Arteriovenous Malformation MAP2K1 Mutation Causes Local Cartilage Overgrowth by a Cell-Non Autonomous Mechanism

Abstract

Extracranial arteriovenous malformation (AVM) is most commonly caused by MAP2K1 mutations in the endothelial cell. The purpose of this study was to determine if local tissue overgrowth associated with AVM is caused by direct or indirect effects of the MAP2K1 mutation (i.e., cell-autonomous or cell-non autonomous). Because cartilage does not have blood vessels, we studied ear AVMs to determine if overgrown cartilage contained AVM-causing mutations. Cartilage was separated from its surrounding tissue and isolated by laser capture microdissection. Droplet digital PCR (ddPCR) was used to identify MAP2K1 mutations. MAP2K1 (p.K57N) variants were present in the tissue adjacent to the cartilage [mutant allele frequency (MAF) 6-8%], and were enriched in endothelial cells (MAF 51%) compared to non-endothelial cells (MAF 0%). MAP2K1 mutations were not identified in the overgrown cartilage, and thus local cartilage overgrowth likely results from the effects of adjacent mutant blood vessels (i.e., cell-non autonomous).

Figure 1

Study cohort of patients with auricular AVMs. All subjects have diffuse AVMs involving all components of the entire ear (i.e., skin, subcutaneous adipose, cartilage). MRI shows enlarged cartilage (low signal, stars). Intraoperative images (panels A and C) illustrate overgrown conchal cartilage that was removed as part of an otoplasty procedure to improve the appearance of the ear. Intraoperative photo for panel B shows separation of excised cartilage from surrounding skin and subcutaneous tissue. A = Patient 1. B = Patient 2. C = Patient 3.

Figure 2

Somatic MAP2K1 mutations are isolated to the skin and subcutis of ear AVM tissue. (A) Laser capture microdissection of cartilage from surrounding tissue to minimize inclusion of adjacent microscopic vessels containing mutant endothelial cells (Alcian Blue stain; Patient 2). Top panel = pre-microdissection, bottom panel = post-microdissection. B,C,D = Patient 1, 2, 3 ddPCR graphs of their AVM ear tissue. Top row of graphs = skin and subcutaneous adipose. Bottom row of graphs = cartilage. Left upper blue droplets contain mutant alleles. Right middle orange droplets have mutant and wild-type alleles. Right lower green droplets contain wild-type alleles. Left lower black droplets are empty. Note absence of mutant droplets in the cartilage graphs.

Figure 3

Histological appearance of overgrown AVM cartilage and normal cartilage is similar. Sections of (A) conchal ear cartilage from a patient with an AVM (Patient 3). (B) Control conchal ear cartilage from a patient with a normal ear. Sections show equivalent distribution and cellularity of chondrocytes in a chondromyxoid matrix. The chondrocytes have normal appearance with monomorphic pyknotic nuclei. (Hematoxylin and eosin stain, 20x magnification, scale bar 20 µm).