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

Abstract

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.

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

“…The maximum VAF of a P/LP variant in blood was 4.5%. These results reinforce previous findings that testing blood alone is not a suitable strategy for diagnosis of somatic overgrowth, although some exceptions have been noted (Riviere et al, , Keppler‐Noreuil et al, , Luks et al , Mirzaa et al, , Chang et al, , Kuentz et al, ). The diagnostic yield increased to 56% (36/64) after removing patients where only blood and/or saliva were submitted.…”

“…Finally, we evaluated the likely cause of negative findings in the 40 patients where a P/LP variant was not identified. As mentioned previously, 12/40 of these patients only had blood available for testing, and thus, it is likely that, for a subset of these patients, P/LP variants were present in the targeted regions but not detectable in blood (Riviere et al, , Keppler‐Noreuil et al, , Luks et al, , Mirzaa et al, , Chang et al, , Kuentz et al, ). To determine whether full PIK3CA sequencing would increase the molecular diagnostic rate, we evaluated an expanded panel which included full exonic coverage for PIK3CA in 7 patients where clearly affected tissue was submitted and the phenotype was strongly suggestive of PROS (Supporting information Table ).…”

“…It is not clear whether this high VAF is representative of a very early post‐zygotic variant or whether the variant was selected for during culture. Several cases of germline PIK3CA variants have been reported recently (Chang et al, ; Mirzaa et al, ). Ultimately, the pregnancy did not go to full‐term.…”

“…A heterogeneous group of overgrowth conditions are caused by post‐zygotic pathogenic variants, resulting in segmental mosaicism and give rise to neural, cutaneous and/or lipomatous overgrowth (Akgumus, Chang, & Li, ; Chang et al, ; Keppler‐Noreuil, Parker, Parker, Darling, & Martinez‐Agosto, ). Typically, these variants arise in growth‐promoting pathways, most commonly the PI3K‐AKT‐MTOR pathway, leading to cellular proliferation and enlargement of tissues arising from the affected cellular lineage.…”

“…Pathogenic post‐zygotic PIK3CA variants are associated with additional mosaic syndromes and isolated findings, now collectively termed PIK3CA ‐related overgrowth spectrum (PROS) (Keppler‐Noreuil et al, ). Discovery of these somatic changes has been facilitated by deep next‐generation sequencing (NGS), which can detect extremely low‐level mosaicism that could not have been identified by traditional sequencing methods or PCR‐based assays (Chang et al, ; Hucthagowder et al, ). Interestingly, these same variants also commonly occur in tumorigenesis, giving a proliferative advantage to the malignant clone.…”