Somatic mosaicism in the MAPK pathway in sporadic brain arteriovenous malformation and association with phenotype.

OBJECTIVE: Sporadic brain arteriovenous malformation (BAVM) is a tangled vascular lesion characterized by direct artery-to-vein connections that can cause life-threatening intracerebral hemorrhage (ICH). Recently, somatic mutations in KRAS have been reported in sporadic BAVM, and mutations in other mitogen-activated protein kinase (MAPK) signaling pathway genes have been identified in other vascular malformations. The objectives of this study were to systematically evaluate somatic mutations in MAPK pathway genes in patients with sporadic BAVM lesions and to evaluate the association of somatic mutations with phenotypes of sporadic BAVM severity. METHODS: The authors performed whole-exome sequencing on paired lesion and blood DNA samples from 14 patients with sporadic BAVM, and 295 genes in the MAPK signaling pathway were evaluated to identify genes with somatic mutations in multiple patients with BAVM. Digital droplet polymerase chain reaction was used to validate KRAS G12V and G12D mutations and to assay an additional 56 BAVM samples. RESULTS: The authors identified a total of 24 candidate BAVM-associated somatic variants in 11 MAPK pathway genes. The previously identified KRAS G12V and G12D mutations were the only recurrent mutations. Overall, somatic KRAS G12V was present in 14.5% of BAVM lesions and G12D was present in 31.9%. The authors did not detect a significant association between the presence or allelic burden of KRAS mutation and three BAVM phenotypes: lesion size (maximum diameter), age at diagnosis, and age at ICH. CONCLUSIONS: The authors confirmed the high prevalence of somatic KRAS mutations in sporadic BAVM lesions and identified several candidate somatic variants in other MAPK pathway genes. These somatic variants may contribute to understanding of the etiology of sporadic BAVM and the clinical characteristics of patients with this condition.

Assessing the association of common genetic variants in EPHB4 and RASA1 with phenotype severity in familial cerebral cavernous malformation.

BACKGROUND: To investigate whether common variants in EPHB4 and RASA1 are associated with cerebral cavernous malformation (CCM) disease severity phenotypes, including intracranial hemorrhage (ICH), total and large lesion counts. METHODS: Familial CCM cases enrolled in the Brain Vascular Malformation Consortium were included (n = 338). Total lesions and large lesions (≥5 mm) were counted on MRI; clinical history of ICH at enrollment was assessed by medical records. Samples were genotyped on the Affymetrix Axiom Genome-Wide LAT1 Human Array. We tested the association of seven common variants (three in EPHB4 and four in RASA1) using multivariable logistic regression for ICH (odds ratio, OR) and multivariable linear regression for total and large lesion counts (proportional increase, PI), adjusting for age, sex, and three principal components. Significance was based on Bonferroni adjustment for multiple comparisons (0.05/7 variants = 0.007). RESULTS: EPHB4 variants were not significantly associated with CCM severity phenotypes. One RASA1 intronic variant (rs72783711 A>C) was significantly associated with ICH (OR = 1.82, 95% CI = 1.21-2.37, p = 0.004) and nominally associated with large lesion count (PI = 1.17, 95% CI = 1.03-1.32, p = 0.02). CONCLUSION: A common RASA1 variant may be associated with ICH and large lesion count in familial CCM. EPHB4 variants were not associated with any of the three CCM severity phenotypes.

Pilot investigation of circulating angiogenic and inflammatory biomarkers associated with vascular malformations.

