Abstract
There are increasing evidences showing the contribution of somatic genetic variants to non-cancer diseases. However, their detection using massive parallel sequencing methods still has important limitations. In addition, the relative importance and dynamics of somatic variation in healthy tissues are not fully understood. We performed high-depth whole-exome sequencing in 16 samples from patients with a previously determined pathogenic somatic variant for a primary immunodeficiency and tested different variant callers detection ability. Subsequently, we explored the load of somatic variants in the whole blood of these individuals and validated it by amplicon-based deep sequencing. Variant callers allowing low frequency read thresholds were able to detect most of the variants, even at very low frequencies in the tissue. The genetic load of somatic coding variants detectable in whole blood is low, ranging from 1 to 2 variants in our dataset, except for one case with 17 variants compatible with clonal haematopoiesis under genetic drift. Because of the ability we demonstrated to detect this type of genetic variation, and its relevant role in disorders such as primary immunodeficiencies, we suggest considering this model of gene mosaicism in future genetic studies and considering revisiting previous massive parallel sequencing data in patients with negative results.
Highlights
There are increasing evidences showing the contribution of somatic genetic variants to non-cancer diseases
In the field of immunerelated diseases, a remarkable number of somatic variants have been described in monogenic autoinflammatory diseases[12,13,14,15,16,17,18,19,20], and a recent work has shown its important contribution to these disorders and other primary immunodeficiencies (PIDs)[21]
We considered that a somatic variant was validated in a given cell type or tissue when the proportion of reads supporting the alternative allele was above 0.30%, a value close to the average error type of sequencing by synthesis technologies, which varies with features such as sequence context or the specific nucleotide change[53,54]
Summary
There are increasing evidences showing the contribution of somatic genetic variants to non-cancer diseases Their detection using massive parallel sequencing methods still has important limitations. Different studies have measured the presence of somatic variation in normal tissues, most assessing the presence of mutations in cancer-driver genes, such as NOTCH1 mutations, which undergo expansion through positive selection[22,23,24]. Other variant calling tools can be applied to non-matched samples[31,32] In this case, allelic imbalance thresholds will need to be relaxed to detect low frequency variants, at the cost of substantially increasing the number of candidate variants. A validation step, such as amplicon-based deep sequencing (ADS), will be required to confirm the presence of a somatic variant and better determine its frequency
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