Molecular diagnosis of somatic overgrowth conditions: A single-center experience

Abstract

Background: Somatic overgrowth conditions, including Proteus syndrome, Sturge-Weber syndrome, and PIK3CA-related overgrowth spectrum, are caused by post-zygotic pathogenic variants, result in segmental mosaicism, and give rise to neural, cutaneous and/or lipomatous overgrowth. These variants occur in growth-promoting pathways leading to cellular proliferation and expansion of tissues that arise from the affected cellular lineage.

Methods: We report on 80 serial patients evaluated for somatic overgrowth conditions in a diagnostic laboratory setting, including three prenatal patients. In total, 166 tissues from these 80 patients were subjected to targeted sequencing of an 8-gene panel capturing 10.2 kb of sequence containing known pathogenic variants associated with somatic overgrowth conditions. Deep next-generation sequencing was performed with the IonTorrent PGM platform at an average depth typically >5,000×.

Results: Likely pathogenic or pathogenic variants were identified in 36 individuals and variants of unknown significance in four. The overall molecular diagnostic yield was 45% but was highly influenced by both submitted tissue type and phenotype. In the prenatal setting, two patients had pathogenic variants identified in cultured amniocytes but in a third patient, the pathogenic variant was only present in post-natal tissues. Finally, expanding the test to include full gene sequencing of PIK3CA in contrast to targeted sequencing identified likely pathogenic variants in 3 of 7 patients that tested negative on the original panel.

Conclusion: Next-generation sequencing has enabled sensitive detection of somatic pathogenic variants associated with overgrowth conditions. However, as the pathogenic variant allele frequency varies by tissue type within an individual, submission of affected tissue(s) greatly increases the chances of a molecular diagnosis.

Keywords: PIK3CA-related overgrowth spectrum; mosaicism; somatic overgrowth.

Figure 1

Pathogenic or likely pathogenic variant detection rate. (a) The diagnostic rate was 45% in this case series and was influenced by sample type. (b) The majority of likely pathogenic or pathogenic variants were identified in PIK3CA (GenBank Accession NM_006218.2)

Figure 2

Frequency of the most common test indications (a) and symptom groups (b). For example, asymmetric limb overgrowth was the most common phenotype, and a suspicion of CLOVES syndrome or lipomatous findings were associated with the highest diagnostic yields

Figure 3

Diagnostic rate and variant allele frequency observed per specimen type. Diagnostic rate per patient and per specimen type are closely related (top panel). Variant allele frequency observed in 36 positive patients' (bottom panel), where each. Each value corresponds to one technical replicate per specimen

Figure 4

IGV screenshot for pathogenic variants observed in distinct tissues from one individual

Figure 5

PIK3CA variants identified in the current study (top) and in the COSMIC database (bottom). Missense changes are shown in green, truncating changes in black, in‐frame indels in brown, and “other changes” in purple. The domains shown are the adaptor‐binding domain (green), the Ras‐binding domain (red), the membrane‐binding domain (blue), the helical domain (yellow), and the kinase catalytic domain (purple). COSMIC mutations accessed August 22, 2017. Nomenclature in reference to GenBank Accession NM_006218.2