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P34: Analysing DNA copy number changes in lung carcinomas and adjacent bronchial epithelium by means of high-resolution array CGH
500
A. Muradyan et al. / European Journal of Medical Genetics 48 (2005) 477–536
NF2, schwannomatosis and sporadic schwannomas to identify putative gene(s) involved in the development of these disorders. Our chromosome 22 array covers 34.7 Mb of 22q with an average resolution of 75 kb. It contains a set of 460 clones derived from chromosome 22, together with chromosome X and autosomal control clones. Profiling of schwannomas revealed a predominant pattern of monosomy 22, followed by terminal and overlapping interstitial deletions encompassing the NF2 gene. We also identified two regions with genomic imbalances that were located within the schwannomatosis candidate region, not affecting the NF2 locus. The first region displayed a regional gain, which was detected both in tumor- and blood-derived DNA from one patient. Analysis of this locus revealed the presence of two candidate genes; TOP3B and PPM1F. The TOP3B gene encodes a DNA topoisomerase that plays a role in recombination and maintenance of genome stability. The protein encoded by PPM1F gene is a Ser/Thr protein phosphatases that functions as a negative regulator of cell stress response pathways. The second candidate region mapped to the immunoglobulin lambda (IGL) locus. Two overlapping deletions, in two unrelated patients, with telomeric breakpoints overlapping with IGL-joining and IGL-constant gene clusters were identified. The role of these imbalances in relation to schwannoma development is an object for further studies. Furthermore, we performed a mutation analysis of the CABIN1 gene that is encompassed within the locus displaying the highest LOD score upon linkage analysis of schwannomatosis families. Missense mutations that were specific for schwannomatosis and NF2 patients were identified, which make this gene a plausible candidate for schwannoma development. Finally, we have developed a PCR product-based, repeat free and non-redundant approach for the construction of high-resolution arrays. Using this approach, we are constructing an array which covers the entire 9 Mb schwannomatosis candidate region (including the above mentioned candidate loci) on 22q11 with ~5000 measurement points. Profiling of schwannomas with this array will enable us to detect minimal 22q aberrations, which may lead towards the identification of a schwannoma/ schwannomatosis gene.
P34: Analysing DNA copy number changes in lung carcinomas and adjacent bronchial epithelium by means of high-resolution array CGH Artur Muradyan a, Helmut Popper b, Iris Halbwedl b, Wei Chen a, Claus Hultschig a, Hilger Ropers a, Reinhard Ullmann a a Max Planck Institute for Molecular Genetics, Germany b Institute of Pathology, Graz, Austria Lung carcinomas develop through the consecutive accumulation of genetic aberrations. Previously, loss of heterozygosity studies have identified chromosomal aberrations in morphological inconspicuous epithelium adjacent to the tumor. We have used laser-microdissection in combination with high-resolution array CGH in order to investigate the possible occurrence and quality of chromosomal aberrations in lung carcinomas and its neighbouring bronchial epithelium. Array CGH was performed on a 36k BAC DNA array consisting of the 1 Mb Sanger set (Fiegler et al., 2003) and the human 32k Re-Array set (Osoegawa et al., 2001; Krzywinski et al., 2004). BAC DNA was amplified by linker-adapter PCR and spotted on epoxycoated slides. Tumor and epithelial DNA was isolated from about 500 cells, which were lasermicrodissected from 10 μm sections of frozen tissue. Amplification was performed by means of a commercial kit (GenomePlex; Rubicon). Feasibility of the amplification procedure was verified by analysing tumor cell lines with known aberrations and by the use of DNA copy
P. Chiarappa et al. / European Journal of Medical Genetics 48 (2005) 477–536
501
number polymorphisms as internal controls. Array CGH data were analysed using our software package CGHPRO. Based on a comparison of median absolute deviation (MADs), no additional experimental noise was introduced by the amplification procedure. Up to now we have successfully analysed eight (out of eight tested) pairs of tumor and associated epithelium.
