelet hypersensitivity to ADP and aspirin (160 mg/day) non-response. The differential sensitivity to aspirin may have potential clinical implications, where adaptation of antiplatelet therapy is necessary according to a patient's clinical and genetic characteristics.

Persistence of transcriptionally silent BCR-ABL rearrangements in chronic myeloid leukemia patients in sustained complete cytogenetic remission.

Persistence of BCR-ABL rearrangements was demonstrated by D-FISH technique in chronic myeloid leukemia (CML) patients in complete cytogenetic response (CCR) after allogeneic bone marrow transplantation (BMT) or interferon-alpha therapy (IFN-alpha). Samples from bone marrow aspirate or peripheral blood or both were analyzed by conventional cytogenetics, Southern blot, fluorescent interphase in situ hybridization (FISH), and quantitative reverse transcription polymerase chain reaction (Q-RT-PCR). In all patients, FISH detected 1% to 12% nuclei with a BCR-ABL fusion gene, whereas Q-RT-PCR were negative or weakly positive. Based on these results, we hypothesize that the BCR-ABL genomic rearrangement remains unexpressed in a small percentage of cells whatever the treatment (IFN-alpha or BMT), and this in spite of the negativity of the RT-PCR-based classical molecular remission criterion. These data corroborate those obtained by other investigators and point to the need for follow-up of CML patients in CCR over an extensive period, at the DNA level to evaluate the residual disease and at the RNA level (Q-RT-PCR) to estimate the risk of relapse and guide the therapeutic decision. Experimental models suggesting the persistence of positive BCR-ABL cells are discussed and tentative explanations of tumor "dormancy" are proposed.

Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.

We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7, 420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Only 11 mutations had relative frequencies >0. 4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of coding/flanking sequences, 516 of 654 (78. 90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations.

Persistence of BCR-ABL genomic rearrangement in chronic myeloid leukemia patients in complete and sustained cytogenetic remission after interferon-alpha therapy or allogeneic bone marrow transplantation.

In recent years, the prognosis of chronic myeloid leukemia (CML) has been greatly improved either with interferon-alpha (IFN-alpha) therapy or allogeneic bone marrow transplantation (BMT). In the present study, minimal residual disease was evaluated in 21 patients in complete cytogenetic response (CCR) after such treatments. Samples from bone marrow aspirates or peripheral blood or both were analyzed by conventional cytogenetics, Southern blot, interphase fluorescent in situ hybridization (FISH), and quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). In all patients, FISH detected 1% to 12% nuclei with a BCR-ABL fusion gene, whereas Q-RT-PCR experiments were negative or weakly positive. Based on these results, we hypothesize that the BCR-ABL genomic rearrangement persists unexpressed in nonproliferating cells whatever the treatment (IFN-alpha or BMT). These data point to the need for follow-up of CML patients in CCR over an extensive period at the DNA level (FISH) to evaluate the residual disease and at the RNA level (Q-RT-PCR) to estimate the risk of relapse. (Blood. 2000;95:404-408)

Molecular cloning, expression analysis, and chromosomal localization of human syntaxin 8 (STX8).

We report the cloning of a cDNA encoding human syntaxin 8 (STX8), using the regulator (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) as a bait to screen a human fetal lung cDNA library by the yeast two-hybrid system. This gene was found broadly transcribed and its mRNA size is about 1.3 kb. The STX8 gene maps to chromosomal band 17p12 and it encodes a 236-amino-acid protein. Syntaxin 8 contains in its C-terminal half a coiled-coil domain found highly conserved in the t-SNARE (SNAP receptor on target membrane) superfamily of proteins, which are involved in vesicular trafficking and docking. In syntaxin 8, a C-terminal hydrophobic domain may constitute a transmembrane anchor. It was recently shown that CFTR-mediated chloride currents can be regulated by syntaxin 1A, a t-SNARE family member, through direct protein-protein interaction. This raises the possibility that syntaxin 8 may also be involved in such regulations.

Does BCR-ABL genomic rearrangement persist in CML patients in complete remission after interferon alpha therapy?

Cytogenetic, interphase fluorescent in situ hybridization (FISH) and RT-PCR methods were used to study minimal residual disease in peripheral blood stem cells collected for autografting in three chronic myeloid leukemia (CML) patients in sustained complete cytogenetic remission after treatment with interferon alpha (IFNalpha). Karyotypic analysis failed to reveal Ph-positive metaphases. FISH detected 9-16% nuclei with a BCR-ABL fusion gene, contrasting with RT-PCR, performed in two cases, which was negative in one case and weakly positive in the other. RT-PCR was also subsequently weakly positive in the third patient. This discrepancy suggests that the BCR-ABL genomic rearrangement persists unexpressed in quiescent cells. These preliminary results, which need to be confirmed in larger series, suggest that monitoring residual disease in CML should be performed both at DNA and RNA levels. Moreover, autografting following IFNalpha therapy should be considered with caution because of the persistence of the BCR-ABL genomic rearrangement in a sizeable proportion of the cells.

Identification of several genes differentially expressed during progression of chronic myelogenous leukemia.

