Detection of complex mutations in Swedish FAP familes

1. Anna Rohlin,1Yvonne Engwall,1Josephine Wernersson 1,Jan Björk,2 and Margareta Nordling1 1Department of Molecular and Clinical Genetics, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.2The Swedish Polyposis Registry, Department of Medicine, Karolinska Institute, Stockholm, Sweden. Detection of complex mutations in Swedish FAP familes A C1466 B C1929 C C2348 D C2581 • Introduction • Familial adenomatous polyposis (FAP) has been linked to germline mutations in the APC tumor suppressor gene. Classical FAP and AFAP (attenuated familial polyposis) show a phenotypic heterogeneity, which is partially related to the mutation site or type. • In our material, 96 unrelated FAP patients from the Swedish polyposis register were screened for mutations in the APC gene. • By using a combination of several different mutation detection techniques, the mutations were revealed in >95% in families with a classical FAP phenotype (Kanter-Smoler et al. 2008). • Around 12%-15% of our families with a classical phenotype are shown to have larger deletions including parts of the APC gene and or the whole APC gene(identified with mlpa). In several studies larger deletion/rearragements of the APC gene seem to be more common than previous expected. • In this study we used the Exon- and SNP arrays from Affymetrix to investigate more complex larger deletions including the whole gene and the promotor regions 1A and 1B of the APC gene. • Material and Methods • Four families with larger deletions (previously found with mlpa) were analyzed with the SNP arrays 6.0 and the 1.0 HuEx arrays both from Affymetrix. • The GeneChip 6.0 platform consists of about 906 600 SNP sequences and about 900 000 nonpolymorphic pobes, which cover the whole genome with an average spacing of 0.7Kb. • The exon-arrays include over 40 probes for each gene and four probes (one probeset) for every exon for all well annotated genes. The exon-arrays reveal the expression levels and the differences in isoforms generated by alternative splicing events. The expression level were also invesigated with TAGman analysis using three different probes for the APC transcripts. Fig1 Fig2 Results The results of the SNP array and exon array analysis for the four large deletions are shown in fig1A-D and table1. Table 1 gives the extensions of the abbreviations, the deleted regions nand the results of the exon expression analysis. The exon expression arrays results include the three different transcripts generated from the promotor 1A and 1B (expression fold change). In fig 2 the analysis (Partek GS) shows the -1.8 times (fold change) reduced APC expression for patient C2348 having a deletion including both the promotor 1A and 1B. The TAGman results are showing the reduced expression levels with three different probes were probe 3 only includes the transcript generated from promotor 1B and probe1 and 2 all the transcripts generated from both the promotors (Fig3). In table 2 the TAGman results for two deletions including promotor 1A and promotor 1A and 1B are shown. Fig3 Table2 Table1 Conclusions A combination of exon-arrays and SNP-arrays can be used in order to get a more detailed picture of copy number changes and correlations with differentially expressed/alternative spliced transcripts. In this study the reduced expression reveals the importance of both the promotor1A and 1B, although a higher overall reduced expression was seen for promotor 1A only. This will give valuable information about regulation of the APC gene and add information regarding new mutational mechanisms. anna.rohlin@vgregion.se Reference: "Clinical characterization and the mutation spectrum in Swedish adenomatous polyposis families"Kanter-Smoler, G., Fritzell, F., Rohlin, A,. Engwall, Y., Hallberg, B., Bergman, A., Meuller, J., Grönberg, H., Karlsson, P., Björk, J. and Nordling, M. BMC Med. (2008) Apr 24;6:10.