P35: Frequent loss of chromosome 1p and 22q loci in insulinomas Yvonne M.H. Jonkers a, Sandra M.H. Claessen a, Ton Feuth b, Ad Geurts van Kessel b, Frans C.S. Ramaekers a, Joris A. Veltman b, Ernst-Jan M. Speel a a Department of Molecular Cell Biology, University of Maastricht, Maastricht, The Netherlands b Department of Human Genetics, University Medical Center Nijmegen, The Netherlands Endocrine pancreatic tumors (EPTs) comprise a highly heterogeneous group of tumors with different clinical behavior and genetic make up. Insulinomas represent the predominant syndromic subtype of EPTs. Our previous molecular studies have shown that gain of chromosome 9q is an early event in tumor development, and that a clear distinction between benign and malignant insulinomas can be made on basis of chromosomal instability (Cancer Res. 2001; 61(13): 5186–92, Endocrine Related Cancer 2005; 12(2): 435–47). In order to quantify and further narrow down the identified regions of interest, we investigated 23 insulinomas by array CGH (3.7 k genomic BAC array). This high resolution approach moreover allows the detection of small, previously unidentified copy number changes as well as the analysis of chromosomal regions 1p32-pter, 16p, 19 and 22. These regions are usually excluded from conventional CGH analyses, because they may show DNA gains in negative control hybridizations. In addition to previously identified alterations in large chromosomal regions, including 9q gain in 65% of the cases, novel copy number changes were identified in many small genomic regions. Loss of chromosome 1p36 (65%) and 22q13.3 (52%) were most frequently detected. In addition, two malignant insulinomas harbored a single amplification, i.e. one at chromosome 4p15 and one at 9q22. If patients with and without metastatic disease were compared with each other, genomic complexity was strongly enhanced in tumors of the first group, as determined by the average number of chromosomal alterations (regions > 10 MB) and loss of chromosomal ends (P < 0.0001 and 0.004, respectively). Our study revealed that the most optimal predictors of malignancy are, amongst others, more than 15 chromosomal alterations (> 10 Mb), gain of chromosome 7p, and loss of chromosomes 10q and 22q. The presented findings indicate that chromosomal instability is a very powerful indicator for the development of metastatic disease in patients with sporadic insulinoma and that the newly identified chromosomal targets are likely to contain genes important in insulinoma tumorigenesis.
P36: Study of genetic pattern in BRCAx hereditary breast tumors by CGH Patrizia Chiarappa, Stefania Tommasi, Anita Mangia, Michele Bruno, Alessandro Monaco, Annalisa Chiriatti, Anna Trentadue, Francesco Schittulli, Angelo Paradiso NCI Bari, Clinical Experimental Oncology Laboratory Hereditary breast cancer has been partly attributed to germline mutations in the BRCA1 and BRCA2 genes that are deleterious for these proteins activity. Thus far, very little is known about the genomic alterations of hereditary breast cancers that are not BRCA1/2-associated
A. Muradyan et al. / European Journal of Medical Genetics 48 (2005) 477–536
NF2, schwannomatosis and sporadic schwannomas to identify putative gene(s) involved in the development of these disorders. Our chromosome 22 array covers 34.7 Mb of 22q with an average resolution of 75 kb. It contains a set of 460 clones derived from chromosome 22, together with chromosome X and autosomal control clones. Profiling of schwannomas revealed a predominant pattern of monosomy 22, followed by terminal and overlapping interstitial deletions encompassing the NF2 gene. We also identified two regions with genomic imbalances that were located within the schwannomatosis candidate region, not affecting the NF2 locus. The first region displayed a regional gain, which was detected both in tumor- and blood-derived DNA from one patient. Analysis of this locus revealed the presence of two candidate genes; TOP3B and PPM1F. The TOP3B gene encodes a DNA topoisomerase that plays a role in recombination and maintenance of genome stability. The protein encoded by PPM1F gene is a Ser/Thr protein phosphatases that functions as a negative regulator of cell stress response pathways. The second candidate region mapped to the immunoglobulin lambda (IGL) locus. Two overlapping deletions, in two unrelated patients, with telomeric breakpoints overlapping with IGL-joining and IGL-constant gene clusters were identified. The role of these imbalances in relation to schwannoma development is an object for further studies. Furthermore, we performed a mutation analysis of the CABIN1 gene that is encompassed within the locus displaying the highest LOD score upon linkage analysis of schwannomatosis families. Missense mutations that were specific for schwannomatosis and NF2 patients were identified, which make this gene a plausible candidate for schwannoma development. Finally, we have developed a PCR product-based, repeat free and non-redundant approach for the construction of high-resolution arrays. Using this approach, we are constructing an array which covers the entire 9 Mb schwannomatosis candidate region (including the above mentioned candidate loci) on 22q11 with ~5000 measurement points. Profiling of schwannomas with this array will enable us to detect minimal 22q aberrations, which may lead towards the identification of a schwannoma/ schwannomatosis gene.
