Scientific Poster Presentations

s of the 1 MC-GARD Meeting, 3–5 May 2007: Scientific Poster Presentations 130 inflammatory, oxidative stress and DNA damage responses. Microarray analysis could be an effective tool for identifying genes differently involved in PPN and for better understanding of the pathogenetic mechanisms of PPN. P3 A DELETION IN CHROMOSOME 7Q21.13 DETECTED IN A PATIENT PRESENTING WITH DYSMORPHIC FACIAL FEATURES, REDUNDANT SKIN ON HANDS AND FEET, AND ANTENATAL CARDIAC ABNORMALITIES Joo Wook Ahn; Sally Walsh; Neil Dixon; Charu Despande; Kathy Mann; Caroline Ogilvie. Guy's and St Thomas' NHS Foundation Trust, London, UK E-mail: [email protected] A male patient (aged 15 months) was referred for molecular karyotyping using a BAC array of ~1Mb resolution. G-banded chromosome analysis and MLPA for subtelomeric abnormalities had previously detected no abnormalities. Antenatal ultrasound had previously detected bilateral superior vena cava and a variant Dandy-Walker malformation. However, a postnatal echocardiogram detected no major cardiac abnormalities. The patient has unusual, distinctive facial features and redundant skin on the dorsum of his hands and feet, as well as brachydactyly. Array analysis detected a single probe deletion (RP5-998H4) which maps to 7q21.13 (88.6 – 88.7Mb). Adjacent clones indicating normal copy number delineated the maximum deletion interval as 87.9 – 89.3Mb. This patient therefore has monosomy for between 0.1 and 1.4Mb of material. This region contains a single gene (ZNF804B) with a putative zinc finger domain, but currently of unknown function. In situ hybridisation using the same BAC confirmed this finding. There are no previously reported copy number variants corresponding to this region. The results of inheritance studies and higher resolution array analysis using oligo arrays will be reported. If this finding represents a de novo abnormality, it seems likely that it is contributing to the patient's phenotype. As this was the only abnormality detected by the 1Mb BAC array, other large scale chromosome imbalance is unlikely. The implications of this finding in light of the inheritance and higher resolution investigations will be discussed. P4 AN EXPERIMENTAL LOOP DESIGN IMPROVES THE DETECTION OF CONSTITUTIONAL CHROMOSOMAL ABERRATIONS BY ARRAY CGH Joke Allemeersch; Steven W. Van Vooren; Femke Hannes; Joris Robert Vermeesch; Yves Moreau. VIB Microarray Facility, Belgium E-mail: [email protected] Introduction. Comparative genomic hybridization microarrays (array CGH or molecular karyotyping) for the detection of constitutional chromosomal aberrations is the application of microarray technology that is coming fastest into routine clinical application. Through genotype-phenotype association, it is also an important technique towards the discovery of disease causing genes and genowewide functional annotation in human. When using a two-channel microarray of genomic DNA probes for array CGH, the basic setup consists in hybridizing a patient sample against a normal reference sample and detecting copy number variations through the deviation of fluorescent signal intensity between patient and normal reference. Two major disadvantages of this setup are (1) the use of half of the resources to measure a (little informative) reference sample and (2) the possibility that deviating signals are caused by benign copy number variation in the "normal" reference instead of a patient aberration. Results. We propose a new experimental loop design that compares three patients in three hybridizations (Patient 1 vs. Patient 3, Patient 3 vs. Patient 2, and Patient 2 vs. Patient 1). We develop and compare two statistical methods (linear models of log ratios and mixed models of absolute measurements). In an analysis of data from 27 patients seen at our genetics center, this new setup together with the linear model analysis significantly overcomes the limitations of the classical setup. Furthermore, we observed that the linear models of the log-ratios had a higher signal-to-noise ratio than the mixed models of the absolute intensities. Conclusions. The improvements of the loop design are important to guarantee a maximal efficiency of array CGH in a clinical setting and will therefore contribute to its quick adoption as a routine diagnostic tool. The