Identification of novel intra-genic deletions of CTNNB1 gene in WT desmoid-type fibromatosis

Background: Desmoid-type fibromatosis (DT) are characterized by molecular alterations of CTNNB1 or APC genes. These abnormalities have been described in ∼85% of DT using Sanger sequencing. Recently, whole-exome sequencing allowed to detect molecular aberrations in CTNNB1 or APC in approximately 95% of DT. In order to characterized the true WT DT, we performed this study on DT WT Sanger-sequenced samples. Methods: Deep sequencing of CTNNB1 and of APC was performed using TruSeq Custom Amplicon Low Input kit (Illumina) on 11 WT FFPE DT samples from patients treated with surgery. Whole Exome sequencing (WES) was performed using Nextseq500 (Illumina, CA) sequencer. Mutations were validated through Sanger sequencing, and expression of CTNNB1 mutated alleles was evaluated through RT-PCR. Results: APC mutation was found in 2 cases, while low-frequency CTNNB1 mutations were discovered in 5 samples (45%) (mean of 16% reads) and 4 cases remained WT. WES was performed in 6 cases (3 CTNNB1 WT and 3 with low frequency mutations). Through in-depth analysis of reads spanning exon 3 of CTNNB1, 2 large intra-genic deletions were detected in 2 cases, one carrying also a T41A low frequency mutation. Both deletions were approximatively of 190bp and led to the loss of the T41 codon hotspot of mutation. In one case, the deletion led to the loss of the second half of exon 3 and part of intron 3, in the other it involved part of intron 2 and the 5’ part of exon 3 losing the splicing acceptor site. We demonstrated that even if the acceptor splicing site was lost, the deleted allele of CTNNB1 was expressed, with the retention of 32 bp of intron 2 in the mature mRNA. Moreover, we demonstrated that, at mRNA level, low frequency T41A mutated allele was the prevalent isoform expressed with respect to the WT allele. The importance of these molecular alterations in the natural history of DT needs further investigations. Conclusions: A minority of DT is WT for either CTNNB1, APC or any other gene involved in the WNT pathway. In this subgroup novel and hard to be detected molecular deletions of CTNNB1 were discovered, contributing to explain a portion of the allegedly WT DT cases.