Integrating deep-exome sequencing and droplet digital PCR for genetic diagnosis of 142 patients with somatic overgrowth and vascular malformation

Background A genetic diagnosis is important to patients with somatic overgrowth and vascular anomalies to enable accurate genetic counselling and precision medicine leading to improved quality of life. It also gives a clearer understanding of the natural history and clinical course of the disease. Lack of sensitive diagnostic methods for detection of variants with low frequencies can cause low diagnostic yield for patients with mosaic conditions. Methods We set up a diagnostic pipeline with deep-exome sequencing followed by droplet digital PCR (ddPCR) in order to detect and confirm low-frequency alleles for conditions known to be caused by somatic variants. Sequencing data were analysed with an in silico gene panel related to somatic overgrowth and vascular malformations, and ddPCR was performed on variants with low allele frequencies to exclude false-positive results. Results The cohort, consisting of 142 patients, had an overall diagnostic yield of 63%, with pathogenic variants in 16 different genes. The diagnostic yield varied substantially between affected tissue (68%; 84/123 patients) and blood samples (26%; 5/19 patients). The variant allele frequency (VAF) ranged from 0.75% to 50%, and 79% (70/89 patients) displayed a VAF below 15%. Conclusion The study demonstrates that applying deep-exome sequencing and ddPCR can achieve a high diagnostic yield in patients with somatic overgrowth and/or vascular malformations. The combination of the two methods strengthens the sensitivity and specificity in patients with low VAF by excluding false-positive results, making it possible to give more patients a genetic diagnosis and accurate clinical management.