Molecular Diagnosis of Mosaic Overgrowth Syndromes Using a Custom-Designed Next-Generation Sequencing Panel

Recent studies have discovered a group of overgrowth syndromes, such as congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) syndrome, Proteus syndrome, and megalencephaly-capillary malformation-polymicrogyria (MCAP) syndrome, are caused by somatic activating variants in genes involved in the phosphatidylinositol 3-kinase/AKT/mechanistic target of rapamycin pathway. Because of the low-abundance nature of these variants, Sanger sequencing often yields negative results. We have developed and validated a next-generation sequencing (NGS) panel that targets all known variants associated with these syndromes. Fifty cases, including two prenatal cases, were tested using the panel. A pathogenic variant in the PIK3CA, PIK3R2, or AKT1 gene was identified in 28 of the 50 cases with the variant allele frequencies ranging from 1.0% to 49.2%. These variants were only present in the affected tissues in most of the cases, demonstrating a causal role in the development of these diseases. In vitro cell culture showed significant enrichment of the cells harboring variant alleles, suggesting that these variants render growth advantages to mutant cells. Phenotype-genotype correlation analysis showed PIK3CA mutation hotspots at residues E542, E545, and H1047 are often associated with CLOVES syndrome, whereas PIK3CA G914R is preferentially related to MCAP. We thus demonstrate that NGS technology is highly sensitive for detecting low-level mosaicism and can facilitate clinical diagnosis of mosaic overgrowth syndromes in both prenatal and postnatal settings.