BACKGROUND: Vascular malformations in the central nervous system are difficult to monitor and treat due to their inaccessible location. Angiogenic and inflammatory proteins are secreted into the bloodstream and may serve as useful biomarkers for identifying patients at risk for complications or with certain disease phenotypes. METHODS: A validated multiplex protein array consisting of 26 angiogenic and inflammatory biomarkers (Angiome) was assessed in plasma isolated from healthy controls and patients with either sporadic brain arteriovenous malformation (BAVM), familial cerebral cavernous malformation (CCM), or hereditary hemorrhagic telangiectasia (HHT). These samples were obtained from archives of ongoing research studies at the University of California San Francisco and through prospective collection at the Toronto HHT Centre at St. Michael's Hospital. RESULTS: We compared circulating biomarker levels from each patient group to healthy controls and analyzed each pairwise combination of patient groups for differences in biomarker levels. Additionally, we analyzed the HHT samples to determine the association between biomarker levels and the following HHT-specific phenotypes, BAVM, pulmonary arteriovenous malformation (PAVM), liver vascular malformation (LVM), and gastrointestinal (GI) bleeding. Compared to controls, levels of SDF1 were significantly elevated in HHT patients (Proportional Increase [PI] = 1.87, p < 0.001, q = 0.011). Levels of sENG were significantly reduced in HHT patients compared to controls (PI = 0.56, p < 0.001, q < 0.001), reflecting the prevalence of HHT1 patients in this cohort. Levels of IL6 (PI = 3.22, p < 0.001, q < 0.001) and sTGFßR3 (PI = 0.70, p = 0.001, q < 0.029) differed significantly in CCM patients compared to controls. Compared to controls, ten of the biomarkers were significantly different in sporadic BAVM patients (q-values < 0.05). Among the pairwise combinations of patient groups, a significant elevation was observed in TGFß1 in CCM patients compared to sporadic BAVM patients (PI = 2.30, p < 0.001, q = 0.034). When examining the association of circulating biomarker levels with HHT-specific phenotypes, four markers were significantly lower in HHT patients with BAVM (q-values < 0.05), and four markers were significantly higher in patients with LVM (q-values < 0.05). CONCLUSIONS: This pilot study suggests that the profile of circulating angiogenic and inflammatory biomarkers may be unique to each type of vascular malformation. Furthermore, this study indicates that circulating biomarkers may be useful for assessing phenotypic traits of vascular malformations.

Seizure Incidence Rates in Children and Adults With Familial Cerebral Cavernous Malformations.

BACKGROUND AND OBJECTIVES: Seizure incidence rates related to Familial Cerebral Cavernous Malformation (FCCM) are not well described, especially for children. To measure the seizure incidence rate, examine seizure predictors and characterize epilepsy severity, we studied a cohort of children and adults with FCCM enrolled in the Brain Vascular Malformation Consortium (BVMC). METHODS: Seizure data were collected from participants with FCCM in the BVMC at enrollment and during follow-up. We estimated seizure probability by age, and tested whether cerebral cavernous malformation (CCM) counts or genotype were associated with earlier seizure onset. RESULTS: The study cohort included 479 FCCM cases. Median age at enrollment was 42.5 years (Interquartile Range [IQR] 22.5-55.0) and 19% were children (<18 years old). Median large CCM count was 3 (IQR: 1-5). Among 393 with genotyping, mutations were: CCM1-Common Hispanic Mutations (88%), another CCM1 mutation (5%), CCM2 mutations (5%), and CCM3 mutations (2%). Prior to or during the study, 202 (42%) had a seizure. The cumulative incidence of a childhood seizure was 20.3% (95% CI 17.0 - 23.4) and by age 80 years was 60.4% (95% CI 54.2-65.7). More total CCMs (Hazard Ratio [HR] 1.24 per SD unit increase, 95% CI 1.1 - 1.4) or more large CCMs (HR=1.5 per SD unit increase, 95% CI 1.2-1.9) than expected for age and sex increased seizure risk. A CCM3 mutation also increased risk compared to other mutations (HR 3.11, 95% CI 1.15-8.45). Individuals with a seizure prior to enrollment had increased hospitalization rates during follow-up (Incidence Rate Ratio 10.9, 95% CI 2.41 - 49.32) compared to patients without a seizure history. DISCUSSION: Individuals with FCCM have a high seizure incidence, and those with more CCMs or CCM3 genotype are at greater risk. Seizures increase health care utilization in FCCM.

