Exceptional aggressiveness of cerebral cavernous malformation disease associated with PDCD10 mutations.

PurposeThe 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.MethodsWe 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.ResultsWe 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.ConclusionThese 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

Single-nucleus DNA sequencing reveals hidden somatic loss-of-heterozygosity in Cerebral Cavernous Malformations

Cerebral Cavernous Malformations (CCMs) are vascular malformations of the central nervous system which can lead to moderate to severe neurological phenotypes in patients. A majority of CCM lesions are driven by a cancer-like three-hit mutational mechanism, including a somatic, activating mutation in the oncogene PIK3CA, as well as biallelic loss-of-function mutations in a CCM gene. However, standard sequencing approaches often fail to yield a full complement of pathogenic mutations in many CCMs. We suggest this reality reflects the limited sensitivity to identify low-frequency variants and the presence of mutations undetectable with bulk short-read sequencing. Here we report a single-nucleus DNA-sequencing approach that leverages the underlying biology of CCMs to identify lesions with somatic loss-of-heterozygosity, a class of such hidden mutations. We identify an alternative genetic mechanism for CCM pathogenesis and establish a method that can be repurposed to investigate the genetic underpinning of other disorders with multiple somatic mutations

Natural genetic variation of integrin alpha L (Itgal) modulates ischemic brain injury in stroke.

During ischemic stroke, occlusion of the cerebrovasculature causes neuronal cell death (infarction), but naturally occurring genetic factors modulating infarction have been difficult to identify in human populations. In a surgically induced mouse model of ischemic stroke, we have previously mapped Civq1 to distal chromosome 7 as a quantitative trait locus determining infarct volume. In this study, genome-wide association mapping using 32 inbred mouse strains and an additional linkage scan for infarct volume confirmed that the size of the infarct is determined by ancestral alleles of the causative gene(s). The genetically isolated Civq1 locus in reciprocal recombinant congenic mice refined the critical interval and demonstrated that infarct size is determined by both vascular (collateral vessel anatomy) and non-vascular (neuroprotection) effects. Through the use of interval-specific SNP haplotype analysis, we further refined the Civq1 locus and identified integrin alpha L (Itgal) as one of the causative genes for Civq1. Itgal is the only gene that exhibits both strain-specific amino acid substitutions and expression differences. Coding SNPs, a 5-bp insertion in exon 30b, and increased mRNA and protein expression of a splice variant of the gene (Itgal-003, ENSMUST00000120857), all segregate with infarct volume. Mice lacking Itgal show increased neuronal cell death in both ex vivo brain slice and in vivo focal cerebral ischemia. Our data demonstrate that sequence variation in Itgal modulates ischemic brain injury, and that infarct volume is determined by both vascular and non-vascular mechanisms

The strain-specific difference in <i>Itgal</i> message level is determined by <i>cis</i>-acting elements at the <i>Civq1</i> locus.

<p>A. mRNA levels of the canonical isoform of <i>Itgal (Itgal-002)</i> in adult cortex from B6, BALB/c, SWR, and CSS7 strains as determined by qRT-PCR. Each bar represents mean ± SEM. ** <i>P<0.001</i> vs. B6. B. The allele-specific expression ratio of <i>Itgal</i> transcript (<i>Itgal</i>-002) levels in F1 (B6×BALB) mice. A coding SNP (rs107634043) in exon 30 was used to monitor the B6-allele and BALB/c-allele transcripts of <i>Itgal</i> in macrophages (MΦ), endothelial cells (EC), P1 and the adult cerebral cortices. The level of the B6-specific <i>Itgal</i> transcript was approximately 6-fold higher than BALB/c-transcript in adult cortex. Each bar represents the allele-specific expression ratio (B6/BALB) averaged for three F1 animals. C. Surface expression of CD11a (ITGAL) on CD11a+ brain immune cells. CD45+ CD11a+ brain cells from BALB/c mice express significantly less surface CD11a as assessed by flow cytometric staining than B6 CD45+ CD11a+ brain cells (** <i>P<0.001</i>).</p

<i>Civq1</i> on chromosome 7 re-identified in intercrosses between multiple mouse strains.

