- Email: [email protected]
Prospective validation of a prognostic gene expression signature and identification of EGFR as a drug target in uveal melanoma
New Biotechnology · Volume 27S · April 2010
POSTERS ABSTRACTS 2
However, increasing evidence challenges the model that bipolar disorder and schizophrenia would be independent syndromes. We performed meta-analysis of gene expression microarray data obtained from post mortem prefrontal cortex of bipolar disorder and schizophrenia patients and matched controls from independent experiments. Comprehensive integration of microarray data has been carried out on the single probe level based on the sequence of each probe assayed on the microarrays. A number of computational techniques have been utilized to carefully check the quality of the data and to infer and interpret the expression differences between the investigated conditions. Additionally, several techniques, including a novel Bayesian algorithm, have been used to cluster the patients according to their gene expression profile. We found 535 and 213 genes specifically altered in bipolar disorder and schizophrenia, respectively. Of these, only 30 genes were shared between the syndromes. In addition, we identified genes of the transcriptional and post-transcriptional machineries altered in BD and genes of the development changed in SZ, suggesting that these functions can be distinctive biological markers of the two conditions. However, we failed to cluster the samples according to the diagnosis, probably due to the complex anatomical architecture of the prefrontal cortex and the resolution levels allowed by the microarray technology. Our findings suggest that, if SZ and BD share a common genetic pattern, the interaction with the environment and consequently the modulation of the gene expression can lead to different clinical features. The current classification of BD and SZ can be thus considered not only a useful model in the clinical practice, but it underlies real biological entities. doi:10.1016/j.nbt.2010.01.183
a robust method for identifying regulatory elements in human chromatin, on NimbleGen 385K tiling microarrays (9.6 Mb) in a megakaryocytic (MK) and erythrocytic cell line. In the MK cell line, we found 259 FAIRE peaks in 55 of 61 tested association loci. The peaks were prevalently located in intergenic and intronic genomic regions (96%), and within 20 kb to the closest transcription start site (64%). Our experiments showed evidence of cell type specificity (P = 0.013), when comparing the number of FAIRE peaks in exclusively expressed genes in the megakaryocytic, erythroid, and monocytic lineages. At a genetic locus associated with mean platelet volume and function, we found megakaryocyte-specific FAIRE enrichment at the region of the lead SNP. This finding provides a first step in shedding light on a possible genetic mechanism. Investigating the sequence variation underlying nucleosomedepleted regions could guide the search for functionally relevant SNPs. In vitro assays could confirm the presence of regulatory elements, and unravel its potentially altered function depending on genotype. doi:10.1016/j.nbt.2010.01.184
[P2.22] Prospective validation of a prognostic gene expression signature and identification of EGFR as a drug target in uveal melanoma V. Mirisola 1,∗ , P. Perri 1 , S. Coupland 2 , C. Mosci 1 , M. Truini 1 , U. Pfeffer 1 1
2
[P2.21] Elucidating the chromatin architecture of loci associated with blood traits and coronary artery disease D.S. Paul 1,∗ , N. Soranzo 1,2 , W.H. Ouwehand 1,3 , P. Deloukas 1 1
Wellcome Trust Sanger Institute, UK King’s College London, UK 3 University of Cambridge and National Health Service Blood and Transplant, UK 2
Genome-wide association studies (GWAS) have identified common genetic variants associated with common diseases, including coronary artery disease (CAD) and its main complication, myocardial infarction (MI). In parallel, GWAS on quantitative haematological traits, such as the count and volume of blood cells, have further elucidated through which biological process such disease-associated loci are likely to act. However, in most of these identified loci, which have usually modest effects, the causative variants remain unknown. Causal variants are very likely to impact regulation of gene expression. Nucleosome depletion is a common marker for active regulatory elements. Mapping accessible chromatin regions at loci associated with CAD/MI and relevant quantitative traits can thus aid identification of functional variants. We performed FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements),
