Targeted Resequencing Of Germline Pten-Negative Patients With Cowden Disease Reveals Alternate Mechanism Of Molecular Alteration

Abstract Introduction Cowden disease belongs to the PTEN hamartoma tumor syndrome (PHTS) group, defined by germline PTEN heterozygous inactivation. Describing the PTEN germline mutation in Cowden disease patients is of particular importance for their breast cancer and thyroid carcinoma predisposition evaluation. Material & methods Targeted resequencing was done on germline DNA of 22 index cases patients with a Cowden disease clinically well established since they do not demonstrate any PTEN mutation analyzed using our current screening method (EMMA, Enhanced Mismatch Mutation Analysis, similar to high resolution melting). The 565 exons of 34 genes of interest together with the entire genomic locus of PTEN were captured using the SureSelect oligonucleotide library capture (Agilent technologies). The bioinformatics resources Suredesign, Ensembl human genome v70 and a UCSC repeat masker least stringent were used to create the capture design. This gave us 28405 unique 120-mers oligonucleotide probes which were duplicated 1 to 16 times depending on the GC content or the size of the targeted regions (maximum boosting performance criteria). For each gene of interest, an exon of the surrounding gene was also captured in order to define the extent of gross gene rearrangement. Samples were prepared according the manufacturer's recommendations. Results Each of the 22 Cowden disease patients demonstrates a mean rate of 70 intronic mutations in the entire genomic locus of PTEN. All of those for which the minor allelic frequency is unknown, were subjected to splicing site bioinformatic prediction. For one patient, the prediction reveals the apparition of a splicing site in a deep intronic mutation of the large intron 1 of PTEN. Furthermore, next generation sequencing (NGS) seems to be more sensitive than our current screening method since for two patients, a germline mosaic exonic heterozygous inactivating mutation of PTEN was detected and confirmed by Sanger sequencing on different biological samples. The level of mosaicism was evaluated by NGS at 3 to 12% depending on the sample and confirmed by pyrosequencing. In addition, some mutations were identified in exons of genes encoding proteins of the PI3Kinase pathway, which could be involved in disease belonging to the PHTS group. With an increasing sensitivity, NGS is a powerful tool to detect low frequency variant. This could used to explain at a molecular level Cowden disease with no initially detectable PTEN mutation. Clinical and biological rationale, bioinformatics workflow and comprehensive results will be presented. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-04-04.