Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System.

BRCA2 is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all BRCA2 VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL BRCA2 transcripts. Of 100 BRCA2e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL BRCA2 transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL BRCA2 with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.

Digestive calcitonin-secreting tumors of the foregut: comparison with non-calcitonin-secreting tumors.

OBJECTIVES: Digestive calcitonin-secreting endocrine tumors are very rare lesions of the foregut. This study was undertaken to compare the characteristics and the prognosis of these tumors and those of non-calcitonin-secreting endocrine tumors. METHODS: All patients with a digestive endocrine tumor of the foregut followed up in Reims University Hospital and whose serum calcitonin levels were determined between 1988 and 2004 were included. Clinical and tumor characteristics of calcitonin-positive and calcitonin-negative patients were compared. RESULTS: Thirty-two patients were included. Among the five (15.6%) with high calcitonin levels (median: 340 pg/ml, range: 42-7460 pg/ml), only one tumor was functioning (diarrhea). Significant differences between patients with positive and negative calcitonin levels were, respectively: liver metastases [5 (100%) versus 11 (40.7%); P=0.04], type according to the World Health Organization 2000 histological classification [notably 4 (80%) versus 3 (12.5%) poorly differentiated endocrine carcinomas; P=0.02] and Ki67 proliferation index [median: 25% (range: 20-30%) versus 7% (0-80%); P=0.03]. The only calcitonin-positive well-differentiated endocrine carcinoma had a high proliferation index (30%). Survival also differed significantly (P=0.001), as all calcitonin-positive patients died, with a median survival of 22.6 months (range: 1.2-27.2 months), versus five (18.5%) calcitonin-negative patients. Median follow-up period for the latter was 42.3 months (range: 3.4-208 months). CONCLUSIONS: The secretion of calcitonin appears predictive of a poor prognosis. Digestive endocrine calcitonin-secreting tumors correspond histopathologically to poorly differentiated or well-differentiated carcinomas with high proliferation indexes.