Cyclo-oxygenase 2, a putative mediator of vessel remodeling, is expressed in the brain AVM vessels and associates with inflammation.

BACKGROUND: Brain arteriovenous malformations (bAVM) may rupture causing disability or death. BAVM vessels are characterized by abnormally high flow that in general triggers expansive vessel remodeling mediated by cyclo-oxygenase-2 (COX2), the target of non-steroidal anti-inflammatory drugs. We investigated whether COX2 is expressed in bAVMs and whether it associates with inflammation and haemorrhage in these lesions. METHODS: Tissue was obtained from surgery of 139 bAVMs and 21 normal Circle of Willis samples. The samples were studied with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR). Clinical data was collected from patient records. RESULTS: COX2 expression was found in 78% (109/139) of the bAVMs and localized to the vessels' lumen or medial layer in 70% (95/135) of the bAVMs. Receptors for prostaglandin E2, a COX2-derived mediator of vascular remodeling, were found in the endothelial and smooth muscle cells and perivascular inflammatory cells of bAVMs. COX2 was expressed by infiltrating inflammatory cells and correlated with the extent of inflammation (r = .231, p = .007, Spearman rank correlation). COX2 expression did not associate with haemorrhage. CONCLUSION: COX2 is induced in bAVMs, and possibly participates in the regulation of vessel wall remodelling and ongoing inflammation. Role of COX2 signalling in the pathobiology and clinical course of bAVMs merits further studies.

Angiopoietin-2 predicts morbidity in adults with Fontan physiology.

Morbidity in patients with single-ventricle Fontan circulation is common and includes arrhythmias, edema, and pulmonary arteriovenous malformations (PAVM) among others. We sought to identify biomarkers that may predict such complications. Twenty-five patients with Fontan physiology and 12 control patients with atrial septal defects (ASD) that underwent cardiac catheterization were included. Plasma was collected from the hepatic vein and superior vena cava and underwent protein profiling for a panel of 20 analytes involved in angiogenesis and endothelial dysfunction. Ten (40%) of Fontan patients had evidence of PAVM, eighteen (72%) had a history of arrhythmia, and five (20%) were actively in arrhythmia or had a recent arrhythmia. Angiopoietin-2 (Ang-2) was higher in Fontan patients (8,875.4 ± 3,336.9 pg/mL) versus the ASD group (1,663.6 ± 587.3 pg/mL, p < 0.0001). Ang-2 was higher in Fontan patients with active or recent arrhythmia (11,396.0 ± 3,457.7 vs 8,118.2 ± 2,795.1 pg/mL, p < 0.05). A threshold of 8,500 pg/mL gives Ang-2 a negative predictive value of 100% and positive predictive value of 42% in diagnosing recent arrhythmia. Ang-2 is elevated among adults with Fontan physiology. Ang-2 level is associated with active or recent arrhythmia, but was not found to be associated with PAVM.