The Bcr-Abl fusion protein plays a crucial role in the initiation and maintenance of chronic myelogenous leukemia (CML). However, additional events are necessary for the transition from the chronic phase to the terminal phase of the disease. To identify genes involved in the disease progression, we constructed a subtractive library from enriched K562 cell mRNA. We obtained 1084 cDNA clones. After a specific hybridization of these clones with a cDNA probe from either chronic phase or K562 cells, 43 clones which present a differential hybridization level have been selected. Among them, several clones corresponded to ribosomal protein genes showing an increased transcription level during the blast crisis. We observed variations in the expression of a cellular adhesion molecule, a laminin-binding protein. An increased transcription level of the MAZ gene has been shown in the terminal phase of the disease. This gene encodes a protein that regulates the transcription of myc.

A YAC contig spanning the blepharophimosis-ptosis-epicanthus inversus syndrome and propionic acidemia loci.

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant condition consisting of congenital dysplasia of the eyelids with a reduced horizontal diameter of the palpebral fissures, droopy eyelids and epicanthus inversus. Two clinical entities have been described: type I and type II. The former is distinguished by female infertility, whereas the latter presents without other symptoms. Both type I and type II were recently mapped on the long arm of chromosome 3 (3q22-q23), suggesting a common gene may be affected. The centromeric and the telomeric limits of this region are well defined between loci D3S1316 and D3S1615, which reside approximately 5 cM apart. Here, we present the construction of a YAC contig spanning the entire BPES locus using 17 polymorphic markers, 2 STS and 28 ESTs. This region of approximately 5 Mb was covered by 31 YACs, and was supported by detailed FISH analysis. In addition, we have precisely mapped the propionyl-CoA carboxylase beta polypeptide (PCCB), the gene mutated in propionic acidemia, within this contig. Apart from providing a framework for the identification of the BPES gene, this contig will also be useful for the future identification of defects and genes mapped to this region, and for developing template resources for genomic sequencing.

Monitoring and prognostic evaluation of sex-mismatched bone marrow transplantation by competitive PCR on Y chromosome sequences.

In one case out of four, allogeneic BMT concerns a male recipient and a female donor. The monitoring of sex-matched BMT can be carried out by PCR amplification on Y-specific chromosome sequences (YCS), whatever the hematological disease. Twelve patients with sex-mismatched non-T-depleted BMT were first studied through a qualitative PCR, which gave semi-quantitative results. When the qualitative PCR revealed YCS, a competitive amplification was performed in order to estimate the YCS amount in the patient blood sample. For the purpose of the study, we classified the patients in two categories according to the results obtained 9 months after BMT. For 10 patients, we did not detect any YCS amplification after this time. These patients were in complete cytogenetic and clinical remission. For the remaining two patients, we always found male DNA in their blood samples. These patients were in cytogenetic remission but relapsed and died 21 and 25 months after BMT. Our results suggest that the persistence of male cells in peripheral blood, even at the low rate of 1% or 0.1%, 1 year after sex-mismatched BMT, is a bad prognosis.

Molecular remission after allogeneic bone marrow transplantation for chronic lymphocytic leukemia.

A 51-year-old man with previously treated CLL received an allogeneic sex mismatched BMT after total body irradiation and high dose chemotherapy. Residual disease was studied at phenotypic and molecular levels including Y chromosome DNA amplification by PCR assay. The patient was clinically disease-free 20 months after BMT with disappearance of the leukemic clone assessed by the most sensitive methods of detection. Long-term follow-up is necessary to ascertain the relevance of Y DNA amplification in predicting outcome in this patient.

CFTR illegitimate transcription in lymphoid cells: quantification and applications to the investigation of pathological transcripts.

Since the isolation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) and the characterization of the main mutation (delta F508) in 1989, a large number of rare mutations has been found. Full screening of the CFTR gene is difficult because it is split into 27 exons covering 250 kb of genomic DNA. This gene is essentially expressed in the lung and intestinal tract, neither of which are easily accessible for routine investigations. The recent description of a faint transcription of highly tissue-specific genes in any cell, a phenomenon known as illegitimate transcription, would facilitate the research of mutations and the characterization of truncated m-RNA caused by splicing mutations. Using the polymerase chain reaction on cDNA (cDNA-PCR), we detected transcripts of the CFTR gene in lymphocytes and lymphoblast cells at a very low level (about 300 times less than in lung or intestine). This strategy allowed us to obtain a sufficient amount of cDNA-PCR product compatible with further molecular analyses. We have, therefore, analyzed a cDNA fragment overlapping exons 10 and 11 by polyacrylamide gel electrophoresis and direct sequencing, and detected the delta F508 mutation at this level. Our protocol can be generalized to the investigation of the total 4.5-kb CFTR coding sequence.

Cystic fibrosis in the population of Reunion Island.

The frequencies of the delta F 508 mutation and haplotypes linked to the cystic fibrosis (CF) gene and detected with DNA probes XV-2C and KM-19 have been studied in the population of Reunion Island, a French province located in the Indian Ocean. The deletion was present in 41.3% of CF chromosomes, whereas this proportion is about 70% in the French population. The delta F 508 mutation was associated with the haplotype B defined by the DNA markers XV-2C (allele 1) and KM-19 (allele 2) in 76.4% of CF chromosomes, while this proportion is over 90% in the French population. Founder effect, genetic drift and admixture can explain these differences.