Identification of NPM1 as a potential high-penetrance gene in hereditary breast cancer.

Abstract Abstract #1042 Background: In a major proportion of breast cancer patients with a strong familial cancer history no BRCA1 or BRCA2 mutations are detected which prompted us to explore other proteins on the BRCA1/2 pathway. Previously we have identified BARD1 (De Brakeleer et al, SABC 2006) as a rare high penetrance breast cancer predisposing gene. Nucleophosmin (NPM1) is a multifunctional protein that is ubiquitylated and stabilized by the BRCA1-BARD1 complex and thus is part of the BRCA1 pathway. Somatic protein truncating mutations in NPM1 are frequent events in acute myeloid leukemia (AML).
 Aim: This study was aimed at determining whether NPM1 germline mutations may contribute to the breast cancer predisposition in families where no BRCA1 or BRCA2 mutation is found.
 Methods: NPM1 mutation analysis was performed on genomic DNA extracted from blood of 198 breast cancer patients with strong indications for a hereditary form of the disease and 110 controls. All the 11 exons of the NPM1 gene were separately amplified by PCR. Since a high number of processed NPM1 pseudogenes are dispersed throughout the human genome, PCR primers had to be designed carefully. The PCR products were subsequently analyzed by denaturing gradient gel electrophoresis (DGGE) and when an abnormal migration pattern occurred, sequencing analysis was performed to determine the exact nucleotide-sequence alteration which was verified against the pseudogene sequences.
 Results: Out of the six novel sequence variations, only one occurred in an exon: a duplication of thymidine located in the 3' non-coding sequence of exon 12 (c.1042dupT). The remaining 5 variants were located in intron sequences close to the exon borders. Two variants flanking exon7 (c.460-31G>A and c.524+42G>A) were always linked, and occurred at the same high frequency in breast cancer patients and controls. Two intron sequence variants were only found in one patient. One was a duplication of a sequence in intron 8 (c.582+52dupT) and the other a sequence deletion in intron 3 (c.139-9delT). One sequence variation located close to exon 11 (c.771-46C>A) was found in 7 familial breast cancer patients in 4 high incidence families but not in unaffected family members and in none of the control samples. This mutation leads to an alternative splicing of the NPM1 mRNA and is predicted to modify the carboxyterminal end of the protein, removing a nucleolar locating signal.
 Discussion: A germline mutation in NPM1 was found only in familial breast cancer cases. This mutation may result in the synthesis of a C-terminal truncated NPM1 protein conferring high susceptibility to breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1042.