S64
www.elsevier.com/locate/nbt
National Cancer Research Institute, Italy University of Liverpool, UK
Intraocular melanomas represent 5% of all melanomas and about 30% of patients develop metastases, usually in the liver, during the first five years after diagnosis. Uveal melanoma prognostic factors are histopathological and cytogenetic features. Onken et al. (Cancer Res 2004) showed that gene expression profiling identifies two distinct molecular classes of uveal melanoma with low and high risk of metastasis. We describe here the prospective validation of the classifier and the identification of therapy targets through mRNA and miRNA expression profiling. Expression profiling was performed on 31 samples using Affymetrix HGUPlus2 arrays. Microarrays for miRNA screening were produced using the Exiqon library version 10.0. miRNA analyses were carried out on 28 samples. Differentially expressed miRNA and mRNAs were validated with qRT-PCR. Unsupervised hierarchical clustering of the samples using the classifier genes proposed by Onken et al. yielded three main clusters one of which contained all the six samples derived from tumors that developed metastases after a mean follow-up of two years. However, distance of the clusters was very limited indicating that the discrimination is not robust. A further up-date of the follow-up will be shown at the conference. mRNA expression analysis identified highly differential expression of several drug targets among which EGFR, a target of specific kinase inhibitors and therapeutic antibodies. We found a significant inverse correlation between the expression miR-128 and
New Biotechnology · Volume 27S · April 2010
its target, EGFR (see figure). Chromosome 7 polysomy also contributes to the overexpression of the receptor.
POSTERS ABSTRACTS 2
in GAPDS. Presumably, the deficiency in GAPDS could be due to the absence of some component of the FS that is required for binding GAPDS. The described results can help in finding genes that are responsible for the development of DFS. The work was supported by the Russian Foundation of Basic Research (grant no. 09-04-01122 and grant no. 09-04-92740NNIOM a), RECESS (DFG). doi:10.1016/j.nbt.2010.01.186
[P2.24] Detailed analysis of chromosome rearrangements in patients with autism and mental retardation Figure 1. Our study indicates that the prognostic signature developed by Onken et al. for the assignment of uveal melanoma patients to high or low risk groups might resist in the prospective setting. Moreover, we propose that patients with tumors that express high levels of EGFR mRNA and low levels of miR-128 could be eligible for treatment with anti-EGFR antibodies. doi:10.1016/j.nbt.2010.01.185
[P2.23] Deficiency in the glycolytic enzyme GAPDS in sperms with dysplasia of fibrous sheath Y.L. Elkina ∗ , E.E. Bragina, E.V. Schmalhausen Moscow State University, Russian Federation
The fibrous sheath (FS) is a unique cytoskeletal structure surrounding the axoneme and outer dense fibers of the sperm flagellum. Nearly half of FS protein isolated from mouse sperm is AKAP4 (A Kinase Anchoring Protein). Besides, FS contains several glycolytic enzymes including sperm-specific glyceraldehyde-3phosphate dehydrogenase (GAPDS). GAPDS is important for the sperm motility. Dysplasia of the fibrous sheath (DFS) is a defect of sperms that was described as ‘stump defect’ of the sperm flagella. This anomaly is observed in spermatozoa of severe asthenozoospermic patients and characterized by morphologically abnormal flagella. DFS has a family character, this suggesting a genetic nature of the disorder. Baccetti et al. demonstrated a partial deletion in AKAP4 and AKAP3 genes and the absence of the protein AKAP4 in one of the five patients with DFS. These results suggest a possible multigene nature of DFS. However, no more proteins associated with DFS have been revealed. Considering that GAPDS is associated with FS, it was assumed that defects in the FS formation might affect the interaction of GAPDS with FS proteins. GAPDS content and enzymatic activity was investigated in sperm samples of five DFS patients in comparison with normal sperms. It was shown that the relative intensity of GAPDS bands in DFS sperm samples constituted 9.3 ± 6.02% compared to the control, this being in agreement with the total dehydrogenase activity determined in the same samples (9.6 ± 5.8%). A conclusion was made that DFS sperms are deficient
