Table 2.

Genetic testing recommendations for PROS

Method	The molecular diagnostic approach should utilize techniques optimized for the detection of low-level mosaic variants (low variant allele fraction or VAF, see also Appendix). Because many of the variants that cause PROS are typically absent in peripheral blood, Next Generation Sequencing (NGS) or digital droplet polymerase chain reaction (ddPCR) testing
Sampling	Affected tissues (i.e., during reduction surgery of macrodactyly or for debulking of ectopic accessory muscles) offer the optimal approach. When tissue samples are not available, a biopsy of affected tissue (e.g., skin with a vascular malformation in a region with soft tissue overgrowth) may be considered. DNA should be extracted without previous culture. Genetic testing on formalin-fixed, paraffin-embedded (FFPE) samples from prior surgeries is also possible. Although, in this type of sample, the DNA may be degraded, reducing the diagnostic yield
Optimal depth of NGS	The absolute minimum number of times that a variant must be determined depends on the platform used and its ability to discriminate against background noise. For example, on hybridization-based NGS platforms, a minimum 350x and mean 500x coverage may be sufficient to detect VAFs of 5%. These numbers can be considerably higher on amplicon-based NGS platforms
Detection of low-level mosaicism	Previous studies have shown a lower diagnostic rate in individuals presenting with very localized or tissue-specific features (e.g., isolated macrodactyly) (Mirzaa et al., 2016). In a clinical context, diagnostic techniques able to detect mosaic variants with allele frequencies or VAF as low as 5% (0.05), or even lower at 1% (0.01) should be selected
Variant validation	Validation by orthogonal methods (e.g., Sanger sequencing for VAFs ≥20%, pyrosequencing for VAFs ≥5%, or ddPCR for VAFs ≤5%) or by a second NGS study of mosaic candidate variants may be needed. Variant validation might not be necessary when the candidate variant is detected in more than one different tissue of the same patient (taking into consideration the limit of detection of the technique). When validating variants in PIK3CA, it is necessary to remember that exons 10 to 14 of this gene share 95% identity with another region or pseudogene of the genome located on chromosome 22. Therefore, validation tests must be designed to discriminate the location of these variants (e.g., using long-range PCR upstream of the final PCR amplification) (Rodriguez-Laguna et al., 2018)
Approach to negative testing results	Given that low-level mosaicism is observed in many individuals with PROS, it is recommended to re-evaluate the quality of the sample (e.g., FFPE samples), the sampled tissue, the mosaic detection limit of the technique, and the clinical diagnosis