method is implemented as a web application and is available at www.esat.kuleuven.be/loop. Abstracts of the 1 MC-GARD Meeting, 3–5 May 2007: Scientific Poster Presentations 131s of the 1 MC-GARD Meeting, 3–5 May 2007: Scientific Poster Presentations 131 P5 MOLECULAR CHARACTERIZATION OF THE 437 CODON OF PFDHPS IN GAMETOCYTES EMERGING AFTER TREATMENT WITH SULFADOXINE-PYRIMETHAMINE, AMODIAQUINE AND THE COMBINATION SP/AQ Makoah Nigel Aminake; Evehe Marie Solange Bebandoue. University of Etoug-Ebe, Cameroon E-mail: [email protected] The high mortality of Plasmodium falciparum is attributed to the emergence and the spread of resistant parasites. The transmission of the disease requires the presence of gametocytes in the peripheral blood. In this study, patients attending the district hospital of EtougEbe (Cameroon) with uncomplicated falciparum malaria in and who fulfilled the criteria as recommended by WHO, were randomly assigned to receive sulfadoxine/pyrimethamine plus placebo; amodiaquine plus placebo, or amodiaquine plus sulfadoxine/ pyrimethamine. Patients were followed up for 28 days and children whose blood contains gametocytes in absence of trophozoites during the follow up were considered for the molecular studies. The presence or absence of mutation was investigated by PCR-RFLP. 13.6 %( 36/260) of the patients recruited were gametocyte-positive during the study and the majority came from the group treated with SP (SP: 19/85; AQ: 8/89; SP/AQ: 9/86) p=0, 0208. We observe gametocyte during all the follow up in the group treated with SP, in the group treated with AQ, gametocytes were present only on day 3 and 7 and in the group, which received the combination, we observe gametocytes on days 3, 7 and 14. This study also revealed that most the gametocytepositive samples carry the mutated allele (dhps-437G), and the majority once again came from the group treated with SP. (SP: 53,3 % ; AQ : 13,3 % ; SP/AQ : 13 ,3 %) p=7,823x10. 8 . These results show that SP enhances the production of gametocyte, and could further contribute to the spread of the resistant allele. Amodiaquine could be a good candidate to reduce the transmission of malaria in this area. P6 GENOTYPING MICROARRAY FOR DIAGNOSIS IN 199 FAMILIES AFFECTED WITH AUTOSOMAL RECESSIVE RETINITIS PIGMENTOSA Almudena Avila-Fernandez; Elena Vallespin; Diego Cantalapiedra; Rosa Riveiro-Alvarez; Jana AguirreLamban; Ascension Gimenez; M. Jose Trujillo-Tiebas; Carmen Ayuso. Genetics Fundacion JImenez Diaz CIBERER, ISCIII, Madrid, Spain E-mail: [email protected] Introduction. Retinitis pigmentosa (RP), is a genetically heterogeneous disorder characterized by progressive loss of vision. The disease can be inherent as an autosomaldominant (adRP), autosomal-recessive (arRP), X-linked (xlRP), and rare mitochondrial and digenic forms. Although arRP is the most common form, it accounts for 40% of cases. Genetic heterogeneity is extensive, 24 genes have been identified (18 cloned, 6 mapped). Patients and Methods. 199 unrelated Spanish families affected with arRP were studied. The methodology consists of a genotyping microarray (Asperbio), for screening mutations in a number of genes associated with arRP: CERKL, CNGA1, CNGB1, MERKT, PDE6A, PDE6B, PNR, RDH12, RGR, PLBP1, SAG, TULP1, CRB1, RPE65, USH2A, USH3A. Results. We report 199 unrelated Spanish families, 78 of which had at least one mutated allele (39%). The allele frequencies were: CERKL 3,1% (12/398), CNGA1 1% (4/398), PDE6A 1,8% (7/398), PDE6B 1% (4/398), RGR 4,5% (18/398), RLBP1 0,5% (2/398), SAG 0,25% (1/398), CRB1 0,75% (3/398), USH2A 16,8% (67/398). Discussion. The preliminary mutation frequency rate found was: 121/398 (30%) of mutated alleles and 39% of mutated families. By analysing the results we have corrected our previous figures, eliminating the possible polymorphisms: 42 families (42/199) 21% with at least one mutation and 61/398 (15%) mutated alleles with the following allele frequencies, respectively: 3,1% (12/398), 1% (4/398), 1,8% (7/398), 0,5% (2/398), 0% (0/398), 0,5% (2/398), 0,25% (1/398), 0,75% (3/398), 7,5% (30/398). Conclusions. The genotyping microarray offers a fast and efficient method of screening for mutations in most recessive RP genes and should be the first step in the molecular diagnostic of families affected with arRP. The study has to be