Z. Sedlacek 1,2,∗ , M. Vlckova 1,2 , M. Hancarova 1,2 , J. Drabova 1,2 , Z. Zmitkova 1,2 , P. Hedvicakova 1,2 1
2
Charles University, Czech Republic University Hospital Motol, Czech Republic
A substantial fraction of cases of autism and/or mental retardation have been associated with chromosome rearrangements. These are either cytogenetically visible, or submicroscopic. Array comparative genome hybridisation (aCGH) is a powerful method for the analysis of both types of aberrations. In patients with defects identified using karyotyping, aCGH can specify the extent of the rearrangement and the location of breakpoints. In cytogenetically normal patients aCGH can reveal small gains and losses of chromosome material. In both aCGH can point to genes potentially causing the phenotypes, and identify the mechanisms of the rearrangements. We present the results of aCGH analysis in several patients with autism and/or mental retardation. Detailed analysis of a patient with a cytogenetically visible defect of 3q12–q21 identified a 20 Mb deletion harbouring 135 genes. Karyotypes of two other patients, one mildly and one severely affected, showed deletions of proximal 6q. aCGH analysis revealed nonoverlapping deletions of 15 Mb (45 genes) and 19 Mb (60 genes) in 6q11–q13 and 6q14–q16, respectively. These deletions divided proximal 6q into two parts, and could contribute to the identification of genes responsible for individual symptoms of the proximal 6q deletion syndrome. In another patient with an Xp deletion and a phenotype pointing to a possible GK and DMD defect aCGH revealed a 9 Mb deletion affecting 20 genes. Another patient was found using MLPA to have a duplication of the Xp subtelomere, but aCGH of his chromosome X revealed a small duplication of the MECP2 region likely to be responsible for his phenotype. Twins from another family were found to have a small deletion of 17q, most likely due to homozygosity of their mother for a common inversion of this region. Additional rearrangements have been detected in several other cases. Supported by INCORE, CHERISH and MZO00064203. doi:10.1016/j.nbt.2010.01.187
www.elsevier.com/locate/nbt S65
POSTERS ABSTRACTS 2
However, increasing evidence challenges the model that bipolar disorder and schizophrenia would be independent syndromes. We performed meta-analysis of gene expression microarray data obtained from post mortem prefrontal cortex of bipolar disorder and schizophrenia patients and matched controls from independent experiments. Comprehensive integration of microarray data has been carried out on the single probe level based on the sequence of each probe assayed on the microarrays. A number of computational techniques have been utilized to carefully check the quality of the data and to infer and interpret the expression differences between the investigated conditions. Additionally, several techniques, including a novel Bayesian algorithm, have been used to cluster the patients according to their gene expression profile. We found 535 and 213 genes specifically altered in bipolar disorder and schizophrenia, respectively. Of these, only 30 genes were shared between the syndromes. In addition, we identified genes of the transcriptional and post-transcriptional machineries altered in BD and genes of the development changed in SZ, suggesting that these functions can be distinctive biological markers of the two conditions. However, we failed to cluster the samples according to the diagnosis, probably due to the complex anatomical architecture of the prefrontal cortex and the resolution levels allowed by the microarray technology. Our findings suggest that, if SZ and BD share a common genetic pattern, the interaction with the environment and consequently the modulation of the gene expression can lead to different clinical features. The current classification of BD and SZ can be thus considered not only a useful model in the clinical practice, but it underlies real biological entities. doi:10.1016/j.nbt.2010.01.183
