Somatic PDGFRB Activating Variants in Fusiform Cerebral Aneurysms

Abstract

The role of somatic genetic variants in the pathogenesis of intracranial-aneurysm formation is unknown. We identified a 23-year-old man with progressive, right-sided intracranial aneurysms, ipsilateral to an impressive cutaneous phenotype. The index individual underwent a series of genetic evaluations for known connective-tissue disorders, but the evaluations were unrevealing. Paired-sample exome sequencing between blood and fibroblasts derived from the diseased areas detected a single novel variant predicted to cause a p.Tyr562Cys (g.149505130T>C [GRCh37/hg19]; c.1685A>G) change within the platelet-derived growth factor receptor β gene (PDGFRB), a juxtamembrane-coding region. Variant-allele fractions ranged from 18.75% to 53.33% within histologically abnormal tissue, suggesting post-zygotic or somatic mosaicism. In an independent cohort of aneurysm specimens, we detected somatic-activating PDGFRB variants in the juxtamembrane domain or the kinase activation loop in 4/6 fusiform aneurysms (and 0/38 saccular aneurysms; Fisher's exact test, p < 0.001). PDGFRB-variant, but not wild-type, patient cells were found to have overactive auto-phosphorylation with downstream activation of ERK, SRC, and AKT. The expression of discovered variants demonstrated non-ligand-dependent auto-phosphorylation, responsive to the kinase inhibitor sunitinib. Somatic gain-of-function variants in PDGFRB are a novel mechanism in the pathophysiology of fusiform cerebral aneurysms and suggest a potential role for targeted therapy with kinase inhibitors.

Keywords: PDGFRB; aneurysm; cerebral aneurysm; exome; fusiform; genetics; mosaic; mosaicism; saccular; sequencing.

Figure 1

Index Individual Phenotype and PDGFRB Genotype (A) A Body map of skin mosaicism (in red) and the specimens used for exome sequencing. (B) Cutaneous appearance. (C) An angiogram 3D reconstruction of a right vertebral injection, illustrating a giant vertebral fusiform aneurysm. (D–I) Abnormal extra-cranial soft-tissue vasculature associated with the occipital artery (Specimen MOS-WES-3). Low power (4×) (D) and medium power (10×) (E) magnification of a hematoxylin and eosin (H&E)-stained slide demonstrating a markedly affected vessel with focal evidence of dissection (arrowhead). There is severe intimal hyperplasia, and the tunica media (^∗) becomes markedly attenuated. Low power (4×) (F) and medium power (10×) (G) magnification of a Gomori trichrome (GT)-stained slide with the intima (^∗∗) and the tunica media (^∗) highlighted. Low power (4×) (H) and medium power (10×) (I) magnification of a Verhoeff-Van Gieson (VVG)-stained slide. The internal elastic lamina (^∗) associated with the relatively better-preserved fragment of tunica media, as well several areas with attenuated internal elastic lamina (^∗∗), are highlighted. (J) An H&E-stained slide of an unremarkable left arm radial artery from specimen MOS-WES-6 at medium power magnification (10×). A well-defined intimal layer, the tunica media, and the tunica adventitia, along with an intact internal elastic lamina (^∗), are visualized.(K) Specimens used for exome sequencing and coverage of the p.Tyr562Cys variant. (L) Next-generation sequencing reads across the area of the missense variant; the variant nucleotides (C) are in blue. The reference nucleotide and amino acid sequences are at the bottom.

Figure 2

Sporadic Fusiform Aneurysms Harbor PDGFRB Variants (A) Demographics of individuals with sporadic fusiform aneurysms and their variants, including age at treatment. (B, C, and F) An angiogram and/or 3D reconstruction from angiogram-representative images illustrating the fusiform morphology. (D and E) Representative H&E-stained sections of specimen VAL-44 at low (1.25×) and medium (10×) magnification showing a markedly affected vessel with vascular wall attenuation, an intraluminal thrombus with early organization, and a dissecting hemorrhage. The tunica media (^∗) is focally present and becomes attenuated (^∗∗). (G) H&E-stained sections at low magnification (1.25×) of specimen VAL-61 showing a representative portion of the 3 cm aneurysm with a large, partially organizing thrombus and a markedly attenuated vascular wall. Abbreviations are as follows: AF = allele frequency; ICA = internal carotid artery; MCA = middle cerebral artery; PCA = posterior cerebral artery; and RCCA = right common carotid artery.

Figure 3

Variants in PDGFRB Within the Juxtamembrane Region and the Kinase Activation Loop Found in Fusiform Aneurysms (A) A schematic representation of the PDGFRB protein, the amino acid sequence of the two hotspots, and the location of variants. Germline and somatic PDGFRB variants with known or implied functional consequences in other syndromes and diseases are included for comparison. (B) Homologous juxtamembrane amino acid sequences for KIT, PDGFRA, and PDGFRB and the location of aneurysm variants. (C) Homologous kinase-domain activation-loop amino acid sequences for KIT, PDGFRA, and PDGFRB and the location of aneurysm mutations. (D) All somatic variants with possible activating consequences (missense and in-frame insertions and deletions) reported in the COSMIC database for KIT, PDGFRA, and PDGFRB. Notice the increased frequency of variants in both the juxtamembrane region and the kinase activation loop of KIT and PDGFRA. There is a comparative lack of variants reported in PDGFRB.

Figure 4

PDGFRB Variants are Constitutively Phosphorylated, Sensitive to Sunitinib Kinase Inhibition, and Activate Downstream Signaling Pathways (A) An immunoblot analysis of non-starved normal (wild-type PDGFRB) and mosaic-affected Tyr562Cys fibroblast cells from the index individual. (B) An immunoblot analysis of starved and PDGF-BB-stimulated normal (wild-type PDGFRB) and mosaic-affected Tyr562Cys fibroblast cells from the index individual. (C) An immunoblot analysis of HEK cells stably expressing the described aneurysm variants and Trp566Arg (IM; gain of function) and Asp844Gly (PFBC; loss of function) control variants showing varying levels of phosphorylation and expression of PDGFRB. (D) Sensitivity of PDGFRB auto-phosphorylation to sunitinib. Band intensity was quantified compared to beta-actin, and relative band densitometry is presented as the mean ± SEM of three separate blots in triplicate.