Somatic Activating PIK3CA Mutations Cause Venous Malformation.

Somatic mutations in TEK, the gene encoding endothelial cell tyrosine kinase receptor TIE2, cause more than half of sporadically occurring unifocal venous malformations (VMs). Here, we report that somatic mutations in PIK3CA, the gene encoding the catalytic p110α subunit of PI3K, cause 54% (27 out of 50) of VMs with no detected TEK mutation. The hotspot mutations c.1624G>A, c.1633G>A, and c.3140A>G (p.Glu542Lys, p.Glu545Lys, and p.His1047Arg), frequent in PIK3CA-associated cancers, overgrowth syndromes, and lymphatic malformation (LM), account for >92% of individuals who carry mutations. Like VM-causative mutations in TEK, the PIK3CA mutations cause chronic activation of AKT, dysregulation of certain important angiogenic factors, and abnormal endothelial cell morphology when expressed in human umbilical vein endothelial cells (HUVECs). The p110α-specific inhibitor BYL719 restores all abnormal phenotypes tested, in PIK3CA- as well as TEK-mutant HUVECs, demonstrating that they operate via the same pathogenic pathways. Nevertheless, significant genotype-phenotype correlations in lesion localization and histology are observed between individuals with mutations in PIK3CA versus TEK, pointing to gene-specific effects.

Non-hotspot PIK3CA mutations are more frequent in CLOVES than in common or combined lymphatic malformations.

BACKGROUND: Theragnostic management, treatment according to precise pathological molecular targets, requests to unravel patients' genotypes. We used targeted next-generation sequencing (NGS) or digital droplet polymerase chain reaction (ddPCR) to screen for somatic PIK3CA mutations on DNA extracted from resected lesional tissue or lymphatic endothelial cells (LECs) isolated from lesions. Our cohort (n = 143) was composed of unrelated patients suffering from a common lymphatic malformation (LM), a combined lymphatic malformation [lymphatico-venous malformation (LVM), capillaro-lymphatic malformation (CLM), capillaro-lymphatico-venous malformation (CLVM)], or a syndrome [CLVM with hypertrophy (Klippel-Trenaunay-Weber syndrome, KTS), congenital lipomatous overgrowth-vascular malformations-epidermal nevi -syndrome (CLOVES), unclassified PIK3CA-related overgrowth syndrome (PROS) or unclassified vascular (lymphatic) anomaly syndrome (UVA)]. RESULTS: We identified a somatic PIK3CA mutation in resected lesions of 108 out of 143 patients (75.5%). The frequency of the variant allele ranged from 0.54 to 25.33% in tissues, and up to 47% in isolated endothelial cells. We detected a statistically significant difference in the distribution of mutations between patients with common and combined LM compared to the syndromes, but not with KTS. Moreover, the variant allele frequency was higher in the syndromes. CONCLUSIONS: Most patients with an common or combined lymphatic malformation with or without overgrowth harbour a somatic PIK3CA mutation. However, in about a quarter of patients, no such mutation was detected, suggesting the existence of (an)other cause(s). We detected a hotspot mutation more frequently in common and combined LMs compared to syndromic cases (CLOVES and PROS). Diagnostic genotyping should thus not be limited to PIK3CA hotspot mutations. Moreover, the higher mutant allele frequency in syndromes suggests a wider distribution in patients' tissues, facilitating detection. Clinical trials have demonstrated efficacy of Sirolimus and Alpelisib in treating patients with an LM or PROS. Genotyping might lead to an increase in efficacy, as treatments could be more targeted, and responses could vary depending on presence and type of PIK3CA-mutation.

Characterization of ANGPT2 mutations associated with primary lymphedema.

Primary lymphedema is caused by developmental and functional defects of the lymphatic vascular system that result in accumulation of protein-rich fluid in tissues, resulting in edema. The 28 currently known genes causing primary lymphedema can explain <30% of cases. Angiopoietin 1 (ANGPT1) and ANGPT2 function via the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 and 2) receptor complex and α5ß1 integrin to form an endothelial cell signaling pathway that is critical for blood and lymphatic vessel formation and remodeling during embryonic development, as well as for homeostasis of the mature vasculature. By screening a cohort of 543 individuals affected by primary lymphedema, we identified one heterozygous de novo ANGPT2 whole-gene deletion and four heterozygous ANGPT2 missense mutations. Functional analyses revealed three missense mutations that resulted in decreased ANGPT2 secretion and inhibited the secretion of wild-type (WT)-ANGPT2, suggesting that they have a dominant-negative effect on ANGPT2 signaling. WT-ANGPT2 and soluble mutants T299M and N304K activated TIE1 and TIE2 in an autocrine assay in human lymphatic endothelial cells. Molecular modeling and biophysical studies showed that amino-terminally truncated ANGPT subunits formed asymmetrical homodimers that bound TIE2 in a 2:1 ratio. The T299M mutant, located in the dimerization interphase, showed reduced integrin α5 binding, and its expression in mouse skin promoted hyperplasia and dilation of cutaneous lymphatic vessels. These results demonstrate that primary lymphedema can be associated with ANGPT2 mutations and provide insights into TIE1 and TIE2 activation mechanisms.

No evidence of locus heterogeneity in familial microcephaly with or without chorioretinopathy, lymphedema, or mental retardation syndrome.

BACKGROUND: Microcephaly with or without chorioretinopathy, lymphedema, or mental retardation syndrome (MCLMR) is a rare autosomal dominant disorder with variable expressivity. It is characterized by mild-to-severe microcephaly, often associated with intellectual disability, ocular defects and lymphedema. It can be sporadic or inherited. Eighty-seven patients have been described to carry a mutation in KIF11, which encodes a homotetrameric motor kinesin, EG5. METHODS: We tested 23 unreported MCLMR index patients for KIF11. We also reviewed the clinical phenotypes of all our patients as well as of those described in previously published studies. RESULTS: We identified 14 mutations, 12 of which are novel. We detected mutations in 12 affected individuals, from 6 out of 6 familial cases, and in 8 out of 17 sporadic patients. Phenotypic evaluation of patients (our 26 + 61 earlier published = 87) revealed microcephaly in 91%, eye anomalies in 72%, intellectual disability in 67% and lymphedema in 47% of the patients. Unaffected carriers were rare (4 out of 87: 5%). Family history is not a requisite for diagnosis; 31% (16 out of 52) were de novo cases. CONCLUSIONS: All inherited cases, and 50% of sporadic cases of MCLMR are due to germline KIF11 mutations. It is possible that mosaic KIF11 mutations cause the remainder of sporadic cases, which the methods employed here were not designed to detect. On the other hand, some of them might have another mimicking disorder and genetic defect, as microcephaly is highly heterogeneous. In aggregate, KIF11 mutations likely cause the majority, if not all, of MCLMR.