Several somatic genetic alterations have been described in non-small-cell lung carcinomas (NSCLC)... more Several somatic genetic alterations have been described in non-small-cell lung carcinomas (NSCLC). Recurrent chromosomal deletions have suggested the presence of tumor-suppressor genes specifically involved in lung carcinogenesis. For one of these, 2 non-overlapping regions have been proposed on the short arm of chromosome 8, encompassing the LPL and NEFL genes. The LPL region has been extensively studied in NSCLC and other cancer types. Two genes, N33 and PRLTS, have been identified, but the small number of mutations excludes their involvement in the vast majority of tumors. In order to delineate a reliable region of deletional overlap on chromosome 8p in NSCLC, a series of 77 NSCLC was studied for 34 microsatellite polymorphisms distributed on chromosome 8p, using multiplex-PCR amplification. After purification of tumor nuclei by flow cytometry based on either the abnormal DNA index or the presence of a high expression of cytokeratin, allelic losses on chromosome 8p were observed in 39% of cases. Measurement of DNA index showed that 62% of tumors were hyperploid; allelic losses were more frequent in hyperploid than in diploid tumors (54% vs. 14%; p < 10(-4)). Deletions of part of the short arm were observed in 7 instances. Our data allow definition of an interval of common deletion, flanked by the loci D8S511 and D8S1992, where the putative tumor-suppressor gene might be localized.
Sensitivity of T4 effector-memory (T4EM) lymphocytes to radiation-induced apoptosis shows heritab... more Sensitivity of T4 effector-memory (T4EM) lymphocytes to radiation-induced apoptosis shows heritability compatible with a Mendelian mode of transmission. Using gene expression studies and flow cytometry, we show a higher TNF-Related Apoptosis Inducing Ligand (TRAIL/TNFSF10)mRNA level and a higher level of membrane bound TRAIL (mTRAIL) on radiosensitive compared to radioresistant T4EM lymphocytes. Functionally, we show that mTRAIL mediates a pro-apoptotic autocrine signaling after irradiation of T4EM lymphocytes linking mTRAIL expression to T4EM radiosensitivity. Using single marker and multimarker Family-Based Association Testing, we identified 3 SNPs in the TRAIL gene that are significantly associated with T4EM lymphocytes radiosensitivity. Among these 3 SNPs, two are also associated with acute and subacute dermatitis after radiotherapy in breast cancer indicating that T4EM lymphocytes radiosensitivity may be used to predict response to radiotherapy. Altogether, these results show t...
We have developed a simple and efficient method to construct partial libraries of swine Chromosom... more We have developed a simple and efficient method to construct partial libraries of swine Chromosome (Chr) 11, starting with only 300 flow-sorted copies. DNA is amplified by PARM-PCR with primer containing at the 5'-end the sequence AGCU-. After amplification, digestion of PCR products with uracil DNA glycosylase generates cohesive ends corresponding to the SstI site. The amplified fragments can then be ligated in vector linearized with the SstI enzyme. Using five different primers, we PARM-PCR amplified and cloned swine Chr 11 DNA. These chromosome-specific libraries have been used to develop 14 different (TG)n microsatellites. Ten of these markers were assigned to Chr 11 by PCR analysis of a panel of Pig-Rodent somatic hybrids and by linkage analysis of the 171 individuals of the PiGMaP reference families. A complete linkage map of 147 cM of this chromosome was then realized by integrating existing markers.
Numerous loci can be amplified by PARM-PCR on 300 sorted chromosomes in low-stringency conditions... more Numerous loci can be amplified by PARM-PCR on 300 sorted chromosomes in low-stringency conditions (annealing at 30~ during the two first cycles) to produce a probe that can be used in FISH painting experiments. We demonstrate that, depending on the primer chosen for the amplification, patterns of different quality can be obtained. In order to design a primer that allows amplification of coding sequences, we have shown that motifs of at least seven glutamic acid repeats (GAG or GAA codons) are present in human proteins more frequently than expected. Moreover, these repeats do not correspond to triplet expansion and can be conserved between species. Using probes prepared from sorted chromosomes with (GAG)7 primer, we were able to achieve homologous FISH painting on human, porcine, ovine, and bovine species, and bidirectional heterologous FISH painting between human and porcine species. As an example, using probes for human Chromosome (Chr) 19 and porcine Chrs 1 and 6, we clearly defined the regional homologies existing between those chromosomes.
