Disorders of somatic mosaicism (DoSM) are a heterogeneous group of conditions caused by postzygotic variants in genes within the PI3K/AKT/mTOR and RAS/MAPK signaling pathway. The co-existence of 2 activating variants in this disease group is extremely rare. A deep sequencing next-generation sequencing assay for the molecular diagnosis of DoSM was run on 936 individuals with DoSM. A single pathogenic or likely pathogenic (P/LP) variant was identified in 584 of 617 (94.8%) positive cases; 33 of 617 (5.2%) cases carried 2 P/LP variants. Of these 33 cases, 22 carried 2 P/LP variants in the same gene, including 8 associated with a loss-of-function disease mechanism and 14 associated with a gain-of-function disease mechanism. Eleven cases had P/LP variants in 2 different genes, including PIKC3A variants in 7 cases and 4 cases with 2 P/LP variants in non-PIK3CA genes. To our knowledge, this is the largest cohort with the co-existence of 2 P/LP somatic variants causing DoSM. The study of the co-existence of 2 clinically significant variants in DoSM requires unique considerations regarding variant allelic fractions, the combination of variants, affected tissue types, and the severity of the disease. Investigations into this unique cohort may further our understanding of the disease mechanism and potential therapeutic options.

Germline variants in DEPDC5 are a cause of familial focal epilepsy with variable foci. Affected individuals may have focal cortical dysplasia if a second brain somatic variant occurs. As access to brain tissue is limited, the second somatic hit in the brain is usually presumed if a clear pathogenic germline variant is present. Here, we present a patient with structural focal epilepsy due to focal cortical dysplasia. He underwent epilepsy surgery at age 2 months. Clinical genetic testing revealed a germline variant of uncertain significance (VUS) DEPDC5 c.920T>G, p. (Leu307Arg). We hypothesized that if the proband's germline DEPDC5 variant was disease‐causal, a second somatic variant may be identifiable in affected cortical tissue. DNA was extracted from archived brain formalin‐fixed paraffin‐embedded tissue and subjected to deep gene panel analysis. This revealed an additional somatic pathogenic variant in DEPDC5 , which was confirmed using droplet digital PCR. Identification of the second somatic hit in brain tissue ( DEPDC5 c.982C>T, p.(Arg328*)) confirmed the two‐hit situation in this patient and supported disease causality of the germline variant. In conclusion, testing tissue for somatic variants may be clinically relevant at a patient‐specific level if a germline VUS affects a gene where a two‐hit mechanism is known.

Variants in PIK3CA (encoding p110α; the catalytic subunit of PI3K) characterize some disorders of somatic mosaicism (DoSM) conditions with clinical features, including sporadic overgrowth and vascular malformations. Here, we profile PIK3CA variants in DoSM. We applied a next-generation, sequencing-based, laboratory-developed test, using an average coverage of approximately 2000× for up to 37 genes associated with DoSM, on a cohort of 1197 patients with DoSM referred for clinical genomics services between 2013 and 2022. We identified clinically reportable variants in 747 (62.4%) individuals in this cohort. Notably, 371 clinically reportable variants in PIK3CA were identified in 368 patients, constituting approximately 49.2% of all patients with reportable findings. Variants in the C2 domain of p110α are enriched in DoSM (this cohort) compared with those of cancer (Catalogue of Somatic Mutations in Cancer [COSMIC] database), highlighting the role of the C2 domain in driving uncontrolled cell proliferation in DoSM. Furthermore, we report 17 novel variants in PIK3CA that are not previously reported in DoSM and describe clinical presentation correlation for 4 novel variants. Our findings from the largest single-center cohort of patients with DoSM expand the spectrum of variants in PIK3CA and shed light on the less-studied role of the C2 domain in the pathogenesis of DoSM.