completed with: Family haplotype analysis and complete candidate gene screening. The differences found bring the need to do studies on a Abstracts of the 1 MC-GARD Meeting, 3–5 May 2007: Scientific Poster Presentations 132s of the 1 MC-GARD Meeting, 3–5 May 2007: Scientific Poster Presentations 132 control Spanish population, the purpose being to determine the possible pathological implications of changes described. P7 PRIORITIZATION OF CANDIDATE GENES BASED ON OVER-REPRESENTED FEATURES Roland Barriot; Léon-Charles Tranchevent; Peter Van Loo; Bert Coessens; Yves Moreau. SymBioSys / ESAT-SCD E-mail: [email protected] Array CGH technology allows to detect chromosomal rearrangements in the whole human genome and is a crucial step in identifying genes responsible for congenital diseases. However, given the size of such chromosomal events, researchers still face lists of hundreds of genes that may be causing the investigated disease. Accurate candidate genes prioritization is therefore of utmost importance to help researchers focus on the best candidates. We present improvements to gene prioritization accuracy in the Endeavour system [Aerts et al. 2006] based on feature enrichment profiles. The principle is to provide the system with a set of known disease-related genes (training set), which will be used to build several models that allow to rank the candidate genes based on their similarity to the training set. Enrichment profiles are built for the Gene Ontology annotations, the KEGG pathways, the protein domains (InterPro) and the EST localization (Ensembl ESTs). To measure the enrichment in a given feature, the hypergeometric distribution is used as a dissimilarity index [Barriot et al. 2004]. Then, the False Discovery Rate [Benjamini and Hochberg 1995] is applied to account for multiple testing. We performed cross validation (leave one out) on 29 human disease training sets by applying different FDR thresholds to prioritize the whole genome. Interestingly, the best performance was obtained for an FDR threshold of 50%. This can be explained by the fact that we use the enrichment score to rank the candidates (Fisher's omnibus meta analysis). Thus, keeping low similarity features in the enrichment profiles helps to better discriminate the candidates. We also stress out that the whole genome is prioritized, which includes genes causing the disease that are still unknown and that may rank higher than the left out gene. The accuracy obtained is thus a pessimistic estimate of the real performances of our method. P8 CHROMOSOMAL IMBALANCES IN ANAEMIC AND NON-ANAEMIC HEAD AND NECK SQUAMOUS CELL CARCINOMA (HNSCC) Verena Lucia Bauer; Herbert Braselmann; Axel Walch; Kristian Unger; Cordelia Langford; Michael Henke; Horst Zitzelsberger. GSF-National Research Center for Environment and Health, Institute of Molecular Radiobiology, Cytogenetics Group E-mail: [email protected] Chromosomal imbalances have been identified in 68 anaemic and 49 non-anaemic HNSCC by conventional CGH. Statistical analyses revealed significant differences between the two cohorts in the frequency and appearance of DNA gains on chromosomes 1p, 2q, 3q, 4q, 5q, 11q, 12q, 13q, 19p and of DNA losses on chromosomes 3p, 4p, 4q, 10p, 16q, 17p, 17q, 18q, 20q, and 21q. Additionally, chromosomal aberrations correlating with a poor prognosis have been identified for anaemic cases. Chromosomal gains on 14q, 16q and 18p correlate with a significantly reduced survival after radiotherapy in anaemic HNSCC while no chromosomal prognostic markers were observed in the non-anaemic cases. To identify candidate genes within these altered chromosomal regions in anaemic cases array CGH was performed. Array CGH detected gains on 14q12-13, 16q22 and 16q23-24 as well as on 18p11.2-11.3. This led to the identification of several candidate genes such as PAX9, FOXA1, TTF-1 and SSTR1 on 14q, HAS3, TRF2, CDH3, CES-2, ARC, E2F4, BCAR1, MAF, TUBB3, GAS8 and FANCA on 16q and VAPA, RAB31/12, KNTC2, TYMS, ADCYAP1 and CETN1 on 18p. Various BAC clones indicating gained regions on chromosome 16q have been used as FISH probes on paraffin-embedded tissue sections from one anaemic HNSCC case. FISH confirmed amplifications for BAC clones mapping TUBB3, GAS8 and FANCA. FANCA is of special interest since it belongs to the central part of the Fanconi's