<p>A. Genome-wide EMMA analysis across chromosome 7 for cerebral infarct volume in 32 inbred mouse strains. The plot is drawn for −log(P) against SNP positions on the chromosome. Green crosses represent SNPs over the significance threshold of P = 10⁻⁵. The genomic region (132.35–134.81 Mb) with significant association in chromosome 7 maps within <i>Civq1</i> detected by multiple linkage studies. B. The graph presents the results of a genome-wide linkage scan for infarct volume in 35 (FVB×BALB) F2 progeny. Chromosomes 1 through X are represented numerically on the x-axis and the y-axis represents the LOD score. The significant (<i>P<0.05</i>) level of linkage is determined by 1,000 permutation tests. Only a single genomic region on chromosome 7 displays significant linkage to infarct volume with a LOD score of 5.2. C. <i>Civq1</i> is mapped to distal chromosome 7 by linkage analyses in multiple F2 (B6×BALB/c, B6×SWR, and FVB×BALB/c) populations. The same SNP marker (rs13479513) is located at the peaks of all linkage analyses.</p

Infarct volume is increased in <i>Itgal</i> knockout (KO) mice.

<p>A. Collateral number does not differ between B6 and <i>Itgal</i> KO mice. B. The posterior faces of representative 1-mm coronal sections from B6 and <i>Itgal</i> KO mice are shown. The size of the infarct in <i>Itgal</i> KO is larger than B6 mice. C. <i>Itgal</i> KO mice on the B6 background exhibit infarct volume ∼3-fold larger than that of B6 mice (15.9 vs. 4.7 mm³). ** <i>P<0.001</i>. D. Increased neuronal death in the cortical region of the brain slices from <i>Itgal</i> KO mice compared to control B6 mice 24 hr after 5.5 min OGD. E. Deficiency of <i>Itgal</i> increases neuronal cell death by ∼50% compared to B6 mice. Values represent mean ± SEM from at least 5 animals per group. ** <i>P<0.001</i>.</p

The critical interval of chromosome 7 retains both infarct volume and collateral artery number phenotypes in congenic animals.

<p>A. Schematic map of two <i>Civq1</i> congenic lines carrying segments of B6 chromosome 7 introgressed into the BALB/c background, and heterozygous BALB/c alleles on B6 background. The latter could only be maintained as a heterozygote with B6. B. The posterior faces of representative 1-mm coronal sections from the B6, C.B6-<i>Civq1</i>-3, and BALB/c mice are shown. The size of the infarct is much smaller in C.B6-<i>Civq1</i>-3 than control BALB/c mouse. C. Congenic mice (C.B6-<i>Civq1</i>-1 and -3) exhibit a reduction in infarct volume by ∼30% compared to BALB/c mice. Infarct volume in congenic mice carrying heterozygous BALB/c alleles on B6 background (B6.C-<i>Civq1</i>(HET)) is 2.5-fold larger than that of control B6 mice. ## <i>P<0.001</i> vs. BALB/c; *<i>P<0.05</i>, ** <i>P<0.001</i> vs. B6. Values represent mean ± SEM. D. Superficial angioarchitecture of the brain in 4-week old B6, C.B6-<i>Civq1</i>-3, and BALB/c mice. Magnified images of the box in the upper panels are shown in the lower panels. The red arrowheads indicate the vessels of pial anastomoses between the MCA and ACA. E. C.B6-<i>Civq1</i> congenic lines show an increase in the number of collaterals connecting the distal MCA and ACA compared control BALB/c strains. Heterozygous congenic mice show no difference compared to control B6 mice. ## <i>P<0.001</i> vs. BALB/c; ** <i>P<0.001</i> vs. B6. F. Cortical brain slices from B6, BALB/c, and C.B6-<i>Civq1</i>-3 mice biolistically transfected with an YFP expression plasmid under normal conditions and 24 hr after 5.5 min of OGD. G. C.B6-<i>Civq1</i>-3 mice exhibit ∼50% increased neuronal viability compared to control BABL/c mice. Total numbers of healthy and YFP-positive neurons in the cortical region of the brain slices were counted at 24 hr after transient OGD. Cell viability was normalized from their non-OGD controls. Values represent mean±SEM from at least 5 animals per group. * <i>P<0.05</i> and ** <i>P<0.001</i>.</p

Oxygen deprivation increases cleaved Caspase 3 in an <i>ex vivo</i> brain slice stroke model.

<p>A. Efficiency of <i>Itgal</i> knock-down in <i>ex vivo</i> brain slices using siRNA. Non-target or <i>Itgal</i>-specific siRNA were transfected into cortical brain slices from B6 mice for 3 days. <i>Itgal</i> mRNA levels normalized to <i>Gapdh</i> control were determined by qRT-PCR. Values represent the mean±SEM of 4 mice.***<i>P<0.01</i>. B. Western blots were performed for cleaved Caspase-3 in explanted brain slices transfected with either non-target siRNA or <i>Itgal</i>-specific siRNA in both control and oxygen deprivation (OD) conditions. C. Quantification of panel B. Cleaved Caspase-3 expression level was normalized to an alpha-tubulin control. Values represent the mean±SEM of 4 mice. *** <i>P<0.001</i>.</p