BRCA1 and BRCA2 Next Generation Sequencingwith methodological improvements in HBOC Uruguayan families.

MARQUES JM; REPETTO L; GUGGERI L; GARCIA E; ALEJANDRA T ; CARDOSO F; DELLA VALLE A; ACEVEDO JC; SAPONE M; ESPERON P; AZAMBUJA C; NEFFA F; SOLANO A

Congreso; ASCO 2015; 2015

Background: Among the hereditary causes of breast cancer, hereditary breast and ovarian cancer (HBOC) syndrome, caused by mutations in the BRCA1 or BRCA2 genes, has been considered the most prevalent. We selected patients from our country, analyzed their full sequence, and reported all the variants detected in BRCA1/2, as well as the large rearrangements in both genes. Methods: Patients were recruited from the Uruguayan Collaborative Group of Inherited Cancer (UCG) and Genia Laboratory¢¥s genetic service. A total of 38 patients diagnosed with HBOC, with a positive family history according to NCCN high risk criteria, and ¡Ã 10% mutation probability by BRCAPro software were selected. DNA samples were amplified using the BRCA1/2 Ampliseq Community Panel by multiplex PCR with modifications of standard pools to achieve better equilibrium between amplicons. After clonal amplification, libraries were sequenced on an Ion Torrent PGM. Standard Bioinformatics pipeline was modified with a different alignment argument to allow more accurate detection of small deletions next to the amplicons ends. Rearrangements were first analyzed by normalizing the coverage per amplicon and by comparing among samples of the same NGS run. We performed MLPA to all NGS negative samples. Results: We detected 8 mutated samples: 2 SNV clinically deleterious, 5 frameshift deletions, and one sample with a deletion of exon 14 of BRCA1. The change in alignment arguments shows effectiveness in detecting heterozygous and homozygous polymorphic 4 bp deletion in intron 11 of BRCA2 present at the end of an amplicon. NGS allows us to determine large rearrangements but has limitations to detect short deletions, while MLPA is able to robustly detect one short exon deletion. Conclusions: Improvements had been made regarding a more comprehensive protocol to diagnose germline mutations. We had characterized 8 pathogenic mutations and interesting VUS, but the number of families waiting for molecular diagnose is still high. It is necessary to include these non mutated samples in a panel we are developing with 22 cancer related genes to try to resolve more cases.