Anaemia (FA)/BRCA pathway in homology-directed DNA repair. Disruptions of genes in this pathway result in chromosome instability, cellular hypersensitivity to DNA cross-linking reagents and cancer progression. Thus, it is likely that alterations of the FA/BRCA pathway are involved in mechanisms leading to a poor prognosis of a subset of anaemic HNSCC. The generation of a gene specific BAC array for the FA pathway is on the way to enable the analyses of copy number changes of these genes in one experiment. Furthermore, alterations of candidate genes Abstracts of the 1 MC-GARD Meeting, 3–5 May 2007: Scientific Poster Presentations 133s of the 1 MC-GARD Meeting, 3–5 May 2007: Scientific Poster Presentations 133 have been verified using Multiplex Ligation-dependent Probe Amplification (MLPA). The results indicate a specific pattern of alteration in anaemic and non-anaemic HNSCC. The identification of genes involved in chromosomal gains on 14q, 16q and 18p may help to investigate mechanisms responsible for the poor prognosis of anaemic cancer patients in more detail. P9 DETECTION OF GENOMIC COPY NUMBER CHANGES IN PATIENTS WITH IDIOPATHIC MENTAL RETARDATION BY HIGHRESOLUTION X-ARRAY-CGH: FREQUENT INCREASED GENE DOSAGE OF KNOWN XLMR GENES Marijke Bauters; Guy Froyen; Hilde Van Esch; Joke Nevelsteen; Suzanna G. M. Frints; Joris Robert Vermeesch; Koen Devriendt; Jean-Pierre Fryns; Peter Marynen. Human Genome Laboratory, Dept. Human Genetics, VIB, K.U.Leuven, Belgium E-mail: {guy.froyen;joke.nevelsteen;marijke.bauters} @med.kuleuven.be A tiling X-chromosome-specific genomic array with a theoretical resolution of 80 kb was developed. We first validated the X-array with aberrations previously detected at low resolution in 5 MR patients. This allowed for delineation of the location and extent of the aberration at high resolution and subsequently, more precise genotype-phenotype analyses. Next, we screened a cohort of 108 patients with idiopathic MR consisting of 57 patients suspected of X-linked mental retardation (XLMR), 26 probands of brother pairs, and 25 sporadic patients. In this screened population, we identified 16 copy number changes in 15 patients (13.9%). These include 2 deletions and 14 duplications ranging from 0.2 2.7 Mb. The aberration most likely is associated with the phenotype in 5 patients (4.6%) based on absence in normal control individuals, de novo aberration, segregation with the disease in the family, involvement of a known or candidate MRX(S) gene, and/or skewed X-inactivation in carrier mothers. Presumed causal aberrations include 2 deletions and 3 duplications that contain known MRX(S) genes. In addition, nine novel apparent benign variants on the X chromosome are described. One interesting copy number change found in 3 cases is a 0.3 Mb MRX gene containing duplication that might act as a susceptibility factor for MR. Taken together, our data strongly suggest that not only deletions but also duplications on X might contribute to the phenotype more often than expected, supporting our increased gene dosage mechanism for deregulation of normal cognitive development, as reported for MECP2. P10 MOLECULAR CHARACTERISATION OF PAEDIATRIC AND ADULT GLIOMA CELLLINES Dorine A. Bax; Lynley Marshall; Nathalie Gaspar; Suzie Little; John Swansbury; Darren Hargrave; David Ellison; Andrew Pearson; Chris Jones. Section of Paediatric Oncology, Institute of Cancer Research, Sutton, UK E-mail: [email protected] Paediatric high-grade gliomas (HGG) have the highest mortality of all brain tumours, with 5 year survival rates of less than 20%. Although advances in our understanding of the molecular genetics of adult HGG are providing rationale for new targeted therapies, the corresponding data is lacking in children. Adult and paediatric HGG are histologically similar, but they appear to be distinct genetic entities. One factor hampering work in the paediatric setting is the relative lack of cell lines derived from paediatric HGG. It is currently unclear whether the well-established adult lines are representative of the underlying molecular genetics of childhood tumours. The aim of this project was to characterize 13 cell-lines, derived from paediatric lowgrade gliomas (LGG) and HGG and adult gliomas, in order to identify differences between paediatric and adult gliomagenesis. We