a robust method for identifying regulatory elements in human chromatin, on NimbleGen 385K tiling microarrays (9.6 Mb) in a megakaryocytic (MK) and erythrocytic cell line. In the MK cell line, we found 259 FAIRE peaks in 55 of 61 tested association loci. The peaks were prevalently located in intergenic and intronic genomic regions (96%), and within 20 kb to the closest transcription start site (64%). Our experiments showed evidence of cell type specificity (P = 0.013), when comparing the number of FAIRE peaks in exclusively expressed genes in the megakaryocytic, erythroid, and monocytic lineages. At a genetic locus associated with mean platelet volume and function, we found megakaryocyte-specific FAIRE enrichment at the region of the lead SNP. This finding provides a first step in shedding light on a possible genetic mechanism. Investigating the sequence variation underlying nucleosomedepleted regions could guide the search for functionally relevant SNPs. In vitro assays could confirm the presence of regulatory elements, and unravel its potentially altered function depending on genotype. doi:10.1016/j.nbt.2010.01.184
[P2.22] Prospective validation of a prognostic gene expression signature and identification of EGFR as a drug target in uveal melanoma V. Mirisola 1,∗ , P. Perri 1 , S. Coupland 2 , C. Mosci 1 , M. Truini 1 , U. Pfeffer 1 1
2
[P2.21] Elucidating the chromatin architecture of loci associated with blood traits and coronary artery disease D.S. Paul 1,∗ , N. Soranzo 1,2 , W.H. Ouwehand 1,3 , P. Deloukas 1 1
Wellcome Trust Sanger Institute, UK King’s College London, UK 3 University of Cambridge and National Health Service Blood and Transplant, UK 2
Genome-wide association studies (GWAS) have identified common genetic variants associated with common diseases, including coronary artery disease (CAD) and its main complication, myocardial infarction (MI). In parallel, GWAS on quantitative haematological traits, such as the count and volume of blood cells, have further elucidated through which biological process such disease-associated loci are likely to act. However, in most of these identified loci, which have usually modest effects, the causative variants remain unknown. Causal variants are very likely to impact regulation of gene expression. Nucleosome depletion is a common marker for active regulatory elements. Mapping accessible chromatin regions at loci associated with CAD/MI and relevant quantitative traits can thus aid identification of functional variants. We performed FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements),
S64
www.elsevier.com/locate/nbt
National Cancer Research Institute, Italy University of Liverpool, UK
Intraocular melanomas represent 5% of all melanomas and about 30% of patients develop metastases, usually in the liver, during the first five years after diagnosis. Uveal melanoma prognostic factors are histopathological and cytogenetic features. Onken et al. (Cancer Res 2004) showed that gene expression profiling identifies two distinct molecular classes of uveal melanoma with low and high risk of metastasis. We describe here the prospective validation of the classifier and the identification of therapy targets through mRNA and miRNA expression profiling. Expression profiling was performed on 31 samples using Affymetrix HGUPlus2 arrays. Microarrays for miRNA screening were produced using the Exiqon library version 10.0. miRNA analyses were carried out on 28 samples. Differentially expressed miRNA and mRNAs were validated with qRT-PCR. Unsupervised hierarchical clustering of the samples using the classifier genes proposed by Onken et al. yielded three main clusters one of which contained all the six samples derived from tumors that developed metastases after a mean follow-up of two years. However, distance of the clusters was very limited indicating that the discrimination is not robust. A further up-date of the follow-up will be shown at the conference. mRNA expression analysis identified highly differential expression of several drug targets among which EGFR, a target of specific kinase inhibitors and therapeutic antibodies. We found a significant inverse correlation between the expression miR-128 and
New Biotechnology · Volume 27S · April 2010
its target, EGFR (see figure). Chromosome 7 polysomy also contributes to the overexpression of the receptor.
