Somatic MAP2K1 Mutations Are Associated with Extracranial Arteriovenous Malformation

Abstract

Arteriovenous malformation (AVM) is a fast-flow, congenital vascular anomaly that may arise anywhere in the body. AVMs typically progress, causing destruction of surrounding tissue and, sometimes, cardiac overload. AVMs are difficult to control; they often re-expand after embolization or resection, and pharmacologic therapy is unavailable. We studied extracranial AVMs in order to identify their biological basis. We performed whole-exome sequencing (WES) and whole-genome sequencing (WGS) on AVM tissue from affected individuals. Endothelial cells were separated from non-endothelial cells by immune-affinity purification. We used droplet digital PCR (ddPCR) to confirm mutations found by WES and WGS, to determine whether mutant alleles were enriched in endothelial or non-endothelial cells, and to screen additional AVM specimens. In seven of ten specimens, WES and WGS detected and ddPCR confirmed somatic mutations in mitogen activated protein kinase kinase 1 (MAP2K1), the gene that encodes MAP-extracellular signal-regulated kinase 1 (MEK1). Mutant alleles were enriched in endothelial cells and were not present in blood or saliva. 9 of 15 additional AVM specimens contained mutant MAP2K1 alleles. Mutations were missense or small in-frame deletions that affect amino acid residues within or adjacent to the protein's negative regulatory domain. Several of these mutations have been found in cancers and shown to increase MEK1 activity. In summary, somatic mutations in MAP2K1 are a common cause of extracranial AVM. The likely mechanism is endothelial cell dysfunction due to increased MEK1 activity. MEK1 inhibitors, which are approved to treat several forms of cancer, are potential therapeutic agents for individuals with extracranial AVM.

Keywords: MAP2K1; MEK1; arteriovenous malformation; vascular anomaly.

Figure 1

Solitary Extracranial AVMs (A and B) Photograph of participant 23 during (A) childhood (stage I) and (B) adulthood (stage III). Note progressive growth of the facial AVM. (C) Coronal magnetic resonance image illustrates the extent of the lesion with multiple signal voids consistent with fast-flow (white arrows). (D) Angiogram showing tortuous arteries (white arrows) that feed the AVM, the “nidus” (dotted oval) where there are direct communications between numerous small arteries and veins, and early filling of draining veins (black arrows). (E) Hematoxylin and eosin-stained section of participant 23’s affected tissue, obtained after initial resection. Note the large feeder artery (asterisk), hyper-muscularized veins (arrows), and an area where arteries and veins connect in the absence of a normal capillary bed (dotted oval). (F) Participant 2, scalp AVM (stage III). (G) Participant 6, ear AVM (stage I). (H) Participant 12, upper lip AVM (stage I). (I) Participant 19, abdominal AVM (stage II).

Figure 2

Somatic Mutation Detection in AVMs after Whole-Exome Sequencing (A) Graph depicts the depth-of-coverage across the exome for ten affected and three unaffected tissue samples. Note ≥90-fold coverage for 90% of the exome obtained for each AVM sample. (B) Integrative Genomic Viewer screenshots showing reads containing variant and reference alleles for four AVM samples. Total read depth at the site of the somatic mutation is indicated as “-fold” coverage. Note 4 reads in participant 19 indicate that the (c.159T>G) p.Phe53Leu and c.199G>T (p.Asp67Tyr) somatic mutations are in cis. (C) Schematic diagram of MEK1 with approximate locations of the D, negative regulatory, and core kinase domains indicated. Note AVM somatic mutations cluster near the negative regulatory domain. The orange arrows indicate that p.Phe53Leu and p.Asp67Tyr variant were found in cis in a single individual. All other variants were found in two or more study participants.

Figure 3

AVM Endothelial Cells Enriched for the MAP2K1 Mutation (A) Photograph of an upper labial AVM (participant 13) from which CD31⁺ and CD31⁻ cells were separated. (B) Angiogram prior to resection shows arteriovenous shunting. (C) ddPCR assay results performed on DNA extracted from the AVM (resected tissue), endothelial (CD31⁺), and non-endothelial (CD31⁻) cells, and peripheral blood. Droplets containing mutant only, mutant and wild-type, or wild-type only alleles appear in left upper, right upper, and right lower quadrants, respectively (empty droplets are in the left lower quadrant). Percentages of mutant alleles in each sample are indicated (droplet counts are provided in Table 2).