have employed traditional karyotyping, copy number and SNP analysis on 500K Affymetrix arrays, oligonucleotide array CGH, expression profiling on U133 2.0Plus chips, and specific target gene/protein characterisation. Supervised analysis was able to generate a list of 169 genes that was able to discriminate between LGG and HGG cell lines, and between adult and paediatric cases. The paediatric HGG lines were found to harbour changes in DNA copy number (>70 chromosomes) including gains of 4p, 16p and 17q and losses of 3p, 6q and 11p. A key amplification at 12q14 was observed in one paediatric line (SF188), which led to overexpression of CDK4 and PIKE, and may provide insight into differential drug sensitivity in this model. Mutations in EGFR and PTEN, common in adults, were not found in the paediatric lines. Although this work is ongoing, the characterization of glioma cell-lines provides insight in the differences of Abstracts of the 1 MC-GARD Meeting, 3–5 May 2007: Scientific Poster Presentations 134s of the 1 MC-GARD Meeting, 3–5 May 2007: Scientific Poster Presentations 134 glioma development in adults and children, and it may aid in the development of new drugs for the treatment of paediatric HGG. P11 MOLECULAR PROFILING OF RADIATIONASSOCIATED BREAST TUMORS Linde M Braaf; van Beers.Erik H.; Petra M. Nederlof; Flora E. Van Leeuwen; Nicola S. Russell; Martin Van Vliet; Lodewijk F. A. Wessels; Laura J. Van't Veer; Annegien Broeks. The Netherlands cancer institute, Department of Experimental Therapy, Amsterdam, The Netherlands E-mail: [email protected] Introduction. Women who received radiation treatment for Hodgkin's Lymphoma (HL) have an increased risk of developing breast cancer. It has been estimated that approximately 90% of the breast carcinomas in these patients is a result of their radiation treatment, which makes this series extremely appropriate to determine a potentially radiation-associated genomic profile. Methods and Materials. We have used array-CGH and gene expression profiling (GEP) technology to assess the molecular changes in these radiation-associated breast tumors. For genomic profiling we used a human 3.5K BAC array and for GEP a 37K oligo microarray (NKICMF). DNA and RNA from breast tumors of HL patients (BfHL) and from breast tumor controls were subsequently hybridized to the arrays and statistical microarray analysis was performed. Results.With array-CGH we have analyzed the genomic profile of 20 BfHL cases and 22 breast tumor controls. Frequency plot analysis revealed several distinct chromosomal aberrant regions in the radiation-associated BfHL tumors compared to the control breast tumors. With GEP we have analyzed the RNA profile of 23 BfHL cases and 20 breast tumor controls. Hierarchical clustering of all samples was performed using 5031 significant oligo's, which resulted in a clustering of the so-called radiation-associated tumors apart from the breast tumor controls. Furthermore we performed supervised classification strategies using SAM and PAM and identified respectively differentially expressed genes and gene classes distinguishing the cases from the controls. Existing gene profiles (70-gene prognosis profile, CIN profile) were applied on our data set and correlations were calculated. Ingenuity and Gene Set Enrichment Analysis (GSEA) were used to identify specific networks and genes that were significantly regulated, and a nearest mean classifier was build. These data will be presented. Conclusion. Our preliminary results indicate that radiation-induced breast tumors can be distinguished from breast tumor controls on the basis of their genomic and expression profile. P12 GENE ALTERATION IN PAPILLARY THYROID CARCINOMAS IDENTIFIED BY ARRAY CGH Herbert Braselmann; Eva Malisch; Kristian Unger; Ludwig Hieber; Katrin Heiliger; Axel Walch; Gerry Thomas; Horst Zitzelsberger. GSF National Research Center for Environment and Health, GmbH, Institute of Molecular Radiation Biology, Germany Introduction. RET/PTC rearrangements are frequent alterations in papillary thyroid carcinomas (PTC), however, recent studies have shown that these typical changes appear heterogeneously distributed within tumour tissues. Thus, it is likely that additional gene alterations are present in these tumours. Moreover, RET/PTC negative tumours should exhibit