Abstrakt
During standard molecular diagnostic procedure, two Czech families with APC (Adenomatous polyposis coli gene) mosaicism have been detected. A woman with attenuated familial adenomatous polyposis (AFAP, OMIM #175100) was recently inspected by next generation sequencing.
Standard bioinfonnatics pipeline, restricted to variants with at least 20% of reads (for germline variants) would miss mutation p.G1412X (NM_000038.5) present in 17% of reads. This novel variant was not present in any of her two children.
Another woman with a clinical manifestation of attenuated PAP was tested 16 years ago without conclusive APC mutation found when denaturing gradient gel electrophoresis (DGGE), protein truncation test (PTT) multiplex ligation probe amplification (MLPA) and direct Sanger sequencing were applied. Recent inspection of her son showed clear mutation p.Q1062X (NM_000038.5, NP_000029.2) leading to premature stop codon.
This fording led to re-evaluation of this protein position in his mother and detection of mosaicism (11% of allele, 22% of heterozygous cells in blood), which was primarily overlooked. Mutations in both patients were confirmed by allele-specific real time PCR (AS qPCR).
In both index patients it was possible to detect and quantify the mosaic allele in biological samples of polyps, adjacent colonic mucosa and buccal swabs. In cases of sporadic appearance of FAP, besides blood we plan to preferably inspect also other samples, where mosaic fraction might be under detection limit of bioinformatics pipelines (<3%).
For our future routine NGS sequencing analysis we will apply our in-house somatic variant detection pipeline to minimize the false negative calls when genes with high level of de-novo mutations are analyzed.