Association of common candidate variants with vascular malformations and intracranial hemorrhage in hereditary hemorrhagic telangiectasia.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is caused by mutations in TGFß/BMP9 pathway genes and characterized by vascular malformations (VM) including arteriovenous malformations (AVM) in lung, liver, and brain, which lead to severe complications including intracranial hemorrhage (ICH) from brain VM. The clinical heterogeneity of HHT suggests a role for genetic modifier effects. Common variants in loci that modify phenotype severity in Tgfb knockout mice were previously reported as associated with lung AVM in HHT. Common variants in candidate genes were reported as associated with sporadic brain AVM and/or ICH. We investigated whether these variants are associated with HHT organ VM or with ICH from brain VM in 752 Caucasian HHT patients enrolled by the Brian Vascular Malformation Consortium. METHODS: We genotyped 11 candidate variants: four variants reported as associated with lung AVM in HHT (PTPN14 rs2936018, USH2A rs700024, ADAM17 rs12474540, rs10495565), and seven variants reported as associated with sporadic BAVM or ICH (APOE ε2, ANGPTL4 rs11672433, EPHB4 rs314308, IL6 rs1800795, IL1B rs1143627, ITGB8 rs10486391, TNFA rs361525). Association of genotype with any VM, lung AVM, liver VM, brain VM or brain VM ICH was evaluated by multivariate logistic regression adjusted for age, gender, and family clustering. RESULTS: None of the 11 variants was significantly associated with any phenotype. There was a trend toward association of USH2A rs700024 with ICH (OR = 2.77, 95% CI = 1.13-6.80, p = .026). CONCLUSION: We did not replicate previously reported associations with HHT lung AVM and variants in Tgfb modifier loci. We also did not find significant associations between variants reported in sporadic brain AVM and VM or ICH in HHT.

Outcomes of surgical management of familial intrahepatic cholestasis 1 and bile salt export protein deficiencies.

Progressive familial intrahepatic cholestasis (PFIC) with normal circulating gamma-glutamyl transpeptidase levels can result from mutations in the ATP8B1 gene (encoding familial intrahepatic cholestasis 1 [FIC1] deficiency) or the ABCB11 gene (bile salt export protein [BSEP] deficiency). We investigated the outcomes of partial external biliary diversion, ileal exclusion, and liver transplantation in these two conditions. We conducted a retrospective multicenter study of 42 patients with FIC1 deficiency (FIC1 patients) and 60 patients with BSEP deficiency (BSEP patients) who had undergone one or more surgical procedures (57 diversions, 6 exclusions, and 57 transplants). For surgeries performed prior to transplantation, BSEP patients were divided into two groups, BSEP-common (bearing common missense mutations D482G or E297G, with likely residual function) and BSEP-other. We evaluated clinical and biochemical outcomes in these patients. Overall, diversion improved biochemical parameters, pruritus, and growth, with substantial variation in individual response. BSEP-common or FIC1 patients survived longer after diversion without developing cirrhosis, being listed for or undergoing liver transplantation, or dying, compared to BSEP-other patients. Transplantation resolved cholestasis in all groups. However, FIC1 patients commonly developed hepatic steatosis, diarrhea, and/or pancreatic disease after transplant accompanied by biochemical abnormalities and often had continued poor growth. In BSEP patients with impaired growth, this generally improved after transplantation. Conclusion: Diversion can improve clinical and biochemical status in FIC1 and BSEP deficiencies, but outcomes differ depending on genetic etiology. For many patients, particularly BSEP-other, diversion is not a permanent solution and transplantation is required. Although transplantation resolves cholestasis in patients with FIC1 and BSEP deficiencies, the overall outcome remains unsatisfactory in many FIC1 patients; this is mainly due to extrahepatic manifestations. (Hepatology Communications 2018;2:515-528).

Frequency and characteristics associated with inherited thrombophilia in patients with intracranial dural arteriovenous fistula.

OBJECTIVE: The pathogenesis of dural arteriovenous fistulas (DAVFs) remains poorly defined. Prior studies on thrombophilia as a risk factor for DAVF development are limited by small sample sizes and poor generalizability. METHODS: In this longitudinal observational study, all patients with intracranial DAVFs evaluated at the University of California, San Francisco from December 1994 through April 2014 were identified. After obtaining patient consent, 3 thrombophilic mutations, factor V Leiden (rs6025), MTHFR (rs1801133), and prothrombin G20210A, were genotyped. The authors evaluated the association of thrombophilia status (presence of any thrombophilic mutation) and clinical and angiographic characteristics using either a 2-sample t-test or Fisher's exact test. RESULTS: A total of 116 patients with diagnosed intracranial DAVFs were included in the study. Twenty-five (22%) patients met criteria for thrombophilia. Focal neurological deficits tended to occur more frequently in the thrombophilia group (78% vs 57%, p = 0.09). Angiographic characteristics of DAVFs, including high-risk venous flow pattern, multiplicity of DAVF, and the presence of venous sinus thrombosis, did not differ significantly between the 2 groups but tended to be more common in the thrombophilic than in the nonthrombophilic group. CONCLUSIONS: This study is one of the largest of thrombophilia and DAVF to date. The frequency of mutations associated with thrombophilia in this study was higher than that in the general population.