We present here a new PCR-based technique that allows the production of several micrograms of DNA... more We present here a new PCR-based technique that allows the production of several micrograms of DNA from only 300 flow-sorted chromosomes. During the first two PCR cycles, the annealing temperature is decreased to 30 degrees C, and numerous random loci are amplified under nonspecific conditions. As demonstrated here for pig chromosomes 1 and 18, the PCR products may be used to identify the chromosomal content of the flow-karyotype peaks of any species by fluorescence in situ hybridization.
Flow cytogenetic is widely used since 1975, and essentially contributes to caryotype analysis and... more Flow cytogenetic is widely used since 1975, and essentially contributes to caryotype analysis and chromosome sorting. The principles of experimentation and its possibilities and limitations are now well known. Recently several new technologies have appeared. What attitude should the cytometrist adopt regarding PCR, microdissection of chromosomes, in situ hybridization, slitscan flow cytometry or image analysis? chromosomes / karyolype / cylomelry / genelies * Correspondence and reprints. ** Correspondence concerning either the applications of FCM to large genome studies or the contribution of PCR to the renewal of flow cytogenetics.
The extent and distribution of conserved chromosomal segments between pig and cattle chromosomes ... more The extent and distribution of conserved chromosomal segments between pig and cattle chromosomes were established using hybridization of porcine chromosome painting probes on bovine metaphases. A total of 44 segments of conserved synteny were identified, resulting in a nearly complete coverage of the bovine karyotype. This study provides new data on chromosome evolution of mammals.
Para localizar genes candidatos, QTLs, o para usar la selección asistida par marcadores se necesi... more Para localizar genes candidatos, QTLs, o para usar la selección asistida par marcadores se necesitan mapas génicos integrados y desarrollados. Como la cartografía génica humana está significativamente más avanzada, un cariotipo comparativo entre la especie humana y la porcina, que indicara la localización y extensión de las regiones de homología permitiría obtener beneficios del mapa humano. Para determinar las correspondencias entre segmentos cromosómicos homólogos humanos y porcinos, utilizamos sondas para coloración e hibridación in situ de cromosomas completos de ambas especies, en experimentos bidireccionales de hibridación heteróloga. Esta estrategia permite determinar las homologias segmento a segmento entre los cromosomas de ambas especies. Las sondas para coloración e hibridación in situ específica de cromosomas de ambas especies fueron obtenidas en todos las casos excepto en uno por DOP-POR (sondas comerciales Cambio) o amplificación PARM-PCR de cromosom as seleccionados e...
Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology, 1999
A comparative karyotype of rat (Rattus norvegicus) and mouse (Mus musculus) based on chromosome G... more A comparative karyotype of rat (Rattus norvegicus) and mouse (Mus musculus) based on chromosome G-banding morphology, heterologous chromosome painting results and available gene mapping data is proposed. Whole chromosome painting probes from both species were generated by PARM-PCR amplification of flow sorted chromosomes. Bidirectional chromosome painting identifies 36 segments of syntenic homology and allows us to propose a nearly complete comparative karyotype of mouse and rat (except for RNO 13 p and RNO 19 p12-13). Seven segments completely covered the RNO chromosomes 3, 5, 8, 11, 12, 15 and 18. Eight segments completely covered the MMU chromosomes 3, 4, 6, 7, 9, 12, 18 and 19. The RNO chromosomes 5, 8, 18 show complete homology with the MMU chromosomes 4, 9 and 18, respectively. Bidirectional hybridization results clearly assign 16 segments to subchromosomal regions in both species. Interpretation of the results allows subchromosomal assignment of all the remaining segments apa...
