EVENT: David W. Smith Workshop on Malformations & Morphogenesis – Sept. 9-14 in Lake Arrowhead, CA

WashU physician, and GPS collaborator, Dr. Kathy Grange has been selected for a platform presentation at this prestigious clinical genetics meeting. One of the themes for this year’s meeting is germline and somatic mosaicism, and she will be highlighting data from our SOMA sequencing tests.

For more information about the David W. Smith Workshop, please visit their website.


Detection of Somatic Variants in Segmental Overgrowth Syndromes Utilizing Next Generation Sequencing

Dorothy K. Grange MD1, Meagan M. Corliss CGC2, Jonathan W. Heusel MD PhD2,Vishwanathan Hucthagowder PhD2 and Catherine E. Cottrell PhD2,

1Division of Genetics and Genomic Medicine, Department of Pediatrics and 2Division of Laboratory and Genomic Medicine, Department of Pathology and Immunology, Washington University School of Medicine

Next-generation sequencing (NGS) is widely used in the clinical setting for genetic variant analysis in human disease. It is increasingly recognized that somatic genetic variation is associated with congenital malformation syndromes. NGS technology has utility in the detection of somatic alterations present at low variant allele frequency (VAF) due to its ability to achieve high sequencing read depths. Somatic variation has been described in Proteus and PIK3CA-related overgrowth syndromes. Because of the somatic nature of these disorders, the causative alteration is detected only in limited tissues, and is typically not identifiable in blood. Genomics and Pathology Services at Washington University (GPS@WUSTL) performs clinical testing for somatic overgrowth and related disorders, by single gene analysis or by a panel (SOMA gene set) of 11 genes (AKT1, AKT2, AKT3, GNAQ, MTOR, PIK3CA, PIK3R2, PTEN, RASA1, TSC1 and TSC2).

Case Examples: Patient 1 is a 2-year-old boy with a vascular malformation on his right face and scalp present at birth. The right face and body are larger than the left. He has 3-4 syndactyly of the right hand and bilateral 2-3 toe syndactyly. Brain MRI showed an asymmetrically enlarged right cerebral hemisphere and right lateral ventricle consistent with hemi-megalencephaly. Development has been normal. Two tissue sources, swabbed buccal mucosal cells and blood, were subjected to NGS. A PIK3CA alteration, p.P104L, was identified with a variant allele frequency (VAF) of approximately 37% in the specimen derived from buccal cells. Manual review of the sequencing data from the blood sample revealed a VAF of <1%. This mutation has been previously reported in breast cancer, but not in association with somatic overgrowth syndromes. Notably, this mutation has subsequently been identified in a second patient with suspected MCAP and polymicrogyria. Patient 2 is a 6-year-old girl with overgrowth involving her lower torso, specifically the buttocks, lower extremities and feet. She also has lipomatous masses on her back. She had syndactyly and macrodactyly of the toes. She is normal developmentally. Three tissue types were analyzed by NGS including blood, buccal scraping and fresh tissue, the latter representing lipomatous soft tissue from a toe. A PIK3CA p.C420R alteration was identified only in the affected tissue from the foot with a VAF of 29%. Blood and buccal samples yielded no evidence of the variant allele. The p.C420R variant has been observed in many tumors and in CLOVES. Patient 3 is a 16-year-old female with megalencephaly, hydrocephalus, strabismus, generalized overgrowth, body asymmetry, vascular skin lesions, 2-3-4 toe syndactyly and intellectual disabilities. She had a posterior tongue mass excised that was a lymphatic malformation. A PIK3CA p.M1043I variant at a near heterozygous allelic frequency of 46.8% was identified in formalin fixed tissue derived from the tongue mass. Analysis of blood revealed the variant at an estimated frequency of <20%. This variant has been reported in MCAP and in various cancers. Patient 4 is a 6-year-old boy with hemi-megalencephaly with intractable seizures who underwent right hemispherectomy, massive overgrowth of the lower extremities, extensive cutaneous vascular malformations, subcutaneous soft tissue masses and developmental disabilities. NGS on fresh tissue from the legs showed a pathogenic PIK3CA p.E545K variant in a very low percentage of total reads. However, DNA extracted from formalin fixed brain cortical and white matter tissue confirmed the variant in a higher percentage of reads. Patient 5

NGS testing was performed on 39 total cases through the GPS laboratory, 28 with the broader SOMA gene set (7-11 genes including PIK3CA) and 11 cases on PIK3CA only. Of these, 25/39 (64.1%) had a pathogenic or likely pathogenic variant identified, ascertained in 18/28 SOMA cases and 7/11 PIK3CA only cases. The vast majority of disease-causing mutations (23/25; 92%) were identified in PIK3CA. SOMA testing demonstrates clear clinical utility in the setting of overgrowth syndromes and exhibits a high diagnostic yield when performed on appropriate specimen types using a deep sequencing approach.