alternative changes. Methods and Materials. We therefore investigated 25 papillary thyroid carcinomas (12 adult tumours, 13 infantile, post-Chernobyl tumours) with known RET/PTC status (RET/PTC positive: 5 adult, 10 infantile cases, RET/PTC negative: 7 adult, 3 infantile cases) by array CGH to uncover such unknown gene alterations in PTC. Results. Overall, array CGH revealed most frequent imbalances (>30% of cases) on chromosomes 1, 6, 9, 10, 11, 13, 20, 22 (DNA losses) and on chromosomes 10, 12, 16, 19, 20, 21 (DNA gains). Array CGH profiles also indicated distinct aberration patterns of adult and childhood tumours as well as of RET/PTC positive and – negative tumours. This finding could be supported by statistical analysis (maximum permutation t-test) which revealed significant differences between RET/PTC+ adult and RET/PTC+ infantile cases on chromosome 1p and between adult RET/PTC+ and adult RET/PTCnegative cases on chromosome 1p, 19, 20p and 20q. Losses on chromosome 1p appear to be specific for adults whilst losses on chromosome 1q are specific for adult RET/PTC+ cases. Gain on chromosome 19 is specific for RET/PTC+ cases in common whilst losses on chromosome 19 are specific for adult RET/PTCcases. Deletion of chromosome 13 was shown to be specific for RET/PTC+ infantile cases. A hierarchical Abstracts of the 1 MC-GARD Meeting, 3–5 May 2007: Scientific Poster Presentations 135s of the 1 MC-GARD Meeting, 3–5 May 2007: Scientific Poster Presentations 135 cluster analysis employing the correlations between the array CGH profiles demonstrated that 6 adult RET/PTC negative cases are apart from all other cases which show frequent co-alterations (gains on 19, 21 or losses on 1pq, 6, 9, 13, 20) In contrast, lost chromosomal regions on chromosomes 7q and 22 are most frequently co-altered in the 6 adult RET/PTC negative cases. These alterations revealed by array CGH have been validated by FISH on FFPE tissue sections of the investigated cases. Candidate genes have been identified (e.g., ROBO1, PTK6, RBAK, TGFBR3 and FOXO3A on 12q) and will be tested for altered expression levels (RNA and protein). Conclusions. Our findings suggest that dependent on the RET/PTC status of tumours additional gene alterations are present in papillary thyroid tumours which may point to alternate pathways involved in tumour development. P13 SCREENING FOR GENE DOSE IMBALANCES OF AUTISM CANDIDATE GENES IN PATIENTS WITH AUTISM SPECTRUM DISORDERS (ASD) USING TWO-COLOR MLPA Anna Bremer; Britt Marie Anderlid; Karen BrøndumNielsen; Niklas Dahl; Michela Barbaro; Mahmoud Mansouri; Magnus Nordenskjöld; MaiBritt Giacobini; Jacqueline Schoumans. Department of Molecular Medicine and Surgery, Karolinska University Hospital, Stockholm, Sweden E-mail: {anna.bremer; jacqueline.schoumans}@ki.se Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in communication and social interaction, accompanied by repetitive and stereotyped behaviors and interests. It's a highly heritable and heterogeneous disorder with a complex genetic etiology. The prevalence for autism is in the order of 10 in 10.000, and as high as 60 in 10.000 for all forms of autism [1]. Despite extensive investigations, involving mutation screening along with linkage and association studies, mutations have only been detected in a very few cases and no definite disease genes have yet been identified. However, cryptic chromosome abnormalities, among them maternally derived duplications/triplications of chromosome 15q11-13 have been reported in patients with ASD. In addition, recent high resolution array-CGH studies of syndromatic ASD patients revealed submicroscopic deletions or duplications in >20% of the cases [2]. We hypothesized that microdeletions or microduplications of autism candidate genes, which escape detection by array-CGH and mutation screening, might be involved in the cause of ASD. Therefore, we designed a multiplex ligation-dependent probe amplification (MLPA) synthetic probe set consisting of ~60 probes to screen for gene dose imbalances of candidate genes in patients diagnosed with ASD. By using two-color MLPA we were able to investigate ~30 specific nucleic acid sequences in a single reaction. DNA from 150 syndromatic and nonsyndromatic ASD patients has been collected for screening. The results of this ongoing study will be presented.