POSTERS ABSTRACTS 2
in GAPDS. Presumably, the deficiency in GAPDS could be due to the absence of some component of the FS that is required for binding GAPDS. The described results can help in finding genes that are responsible for the development of DFS. The work was supported by the Russian Foundation of Basic Research (grant no. 09-04-01122 and grant no. 09-04-92740NNIOM a), RECESS (DFG). doi:10.1016/j.nbt.2010.01.186
[P2.24] Detailed analysis of chromosome rearrangements in patients with autism and mental retardation Figure 1. Our study indicates that the prognostic signature developed by Onken et al. for the assignment of uveal melanoma patients to high or low risk groups might resist in the prospective setting. Moreover, we propose that patients with tumors that express high levels of EGFR mRNA and low levels of miR-128 could be eligible for treatment with anti-EGFR antibodies. doi:10.1016/j.nbt.2010.01.185
[P2.23] Deficiency in the glycolytic enzyme GAPDS in sperms with dysplasia of fibrous sheath Y.L. Elkina ∗ , E.E. Bragina, E.V. Schmalhausen Moscow State University, Russian Federation
The fibrous sheath (FS) is a unique cytoskeletal structure surrounding the axoneme and outer dense fibers of the sperm flagellum. Nearly half of FS protein isolated from mouse sperm is AKAP4 (A Kinase Anchoring Protein). Besides, FS contains several glycolytic enzymes including sperm-specific glyceraldehyde-3phosphate dehydrogenase (GAPDS). GAPDS is important for the sperm motility. Dysplasia of the fibrous sheath (DFS) is a defect of sperms that was described as ‘stump defect’ of the sperm flagella. This anomaly is observed in spermatozoa of severe asthenozoospermic patients and characterized by morphologically abnormal flagella. DFS has a family character, this suggesting a genetic nature of the disorder. Baccetti et al. demonstrated a partial deletion in AKAP4 and AKAP3 genes and the absence of the protein AKAP4 in one of the five patients with DFS. These results suggest a possible multigene nature of DFS. However, no more proteins associated with DFS have been revealed. Considering that GAPDS is associated with FS, it was assumed that defects in the FS formation might affect the interaction of GAPDS with FS proteins. GAPDS content and enzymatic activity was investigated in sperm samples of five DFS patients in comparison with normal sperms. It was shown that the relative intensity of GAPDS bands in DFS sperm samples constituted 9.3 ± 6.02% compared to the control, this being in agreement with the total dehydrogenase activity determined in the same samples (9.6 ± 5.8%). A conclusion was made that DFS sperms are deficient
Z. Sedlacek 1,2,∗ , M. Vlckova 1,2 , M. Hancarova 1,2 , J. Drabova 1,2 , Z. Zmitkova 1,2 , P. Hedvicakova 1,2 1
2
Charles University, Czech Republic University Hospital Motol, Czech Republic
A substantial fraction of cases of autism and/or mental retardation have been associated with chromosome rearrangements. These are either cytogenetically visible, or submicroscopic. Array comparative genome hybridisation (aCGH) is a powerful method for the analysis of both types of aberrations. In patients with defects identified using karyotyping, aCGH can specify the extent of the rearrangement and the location of breakpoints. In cytogenetically normal patients aCGH can reveal small gains and losses of chromosome material. In both aCGH can point to genes potentially causing the phenotypes, and identify the mechanisms of the rearrangements. We present the results of aCGH analysis in several patients with autism and/or mental retardation. Detailed analysis of a patient with a cytogenetically visible defect of 3q12–q21 identified a 20 Mb deletion harbouring 135 genes. Karyotypes of two other patients, one mildly and one severely affected, showed deletions of proximal 6q. aCGH analysis revealed nonoverlapping deletions of 15 Mb (45 genes) and 19 Mb (60 genes) in 6q11–q13 and 6q14–q16, respectively. These deletions divided proximal 6q into two parts, and could contribute to the identification of genes responsible for individual symptoms of the proximal 6q deletion syndrome. In another patient with an Xp deletion and a phenotype pointing to a possible GK and DMD defect aCGH revealed a 9 Mb deletion affecting 20 genes. Another patient was found using MLPA to have a duplication of the Xp subtelomere, but aCGH of his chromosome X revealed a small duplication of the MECP2 region likely to be responsible for his phenotype. Twins from another family were found to have a small deletion of 17q, most likely due to homozygosity of their mother for a common inversion of this region. Additional rearrangements have been detected in several other cases. Supported by INCORE, CHERISH and MZO00064203. doi:10.1016/j.nbt.2010.01.187
www.elsevier.com/locate/nbt S65