Recurrent allelic losses on chromosome 22q have been reported in colorectal cancer, distal to the... more Recurrent allelic losses on chromosome 22q have been reported in colorectal cancer, distal to the NF2 gene, suggesting that another tumor suppressor gene might be involved. We report here the typing of 256 sporadic colorectal tumors and 18 colonic cancer cell lines using a set of chromosome 22 polymorphisms, ranging from 20 to 45. A panel of somatic cell hybrids, that allows to distinguish 11 bins in the 22q13 region, was used to localize 19 of the 45 selected markers and the putative tumor suppressor gene BZRP. Allelic-loss was observed in 43% of tumors. The minimal region of deletion that could be determined, telomeric to locus D22S270, refines significantly the position of the gene. The localization of the BZRP gene in this region led to a systematic screening for somatic point mutation. Direct sequencing of its coding sequence in 36 tumors hemizygous for chromosome 22 allowed the identification of three polymorphisms but failed to detect somatic mutation.
A subset of genetic alterations distinguishes two groups of colon cancers. In the first group ins... more A subset of genetic alterations distinguishes two groups of colon cancers. In the first group instability of microsatellite loci due to a defective DNA mismatch repair system is observed. The second group is characterized by recurrent losses of chromosome regions, frequently associated with hyperploidization. We have developed a technique which enables a fine description of allelic losses in this second group of tumours. The typing of 278 loci in 47 hyperploid colon cancers has provided information for an average of 160 loci per tumour. The high frequency of allelic losses on chromosomes 17, 18 and 5 was confirmed thus validating our methodological approach. Several additional chromosome segments were observed lost in over 40% of the cases, suggesting that tumour suppressor genes may map within these regions. Further technical development should contribute to the identification of these genes.
Several somatic genetic alterations have been described in non-small-cell lung carcinomas (NSCLC)... more Several somatic genetic alterations have been described in non-small-cell lung carcinomas (NSCLC). Recurrent chromosomal deletions have suggested the presence of tumor-suppressor genes specifically involved in lung carcinogenesis. For one of these, 2 non-overlapping regions have been proposed on the short arm of chromosome 8, encompassing the LPL and NEFL genes. The LPL region has been extensively studied in NSCLC and other cancer types. Two genes, N33 and PRLTS, have been identified, but the small number of mutations excludes their involvement in the vast majority of tumors. In order to delineate a reliable region of deletional overlap on chromosome 8p in NSCLC, a series of 77 NSCLC was studied for 34 microsatellite polymorphisms distributed on chromosome 8p, using multiplex-PCR amplification. After purification of tumor nuclei by flow cytometry based on either the abnormal DNA index or the presence of a high expression of cytokeratin, allelic losses on chromosome 8p were observed in 39% of cases. Measurement of DNA index showed that 62% of tumors were hyperploid; allelic losses were more frequent in hyperploid than in diploid tumors (54% vs. 14%; p < 10(-4)). Deletions of part of the short arm were observed in 7 instances. Our data allow definition of an interval of common deletion, flanked by the loci D8S511 and D8S1992, where the putative tumor-suppressor gene might be localized.
Sensitivity of T4 effector-memory (T4EM) lymphocytes to radiation-induced apoptosis shows heritab... more Sensitivity of T4 effector-memory (T4EM) lymphocytes to radiation-induced apoptosis shows heritability compatible with a Mendelian mode of transmission. Using gene expression studies and flow cytometry, we show a higher TNF-Related Apoptosis Inducing Ligand (TRAIL/TNFSF10)mRNA level and a higher level of membrane bound TRAIL (mTRAIL) on radiosensitive compared to radioresistant T4EM lymphocytes. Functionally, we show that mTRAIL mediates a pro-apoptotic autocrine signaling after irradiation of T4EM lymphocytes linking mTRAIL expression to T4EM radiosensitivity. Using single marker and multimarker Family-Based Association Testing, we identified 3 SNPs in the TRAIL gene that are significantly associated with T4EM lymphocytes radiosensitivity. Among these 3 SNPs, two are also associated with acute and subacute dermatitis after radiotherapy in breast cancer indicating that T4EM lymphocytes radiosensitivity may be used to predict response to radiotherapy. Altogether, these results show t...
