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Advances in sequencing technologies, such as next-generation sequencing (NGS), represent an opportunity to perform genetic testing in a clinical scenario. In this study, we developed and tested a method for the detection of mutations in the large BRCA1and BRCA2tumor suppressor genes, using long-range PCR (LR-PCR) and NGS, in samples from individuals with a personal and/or family history of breast and/or ovarian cancer. Eleven LR-PCR fragments, between 3000 and 15,300 bp, containing all coding exons and flanking splice junctions of BRCA1and BRCA2, were obtained from DNA samples of five individuals carrying mutations in either BRCA1or BRCA2. Libraries for NGS were prepared using an enzymatic (Nextera technology) method. We analyzed five individual samples in parallel by NGS and obtained complete coverage of all LR-PCR fragments, with an average coding sequence depth for each nucleotide of 30 reads, running from ×7 (in exon 22 of BRCA1) to ×150. We detected and confirmed 100% of the mutations that predispose to the risk of cancer, together with other genomic variations in BRCA1and BRCA2. Our approach demonstrates that genomic LR-PCR, together with NGS, using the GS Junior 454 System platform, is an effective method for patient sample analysis of BRCA1and BRCA2genes. In addition, this method could be performed in regular molecular genetics laboratories.

Mutations in the c‐KIT gene have been identified in many sporadic and familial cases of gastrointestinal stromal tumor (GIST). We report a familial case of GIST with cutaneous hyperpigmentation associated with a novel germline mutation in the c‐KIT gene. Screening for mutations in exon 11 of the c‐KIT gene in genomic DNA from tumors and peripheral blood of the members of a family with GISTs was undertaken by direct genomic sequencing. Tumors from GIST patients were analyzed histologically and immunohistochemically. Clinical examination of GIST patients was also performed to detect other systemic diseases associated with c‐KIT mutations. Histological study showed that the tumors were GISTs expressing CD34 and c‐KIT protein. This GIST‐hyperpigmentation disease was associated in the family with a germline mutation in the c‐KIT gene. The mutation is a duplication of the sequence CAACTT located in exon 11 of the c‐KIT gene, which introduces two extra glutamine and leucine residues in the encoding protein between positions 576 and 577. This Spanish family was affected with GISTs and cutaneous hyperpigmentation associated with a novel germline mutation Leu576_Pro577insGlnLeu in the juxtamembrane domain of the c‐KIT receptor. These types of mutation in the c‐KIT gene activate the tyrosine kinase activity of the c‐KIT receptor and induce constitutive signaling leading to GISTs, in some cases associated with cutaneous hyperpigmentation. © 2004 Wiley‐Liss, Inc.