Genome-wide association study of sporadic brain arteriovenous malformations.

BACKGROUND: The pathogenesis of sporadic brain arteriovenous malformations (BAVMs) remains unknown, but studies suggest a genetic component. We estimated the heritability of sporadic BAVM and performed a genome-wide association study (GWAS) to investigate association of common single nucleotide polymorphisms (SNPs) with risk of sporadic BAVM in the international, multicentre Genetics of Arteriovenous Malformation (GEN-AVM) consortium. METHODS: The Caucasian discovery cohort included 515 BAVM cases and 1191 controls genotyped using Affymetrix genome-wide SNP arrays. Genotype data were imputed to 1000 Genomes Project data, and well-imputed SNPs (>0.01 minor allele frequency) were analysed for association with BAVM. 57 top BAVM-associated SNPs (51 SNPs with p<10(-05) or p<10(-04) in candidate pathway genes, and 6 candidate BAVM SNPs) were tested in a replication cohort including 608 BAVM cases and 744 controls. RESULTS: The estimated heritability of BAVM was 17.6% (SE 8.9%, age and sex-adjusted p=0.015). None of the SNPs were significantly associated with BAVM in the replication cohort after correction for multiple testing. 6 SNPs had a nominal p<0.1 in the replication cohort and map to introns in EGFEM1P, SP4 and CDKAL1 or near JAG1 and BNC2. Of the 6 candidate SNPs, 2 in ACVRL1 and MMP3 had a nominal p<0.05 in the replication cohort. CONCLUSIONS: We performed the first GWAS of sporadic BAVM in the largest BAVM cohort assembled to date. No GWAS SNPs were replicated, suggesting that common SNPs do not contribute strongly to BAVM susceptibility. However, heritability estimates suggest a modest but significant genetic contribution.

Cytochrome P450 and matrix metalloproteinase genetic modifiers of disease severity in Cerebral Cavernous Malformation type 1.

BACKGROUND: Familial Cerebral Cavernous Malformation type 1 (CCM1) is an autosomal dominant disease caused by mutations in the Krev Interaction Trapped 1 (KRIT1/CCM1) gene, and characterized by multiple brain lesions. CCM lesions manifest across a range of different phenotypes, including wide differences in lesion number, size and susceptibility to intracerebral hemorrhage (ICH). Oxidative stress plays an important role in cerebrovascular disease pathogenesis, raising the possibility that inter-individual variability in genes related to oxidative stress may contribute to the phenotypic differences observed in CCM1 disease. Here, we investigated whether candidate oxidative stress-related cytochrome P450 (CYP) and matrix metalloproteinase (MMP) genetic markers grouped by superfamilies, families or genes, or analyzed individually influence the severity of CCM1 disease. METHODS: Clinical assessment and cerebral susceptibility-weighted magnetic resonance imaging (SWI) were performed to determine total and large (≥5mm in diameter) lesion counts as well as ICH in 188 Hispanic CCM1 patients harboring the founder KRIT1/CCM1 'common Hispanic mutation' (CCM1-CHM). Samples were genotyped on the Affymetrix Axiom Genome-Wide LAT1 Human Array. We analyzed 1,122 genetic markers (both single nucleotide polymorphisms (SNPs) and insertion/deletions) grouped by CYP and MMP superfamily, family or gene for association with total or large lesion count and ICH adjusted for age at enrollment and gender. Genetic markers bearing the associations were then analyzed individually. RESULTS: The CYP superfamily showed a trend toward association with total lesion count (P=0.057) and large lesion count (P=0.088) in contrast to the MMP superfamily. The CYP4 and CYP8 families were associated with either large lesion count or total lesion count (P=0.014), and two other families (CYP46 and the MMP Stromelysins) were associated with ICH (P=0.011 and 0.007, respectively). CYP4F12 rs11085971, CYP8A1 rs5628, CYP46A1 rs10151332, and MMP3 rs117153070 single SNPs, mainly bearing the above-mentioned associations, were also individually associated with CCM1 disease severity. CONCLUSIONS: Overall, our candidate oxidative stress-related genetic markers set approach outlined CYP and MMP families and identified suggestive SNPs that may impact the severity of CCM1 disease, including the development of numerous and large CCM lesions and ICH. These novel genetic risk factors of prognostic value could serve as early objective predictors of disease outcome and might ultimately provide better options for disease prevention and treatment.

