Detection of low-level parental somatic mosaicism for clinically relevant SNVs and indels identified in a large exome sequencing dataset.

BACKGROUND: Due to the limitations of the current routine diagnostic methods, low-level somatic mosaicism with variant allele fraction (VAF) < 10% is often undetected in clinical settings. To date, only a few studies have attempted to analyze tissue distribution of low-level parental mosaicism in a large clinical exome sequencing (ES) cohort. METHODS: Using a customized bioinformatics pipeline, we analyzed apparent de novo single-nucleotide variants or indels identified in the affected probands in ES trio data at Baylor Genetics clinical laboratories. Clinically relevant variants with VAFs between 30 and 70% in probands and lower than 10% in one parent were studied. DNA samples extracted from saliva, buccal cells, redrawn peripheral blood, urine, hair follicles, and nail, representing all three germ layers, were tested using PCR amplicon next-generation sequencing (amplicon NGS) and droplet digital PCR (ddPCR). RESULTS: In a cohort of 592 clinical ES trios, we found 61 trios, each with one parent suspected of low-level mosaicism. In 21 parents, the variants were validated using amplicon NGS and seven of them by ddPCR in peripheral blood DNA samples. The parental VAFs in blood samples varied between 0.08 and 9%. The distribution of VAFs in additional tissues ranged from 0.03% in hair follicles to 9% in re-drawn peripheral blood. CONCLUSIONS: Our study illustrates the importance of analyzing ES data using sensitive computational and molecular methods for low-level parental somatic mosaicism for clinically relevant variants previously diagnosed in routine clinical diagnostics as apparent de novo.

Parental somatic mosaicism for CNV deletions - A need for more sensitive and precise detection methods in clinical diagnostics settings.

To further assess the scale and level of parental somatic mosaicism, we queried the CMA database at Baylor Genetics. We selected 50 unrelated families where clinically relevant apparent de novo CNV-deletions were found in the affected probands. Parental blood samples screening using deletion junction-specific PCR revealed four parents with somatic mosaicism. Droplet digital PCR (ddPCR), qPCR, and amplicon-based next-generation sequencing (NGS) were applied to validate these findings. Using ddPCR levels of mosaicism ranged from undetectable to 18.5%. Amplicon-based NGS and qPCR for the father with undetectable mosaicism was able to detect mosaicism at 0.39%. In one mother, ddPCR analysis revealed 15.6%, 10.6%, 8.2%, and undetectable levels of mosaicism in her blood, buccal cells, saliva, and urine samples, respectively. Our data suggest that more sensitive and precise methods, e.g. CNV junction-specific LR-PCR, ddPCR, or qPCR may allow for a more refined assessment of the potential disease recurrence risk for an identified variant.