We have developed a simple and efficient method to construct partial libraries of swine Chromosom... more We have developed a simple and efficient method to construct partial libraries of swine Chromosome (Chr) 11, starting with only 300 flow-sorted copies. DNA is amplified by PARM-PCR with primer containing at the 5'-end the sequence AGCU-. After amplification, digestion of PCR products with uracil DNA glycosylase generates cohesive ends corresponding to the SstI site. The amplified fragments can then be ligated in vector linearized with the SstI enzyme. Using five different primers, we PARM-PCR amplified and cloned swine Chr 11 DNA. These chromosome-specific libraries have been used to develop 14 different (TG)n microsatellites. Ten of these markers were assigned to Chr 11 by PCR analysis of a panel of Pig-Rodent somatic hybrids and by linkage analysis of the 171 individuals of the PiGMaP reference families. A complete linkage map of 147 cM of this chromosome was then realized by integrating existing markers.
Numerous loci can be amplified by PARM-PCR on 300 sorted chromosomes in low-stringency conditions... more Numerous loci can be amplified by PARM-PCR on 300 sorted chromosomes in low-stringency conditions (annealing at 30~ during the two first cycles) to produce a probe that can be used in FISH painting experiments. We demonstrate that, depending on the primer chosen for the amplification, patterns of different quality can be obtained. In order to design a primer that allows amplification of coding sequences, we have shown that motifs of at least seven glutamic acid repeats (GAG or GAA codons) are present in human proteins more frequently than expected. Moreover, these repeats do not correspond to triplet expansion and can be conserved between species. Using probes prepared from sorted chromosomes with (GAG)7 primer, we were able to achieve homologous FISH painting on human, porcine, ovine, and bovine species, and bidirectional heterologous FISH painting between human and porcine species. As an example, using probes for human Chromosome (Chr) 19 and porcine Chrs 1 and 6, we clearly defined the regional homologies existing between those chromosomes.
We present here a new PCR-based technique that allows the production of several micrograms of DNA... more We present here a new PCR-based technique that allows the production of several micrograms of DNA from only 300 flow-sorted chromosomes. During the first two PCR cycles, the annealing temperature is decreased to 30 degrees C, and numerous random loci are amplified under nonspecific conditions. As demonstrated here for pig chromosomes 1 and 18, the PCR products may be used to identify the chromosomal content of the flow-karyotype peaks of any species by fluorescence in situ hybridization.
Flow cytogenetic is widely used since 1975, and essentially contributes to caryotype analysis and... more Flow cytogenetic is widely used since 1975, and essentially contributes to caryotype analysis and chromosome sorting. The principles of experimentation and its possibilities and limitations are now well known. Recently several new technologies have appeared. What attitude should the cytometrist adopt regarding PCR, microdissection of chromosomes, in situ hybridization, slitscan flow cytometry or image analysis? chromosomes / karyolype / cylomelry / genelies * Correspondence and reprints. ** Correspondence concerning either the applications of FCM to large genome studies or the contribution of PCR to the renewal of flow cytogenetics.
The extent and distribution of conserved chromosomal segments between pig and cattle chromosomes ... more The extent and distribution of conserved chromosomal segments between pig and cattle chromosomes were established using hybridization of porcine chromosome painting probes on bovine metaphases. A total of 44 segments of conserved synteny were identified, resulting in a nearly complete coverage of the bovine karyotype. This study provides new data on chromosome evolution of mammals.
