Activating variants in the PIK3CA gene cause a heterogeneous spectrum of disorders that involve congenital or early-onset segmental/focal overgrowth, now referred to as PIK3CA-related overgrowth spectrum (PROS). Historically, the clinical diagnoses of patients with PROS included a range of distinct syndromes, including CLOVES syndrome, dysplastic megalencephaly, hemimegalencephaly, focal cortical dysplasia, Klippel-Trenaunay syndrome, CLAPO syndrome, fibroadipose hyperplasia or overgrowth, hemihyperplasia multiple lipomatosis, and megalencephaly capillary malformation-polymicrogyria (MCAP) syndrome. MCAP is a sporadic overgrowth disorder that exhibits core features of progressive megalencephaly, vascular malformations, distal limb malformations, cortical brain malformations, and connective tissue dysplasia. In 2012, our research group contributed to the identification of predominantly mosaic, gain-of-function variants in PIK3CA as an underlying genetic cause of the syndrome. Mosaic variants are technically more difficult to detect and require implementation of more sensitive sequencing technologies and less stringent variant calling algorithms. In this study, we demonstrated the utility of deep sequencing using the Illumina TruSight Oncology 500 (TSO500) sequencing panel in identifying variants with low allele fractions in a series of patients with PROS and suspected mosaicism: pathogenic, mosaic PIK3CA variants were identified in all 13 individuals, including 6 positive controls. This study highlights the importance of screening for low-level mosaic variants in PROS patients. The use of targeted panels with deep sequencing in clinical genetic testing laboratories would improve diagnostic yield and accuracy within this patient population. PIK3CA-related overgrowth spectrum (PROS) encompasses a range of clinical findings in which the core features are congenital or early-childhood onset of segmental/focal overgrowth with or without cellular dysplasia. Prior to the identification of PIK3CA as the causative gene, PROS was separated into distinct clinical syndromes based on the tissues and/or organs involved (e.g., MCAP [megalencephaly-capillary malformation] syndrome and CLOVES [congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies] syndrome). The predominant areas of overgrowth include the brain, limbs (including fingers and toes), trunk (including abdomen and chest), and face, all usually in an asymmetric distribution. Generalized brain overgrowth may be accompanied by secondary overgrowth of specific brain structures resulting in ventriculomegaly, a markedly thick corpus callosum, and cerebellar tonsillar ectopia with crowding of the posterior fossa. Vascular malformations may include capillary, venous, and less frequently, arterial or mixed (capillary-lymphatic-venous or arteriovenous) malformations. Lymphatic malformations may be in various locations (internal and/or external) and can cause various clinical issues, including swelling, pain, and occasionally localized bleeding secondary to trauma. Lipomatous overgrowth may occur ipsilateral or contralateral to a vascular malformation, if present. The degree of intellectual disability appears to be mostly related to the presence and severity of seizures, cortical dysplasia (e.g., polymicrogyria), and hydrocephalus. Many children have feeding difficulties that are often multifactorial in nature. Endocrine issues affect a small number of individuals and most commonly include hypoglycemia (largely hypoinsulinemic hypoketotic hypoglycemia), hypothyroidism, and growth hormone deficiency. The diagnosis of PROS is established in a proband with suggestive findings and a heterozygous mosaic (or rarely, constitutional) activating pathogenic variant in PIK3CA. Sequence analysis of DNA derived from clinically affected tissue samples ‒ preferably from freshly obtained dermal biopsy overlying an affected area, from surgical excision of the overgrown tissue, or from uncultured tissues (such as skin fibroblasts or other tissues) ‒ should be prioritized for genetic testing. Targeted capture of the entire PIK3CA coding region followed by next-generation sequencing at very deep coverage is recommended for somatic variant detection, as it allows for detection of very low levels of mosaicism throughout the gene. Targeted therapy: Alpelisib (VIJOICE®) 50 mg orally with food once a day (at about the same time every day) for those between age two years and <18 years with PROS. In those age six years or older, the dose may be increased to 125 mg once a day after 24 weeks. A starting dose of 250 mg orally with food once a day (at about the same time every day) has been approved for those age ≥18 years. Alpelisib has been approved specifically for the reduction of overgrowth, vascular lesions, and other functional complications. To date, it is unknown whether this drug has any efficacy in treating the neurologic manifestations of PROS (as, e.g., in MCAP syndrome). Supportive care: Significant or lipomatous segmental overgrowth may require debulking; scoliosis and leg-length discrepancy may require orthopedic care and surgical intervention. Neurologic complications (e.g., obstructive hydrocephalus, increased intracranial pressure, progressive and/or symptomatic cerebellar tonsillar ectopia or Chiari malformation, and epilepsy in those with brain overgrowth/malformations) may warrant neurosurgical intervention. Depending on the type of vascular malformations, sclerotherapy, laser therapy, or oral medications such as sirolimus may be used. Similarly, lymphatic malformations may be treated through oral medications or careful surgical debulking, preferably by a vascular anomalies team. For those with pain, evaluation for the source of pain and treatment of the underlying cause is recommended. For those with growth hormone deficiency, evaluation of the hypothalamic-pituitary-adrenal axis is warranted; a trial of growth hormone therapy may be considered with careful monitoring of linear growth and overgrowth. Severe persistent hypoglycemia has been reported, and requires evaluation and ongoing treatment, which can include cornstarch administration. Routine treatment of the following, when present, is indicated: cardiac and renal abnormalities; intellectual disabilities and behavior issues; polydactyly and foot deformities; coagulopathy or thrombosis; Wilms tumor; and hypothyroidism. Surveillance: At each visit: measurement of growth parameters including head circumference, length of arms, hands, legs, and feet; assess for new neurologic manifestations (seizures, changes in tone, and other signs/symptoms of Chiari malformation); monitor developmental progress and behavior; assess motor skills; clinical assessment for scoliosis and abdominal examination for organomegaly and/or abdominal masses. Serial head MRI imagining is recommended, with frequency based on the severity of findings on initial assessment and the degree of brain maturation. For those with CNS overgrowth or dysplasia, brain MRI every six months until age two years and then annually until age eight years to monitor specifically for progressive hydrocephalus and Chiari malformation. As clinically indicated: clinical assessment and monitoring of any vascular and/or lymphatic malformations; radiographs of the limbs in those with overgrowth of a limb or portion of a limb; ultrasound or MRI follow up in those with truncal overgrowth; spinal MRI in those with scoliosis or deformities that affect the spine; blood glucose monitoring and evaluation of the hypothalamic-pituitary-adrenal axis for those with persistent hypoglycemia, particularly if they require ongoing treatment for hypoglycemia. Hematology consultation with recommendations for assessment for thrombosis and coagulopathy risk after any surgical intervention, particularly in those with the CLOVES phenotype and/or those with vascular malformations. Consideration of renal ultrasound every three months until age eight years (tumor screening for Wilms tumor is controversial). PROS disorders are not known to be inherited, as most identified pathogenic variants are somatic (mosaic). No confirmed vertical transmission or sib recurrence has been reported to date. The risk to sibs of a proband with somatic mosaicism for a pathogenic variant in PIK3CA would be expected to be the same as in the general population. All but a few affected individuals with PROS have had somatic mosaicism for a PIK3CA pathogenic variant, suggesting that mutation occurred post fertilization in one cell of the multicellular embryo. Therefore, the risk for transmission to offspring is expected to be less than 50%.