P34: Analysing DNA copy number changes in lung carcinomas and adjacent bronchial epithelium by means of high-resolution array CGH Artur Muradyan a, Helmut Popper b, Iris Halbwedl b, Wei Chen a, Claus Hultschig a, Hilger Ropers a, Reinhard Ullmann a a Max Planck Institute for Molecular Genetics, Germany b Institute of Pathology, Graz, Austria Lung carcinomas develop through the consecutive accumulation of genetic aberrations. Previously, loss of heterozygosity studies have identified chromosomal aberrations in morphological inconspicuous epithelium adjacent to the tumor. We have used laser-microdissection in combination with high-resolution array CGH in order to investigate the possible occurrence and quality of chromosomal aberrations in lung carcinomas and its neighbouring bronchial epithelium. Array CGH was performed on a 36k BAC DNA array consisting of the 1 Mb Sanger set (Fiegler et al., 2003) and the human 32k Re-Array set (Osoegawa et al., 2001; Krzywinski et al., 2004). BAC DNA was amplified by linker-adapter PCR and spotted on epoxycoated slides. Tumor and epithelial DNA was isolated from about 500 cells, which were lasermicrodissected from 10 μm sections of frozen tissue. Amplification was performed by means of a commercial kit (GenomePlex; Rubicon). Feasibility of the amplification procedure was verified by analysing tumor cell lines with known aberrations and by the use of DNA copy
P. Chiarappa et al. / European Journal of Medical Genetics 48 (2005) 477–536
501
number polymorphisms as internal controls. Array CGH data were analysed using our software package CGHPRO. Based on a comparison of median absolute deviation (MADs), no additional experimental noise was introduced by the amplification procedure. Up to now we have successfully analysed eight (out of eight tested) pairs of tumor and associated epithelium.
P35: Frequent loss of chromosome 1p and 22q loci in insulinomas Yvonne M.H. Jonkers a, Sandra M.H. Claessen a, Ton Feuth b, Ad Geurts van Kessel b, Frans C.S. Ramaekers a, Joris A. Veltman b, Ernst-Jan M. Speel a a Department of Molecular Cell Biology, University of Maastricht, Maastricht, The Netherlands b Department of Human Genetics, University Medical Center Nijmegen, The Netherlands Endocrine pancreatic tumors (EPTs) comprise a highly heterogeneous group of tumors with different clinical behavior and genetic make up. Insulinomas represent the predominant syndromic subtype of EPTs. Our previous molecular studies have shown that gain of chromosome 9q is an early event in tumor development, and that a clear distinction between benign and malignant insulinomas can be made on basis of chromosomal instability (Cancer Res. 2001; 61(13): 5186–92, Endocrine Related Cancer 2005; 12(2): 435–47). In order to quantify and further narrow down the identified regions of interest, we investigated 23 insulinomas by array CGH (3.7 k genomic BAC array). This high resolution approach moreover allows the detection of small, previously unidentified copy number changes as well as the analysis of chromosomal regions 1p32-pter, 16p, 19 and 22. These regions are usually excluded from conventional CGH analyses, because they may show DNA gains in negative control hybridizations. In addition to previously identified alterations in large chromosomal regions, including 9q gain in 65% of the cases, novel copy number changes were identified in many small genomic regions. Loss of chromosome 1p36 (65%) and 22q13.3 (52%) were most frequently detected. In addition, two malignant insulinomas harbored a single amplification, i.e. one at chromosome 4p15 and one at 9q22. If patients with and without metastatic disease were compared with each other, genomic complexity was strongly enhanced in tumors of the first group, as determined by the average number of chromosomal alterations (regions > 10 MB) and loss of chromosomal ends (P < 0.0001 and 0.004, respectively). Our study revealed that the most optimal predictors of malignancy are, amongst others, more than 15 chromosomal alterations (> 10 Mb), gain of chromosome 7p, and loss of chromosomes 10q and 22q. The presented findings indicate that chromosomal instability is a very powerful indicator for the development of metastatic disease in patients with sporadic insulinoma and that the newly identified chromosomal targets are likely to contain genes important in insulinoma tumorigenesis.
P36: Study of genetic pattern in BRCAx hereditary breast tumors by CGH Patrizia Chiarappa, Stefania Tommasi, Anita Mangia, Michele Bruno, Alessandro Monaco, Annalisa Chiriatti, Anna Trentadue, Francesco Schittulli, Angelo Paradiso NCI Bari, Clinical Experimental Oncology Laboratory Hereditary breast cancer has been partly attributed to germline mutations in the BRCA1 and BRCA2 genes that are deleterious for these proteins activity. Thus far, very little is known about the genomic alterations of hereditary breast cancers that are not BRCA1/2-associated