Para localizar genes candidatos, QTLs, o para usar la selección asistida par marcadores se necesi... more Para localizar genes candidatos, QTLs, o para usar la selección asistida par marcadores se necesitan mapas génicos integrados y desarrollados. Como la cartografía génica humana está significativamente más avanzada, un cariotipo comparativo entre la especie humana y la porcina, que indicara la localización y extensión de las regiones de homología permitiría obtener beneficios del mapa humano. Para determinar las correspondencias entre segmentos cromosómicos homólogos humanos y porcinos, utilizamos sondas para coloración e hibridación in situ de cromosomas completos de ambas especies, en experimentos bidireccionales de hibridación heteróloga. Esta estrategia permite determinar las homologias segmento a segmento entre los cromosomas de ambas especies. Las sondas para coloración e hibridación in situ específica de cromosomas de ambas especies fueron obtenidas en todos las casos excepto en uno por DOP-POR (sondas comerciales Cambio) o amplificación PARM-PCR de cromosom as seleccionados e...
Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology, 1999
A comparative karyotype of rat (Rattus norvegicus) and mouse (Mus musculus) based on chromosome G... more A comparative karyotype of rat (Rattus norvegicus) and mouse (Mus musculus) based on chromosome G-banding morphology, heterologous chromosome painting results and available gene mapping data is proposed. Whole chromosome painting probes from both species were generated by PARM-PCR amplification of flow sorted chromosomes. Bidirectional chromosome painting identifies 36 segments of syntenic homology and allows us to propose a nearly complete comparative karyotype of mouse and rat (except for RNO 13 p and RNO 19 p12-13). Seven segments completely covered the RNO chromosomes 3, 5, 8, 11, 12, 15 and 18. Eight segments completely covered the MMU chromosomes 3, 4, 6, 7, 9, 12, 18 and 19. The RNO chromosomes 5, 8, 18 show complete homology with the MMU chromosomes 4, 9 and 18, respectively. Bidirectional hybridization results clearly assign 16 segments to subchromosomal regions in both species. Interpretation of the results allows subchromosomal assignment of all the remaining segments apa...
Recurrent allelic losses on chromosome 22q have been reported in colorectal cancer, distal to the... more Recurrent allelic losses on chromosome 22q have been reported in colorectal cancer, distal to the NF2 gene, suggesting that another tumor suppressor gene might be involved. We report here the typing of 256 sporadic colorectal tumors and 18 colonic cancer cell lines using a set of chromosome 22 polymorphisms, ranging from 20 to 45. A panel of somatic cell hybrids, that allows to distinguish 11 bins in the 22q13 region, was used to localize 19 of the 45 selected markers and the putative tumor suppressor gene BZRP. Allelic-loss was observed in 43% of tumors. The minimal region of deletion that could be determined, telomeric to locus D22S270, refines significantly the position of the gene. The localization of the BZRP gene in this region led to a systematic screening for somatic point mutation. Direct sequencing of its coding sequence in 36 tumors hemizygous for chromosome 22 allowed the identification of three polymorphisms but failed to detect somatic mutation.
A subset of genetic alterations distinguishes two groups of colon cancers. In the first group ins... more A subset of genetic alterations distinguishes two groups of colon cancers. In the first group instability of microsatellite loci due to a defective DNA mismatch repair system is observed. The second group is characterized by recurrent losses of chromosome regions, frequently associated with hyperploidization. We have developed a technique which enables a fine description of allelic losses in this second group of tumours. The typing of 278 loci in 47 hyperploid colon cancers has provided information for an average of 160 loci per tumour. The high frequency of allelic losses on chromosomes 17, 18 and 5 was confirmed thus validating our methodological approach. Several additional chromosome segments were observed lost in over 40% of the cases, suggesting that tumour suppressor genes may map within these regions. Further technical development should contribute to the identification of these genes.
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Papers by Annette Schmitz