Somatic mosaicism is an important mechanism of immune dysregulation and explains a growing proportion of the 70% of patients with suspected IEI who lack a molecular diagnosis. Identifying disease-causing somatic mutations is challenging due to the presence of such variants in a small subset of cells, leading to a low variant allele fraction (VAF).
We developed a custom 71-gene capture-based panel for high-depth sequencing of genes known or hypothesized to be associated with dominant immune dysregulation. Sequencing was performed with samples from 223 patients with immune dysregulation phenotypes and 96 currently healthy individuals. Mean age was 24.5 years for patients and 17.8 years for healthy individuals. 52.5% of patients and 40.6% of healthy individuals were biologic males. Coverage across coding regions of targeted genes had an average depth of 5752×. Our custom analysis pipeline utilized the union of multiple variant callers to call somatic variants.
We identified 36 somatic variants in 21 genes from 28 patients (12.6% of patients) and 6 healthy controls (6.25% of healthy). To validate our assay, an independent library was sequenced for 60 patients and 18 healthy controls, including samples harboring 27 somatic variants. Droplet digital PCR (ddPCR) was used as an orthogonal method to validate 17 variants. All tested variants were confirmed as somatic. Genes with somatic mosaicism in >1 affected patient included FAS, STAT3, CARD11, NRAS, TNFAIP3, NLRP3, and PIK3CD. Three variants were previously described to cause somatic immune dysregulation. Immune cell populations from 7 patients with variants in FAS, NRAS, TNFAIP3, and UBA1 were purified and ddPCR performed, which identified enrichment of somatic variants in different cell types. This included somatic variants in FAS that had a VAF of 5-7% in whole blood but 21-28% in DNTs, all in individuals with a clinical phenotype consistent with ALPS.
Our studies demonstrate the utility of high-depth targeted sequencing of suspected IEI patients to identify pathogenic and potentially pathogenic somatic variants, including low VAF variants directly from whole blood. Ongoing studies include expanding our gene panel, in vitro functional validation of novel variants, and utilizing single-cell technologies to identify the functional impact of somatic variants.
