Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome

Abstract

Congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) is a sporadically occurring, nonhereditary disorder characterized by asymmetric somatic hypertrophy and anomalies in multiple organs. We hypothesized that CLOVES syndrome would be caused by a somatic mutation arising during early embryonic development. Therefore, we employed massively parallel sequencing to search for somatic mosaic mutations in fresh, frozen, or fixed archival tissue from six affected individuals. We identified mutations in PIK3CA in all six individuals, and mutant allele frequencies ranged from 3% to 30% in affected tissue from multiple embryonic lineages. Interestingly, these same mutations have been identified in cancer cells, in which they increase phosphoinositide-3-kinase activity. We conclude that CLOVES is caused by postzygotic activating mutations in PIK3CA. The application of similar sequencing strategies will probably identify additional genetic causes for sporadically occurring, nonheritable malformations.

Figure 1

Clinical Features of CLOVES Syndrome (A) Participant CL5 at age 15 years. Note large, bilateral, posterior thoracic fatty masses with overlying capillary malformation on the right side. (B) Participant CL2 at age 18 months. She has overgrowth of her lower extremities, polydactyly, and wide feet with an expanded first interdigital space. (C) A sagittal T1-MRI (magnetic resonance image) of participant CL5 demonstrates cervicothoracic lipomatous overgrowth (straight arrows) extending into the posterior mediastinum and paraspinal region (bent arrow) and scoliosis. (D) A coronal postcontrast T1-MRI of participant CL3 shows bilateral truncal (short arrows) and mediastinal (bent arrows) fatty overgrowth, phlebectasia (long arrow), scoliosis, and asymmetrical kidneys due to right renal hypoplasia (notched arrows).

Figure 2

Somatic Activating PIK3CA Mutations in CLOVES Syndrome (A) Demonstration of the PIK3CA c.1624G>A somatic mosaic mutation in participant CL4. Massively parallel sequencing of this individual's lesional tissue identified a c.1624G>A mutation in 2 of 16 reads (Table 2). With blood DNA and lesional-tissue DNA as templates, PCR amplimers encompassing the candidate mutation were generated and Sanger sequenced. On the top left is an electropherogram of blood-DNA amplimers showing only a wild-type sequence. On the top right is an electropherogram of lesional-tissue amplimers showing wild-type and mutant sequences. Blood and lesional-tissue amplimers were subcloned, and 48 individual colonies were sequenced. In the middle on the left, all subclones from blood-DNA amplimers contain a wild-type sequence; a representative electropherogram of a wild-type sequence from a single clone is shown. In the middle on the right, 3 of 48 subclones from lesional-DNA amplimers contain a mutant sequence; a representative electropherogram of a mutant sequence from a single clone is shown. With mRNA recovered from lesional tissue as a template, RT-PCR was performed with primers in exons flanking the exon containing the candidate mutation. On the bottom left, a Sanger-sequence electropherogram of the RT-PCR amplimers shows wild-type and mutant sequences. Five of the 48 subclones from the RT-PCR amplimers contain a mutant sequence, one of which is shown at the bottom right. (B) A schematic of the PI3K-AKT signaling pathway indicates the overgrowth syndromes currently associated with mutations in this pathway. Binding of a growth factor to a receptor-tyrosine kinase activates a PI3K family member, including PIK3CA, which converts phosphatidylinositol-3,4-bisphosphate (PIP2) to the −3,4,5-triphosphate (PIP3) in a reaction that is antagonized by PTEN. Membrane-associated PIP3 facilitates the localization and phosphorylation of PDK1, which then activates AKT by phosphorylation at Thr308. AKT is further activated by Ser742 phosphorylation by the PDK2 complex including mTOR (FRAP1 [MIM 601231]). The following abbreviation is used: PTEN-HTS, PTEN Hamartoma Tumor Syndrome. (C) PIK3CA mutations constitutively activate the PI3K-AKT pathway. In the first column are immunoblot luminescence images of protein lysates prepared from normal subcutaneous adipose tissue. In the second column is hamartomatous tissue from an individual with a known PTEN mutation. In the right columns is lipomatous tissue from CLOVES-affected participants CL5 and CL6 (the blank lane between samples was removed). Lysates were separated by 4%–8% SDS-PAGE, transferred to immobilon P, and immunodetected with antibodies (Cell Signaling, Cambridge, MA, USA) that recognize total AKT, phosphorylated forms of PDK1 and AKT1, and beta-actin (as a loading control). Compared to lysates from adipose tissue from an unaffected individual or lysates from lesional tissue from an individual with a heterozygous PTEN mutation, lysates from the CLOVES lipomatous tissue show marked increases in the activated forms of PDK1 and AKT1.

Figure 3

Detection of PIK3CA Somatic Mosaicism in Multiple Tissue Types (A) At 18 years of age, participant CL6 shows overgrowth of the lower limbs, right-foot polydactyly, and lymphatic and venous anomalies of the lower-left extremity. (B) The participant's resected lower limb. The scale bar represents 10 cm. (C) The participant's radiograph (after venous contrast injection) demonstrates a dilated and aberrant venous system. (D–M) Resection specimen and photomicrographs of hematoxylin- and eosin-stained sections from sampled areas used for DNA isolation. The locations of tissue sampling are indicated by boxes. The scale bar indicates 200 μm. (D) A transverse section at the base of the calf shows massive lipomatous overgrowth involving subcutaneous tissue and skeletal muscle (I) and obliteration of tissue planes. The stranded appearance of subcutaneous tissue is due to extensive lymphatic malformation (H). The asterisk shows an abnormally dilated vein that was dissected (E) and sampled (J). (F) A metatarsal-phalangeal joint with degenerative articular changes (top) and synovial expansion by fat and venous malformation. Also shown are sampled sections of the total joint (K), synovium with fat and venous malformation (L), and articular cartilage and bone (M). (G) A transmetatarsal section shows extensive involvement of lipomatous overgrowth and lymphatic malformation. (N) A tube containing adipocytes (yellow layer) freshly isolated after collagenase treatment by centrifugation. DNA was amplified from each of the aforementioned tissues. Amplimers were subcloned, and the frequency of mutant alleles was determined. The numbers and percentages of mutant alleles are listed below the corresponding tissue samples.