Exceptional aggressiveness of cerebral cavernous malformation disease associated with PDCD10 mutations

Abstract

Purpose: The phenotypic manifestations of cerebral cavernous malformation disease caused by rare PDCD10 mutations have not been systematically examined, and a mechanistic link to Rho kinase-mediated hyperpermeability, a potential therapeutic target, has not been established.

Methods: We analyzed PDCD10 small interfering RNA-treated endothelial cells for stress fibers, Rho kinase activity, and permeability. Rho kinase activity was assessed in cerebral cavernous malformation lesions. Brain permeability and cerebral cavernous malformation lesion burden were quantified, and clinical manifestations were assessed in prospectively enrolled subjects with PDCD10 mutations.

Results: We determined that PDCD10 protein suppresses endothelial stress fibers, Rho kinase activity, and permeability in vitro. Pdcd10 heterozygous mice have greater lesion burden than other Ccm genotypes. We demonstrated robust Rho kinase activity in murine and human cerebral cavernous malformation vasculature and increased brain vascular permeability in humans with PDCD10 mutation. Clinical phenotype is exceptionally aggressive compared with the more common KRIT1 and CCM2 familial and sporadic cerebral cavernous malformation, with greater lesion burden and more frequent hemorrhages earlier in life. We first report other phenotypic features, including scoliosis, cognitive disability, and skin lesions, unrelated to lesion burden or bleeding.

Conclusion: These findings define a unique cerebral cavernous malformation disease with exceptional aggressiveness, and they inform preclinical therapeutic testing, clinical counseling, and the design of trials.Genet Med 17 3, 188-196.

Figure 1. PDCD10 suppresses stress fibers, ROCK activity and permeability in vitro

HUVECs were treated with control or PDCD10 siRNA. (A) PDCD10 gene expression is reduced by 80% by PDCD10 siRNA in HUVECs as compared to those treated with control siRNA. Data bars are means ± SE. (B) Increased f-actin stress fibers by PDCD10 depletion is blunted by the ROCK inhibitor H-1152. Bar, 100 μm. (C) Increased pMLC activity by PDCD10 depletion is reversed by H-1152. (D) PDCD10 depletion during 4 and 24 hours increases monolayer permeability in transwell assays. H-1152 treatment reverses this increase, implying that PDCD10 inhibits ROCK-mediated monolayer leak. Data bars are means ± SE of n = 3. Analysis by ANOVA indicates *P<0.05, **P<0.01, ***P<0.001.

Figure 2. ROCK activity in CCM lesions from human subjects

There is greater ROCK activity in human PDCD10 CCM lesions than in human sporadic lesions as shown by brown pMLC and pMBS staining. Bars are 100 μm. The histogram shows that twice as many caverns have at least one endothelial cell stained with pMLC in human PDCD10 CCM lesions than in human KRIT1, CCM2 and sporadic lesions (*P<0.05). Data bars are means ± SE.

Figure 3. PDCD10 patients show a more aggressive phenotype

(A) Percent of bleed free PDCD10 patients versus age, showing high bleeding propensity in the first decade of life, leveling off in the teen years. The time of hemorrhage has been established for every adjudicated bleed. (B) Number of bleeds per PDCD10 patient (mean plotted, with standard error bar) vs. years since birth, after first symptom onset, and after first hemorrhage. (C) The age at first bleed is lower in PDCD10 patients than in KRIT1 and CCM2 patients. Data bars are means ± SE. (D) An SWI scan showing high lesion burden in a PDCD10 patient.

Figure 4. Phenotypic profile of PDCD10 patients

The cases are grouped by their respective proband (F1 to F12). Each subject’s lesion burden and clinical associations is indicated. Each symptomatic bleed is noted by a red vertical bar during the lifespan of each subject. First symptomatic onset is noted by a yellow vertical bar. Sp = spontaneous mutation, PI = parental inheritance, CD = cognitive decline, Sk = skin manifestation, Sc = scoliosis, T = tumor, SWI = number of lesions on susceptibility weighted imaging.