Mitochondrial DNA Heteroplasmy Associations With Neurosensory and Mobility Function in Elderly Adults.

BACKGROUND: Mitochondrial DNA (mtDNA) heteroplasmy is a mixture of normal and mutated mtDNA molecules in a cell. High levels of heteroplasmy at specific mtDNA sites lead to inherited mitochondrial diseases with neurological, sensory, and movement impairments. Here we test the hypothesis that heteroplasmy levels in elderly adults are associated with impaired function resembling mild forms of mitochondrial disease. METHODS: We examined platelet mtDNA heteroplasmy at 20 disease-causing sites for associations with neurosensory and mobility function among 137 participants from the community-based Health, Aging, and Body Composition Study. RESULTS: Elevated mtDNA heteroplasmy at four mtDNA sites in complex I and tRNA genes was nominally associated with reduced cognition, vision, hearing, and mobility: m.10158T>C with Modified Mini-Mental State Examination score (p = .009); m.11778G>A with contrast sensitivity (p = .02); m.7445A>G with high-frequency hearing (p = .047); and m.5703G>A with 400 m walking speed (p = .007). CONCLUSIONS: These results indicate that increased mtDNA heteroplasmy at disease-causing sites is associated with neurosensory and mobility function in older persons. We propose the novel use of mtDNA heteroplasmy as a simple, noninvasive predictor of age-related neurologic, sensory, and movement impairments.

The ACVRL1 c.314-35A>G polymorphism is associated with organ vascular malformations in hereditary hemorrhagic telangiectasia patients with ENG mutations, but not in patients with ACVRL1 mutations.

Hereditary hemorrhagic telangiectasia (HHT) is characterized by vascular malformations (VMs) and caused by mutations in TGFß/BMP9 pathway genes, most commonly ENG or ACVRL1. Patients with HHT have diverse manifestations related to skin and mucosal telangiectases and organ VMs, including arteriovenous malformations (AVM). The clinical heterogeneity of HHT suggests a role for genetic modifiers. We hypothesized that the ACVRL1 c.314-35A>G and ENG c.207G>A polymorphisms, previously associated with sporadic brain AVM, are associated with organ VM in HHT. We genotyped these variants in 716 patients with HHT and evaluated association of genotype with presence of any organ VM, and specifically with brain VM, liver VM and pulmonary AVM, by multivariate logistic regression analyses stratified by HHT mutation. Among all patients with HHT, neither polymorphism was significantly associated with presence of any organ VM; ACVRL1 c.314-35A>G showed a trend toward association with pulmonary AVM (OR = 1.48, P = 0.062). ACVRL1 c.314-35A>G was significantly associated with any VM among patients with HHT with ENG (OR = 2.66, P = 0.022), but not ACVRL1 (OR = 0.79, P = 0.52) mutations. ACVRL1 c.314-35A>G was also associated with pulmonary AVM and liver VM among ENG mutation heterozygotes. There were no significant associations between ENG c.207G>A and any VM phenotype. These results suggest that common polymorphisms in HHT genes other than the mutated gene modulate phenotype severity of HHT disease, specifically presence of organ VM.

Plasma soluble thrombomodulin levels are associated with mortality in the acute respiratory distress syndrome.

OBJECTIVE: Thombomodulin (TM) is an activator of protein C and a biomarker for endothelial injury. We hypothesized that (1) elevated plasma levels would be associated with clinical outcomes and (2) polymorphisms in the TM gene would be associated with plasma levels. PATIENTS: We studied 449 patients enrolled in the Fluid and Catheter Treatment Trial (FACTT) for whom both plasma and DNA were available. We used logistic regression and receiver operator curves (ROC) to test for associations between soluble TM (sTM) and mortality at 60 days. MEASUREMENTS AND RESULTS: Plasma sTM levels were higher in non-survivors than survivors at baseline [median 147 (IQR, 95-218) vs. 89 (56-129) ng/mL, p < 0.0001] and on day 3 after study enrollment [205 (146-302) vs. 127 (85-189), p < 0.0001]. The odds of death increased by 2.4 (95 % CI 1.5-3.8, p < 0.001), and by 2.8 (1.7-4.7, P < 0.001) for every log increase in baseline and day 3 sTM levels, respectively, after adjustment for age, race, gender, severity of illness, fluid management strategy, baseline creatinine, and non-pulmonary sepsis as the primary cause of ARDS. By ROC analysis, plasma sTM levels discriminated between non-survivors and survivors [AUC = 72 % (66-78 %) vs. AUC = 54 % for severity based on Berlin criteria). Addition of sTM improved discrimination based on APACHE III from 77 to 80 % (P < 0.03). sTM levels at baseline were not statistically different among subjects stratified by genotypes of tag SNPs in the TM gene. CONCLUSIONS: Higher plasma sTM levels are associated with increased mortality in ARDS. The lack of association between the sTM levels and genetic variants suggests that the increased levels of sTM may reflect severity of endothelial damage rather than genetic heterogeneity. These findings underscore the importance of endothelial injury in ARDS pathogenesis and suggest that, in combination with clinical markers, sTM could contribute to risk stratification.

Expression quantitative trait loci and receptor pharmacology implicate Arg1 and the GABA-A receptor as therapeutic targets in neuroblastoma.

The development of targeted therapeutics for neuroblastoma, the third most common tumor in children, has been limited by a poor understanding of growth signaling mechanisms unique to the peripheral nerve precursors from which tumors arise. In this study, we combined genetics with gene-expression analysis in the peripheral sympathetic nervous system to implicate arginase 1 and GABA signaling in tumor formation in vivo. In human neuroblastoma cells, either blockade of ARG1 or benzodiazepine-mediated activation of GABA-A receptors induced apoptosis and inhibited mitogenic signaling through AKT and MAPK. These results suggest that ARG1 and GABA influence both neural development and neuroblastoma and that benzodiazepines in clinical use may have potential applications for neuroblastoma therapy.

Polymorphisms in inflammatory and immune response genes associated with cerebral cavernous malformation type 1 severity.

BACKGROUND: Familial cerebral cavernous malformation type 1 (CCM1) is an autosomal dominant disease caused by mutations in the Krev Interaction Trapped 1 (KRIT1/CCM1) gene, and characterized by multiple brain lesions that often result in intracerebral hemorrhage (ICH), seizures, and neurological deficits. Carriers of the same genetic mutation can present with variable symptoms and severity of disease, suggesting the influence of modifier factors. Evidence is emerging that inflammation and immune response play a role in the pathogenesis of CCM. The purpose of this study was to investigate whether common variants in inflammatory and immune response genes influence the severity of familial CCM1 disease, as manifested by ICH and greater brain lesion count. METHODS: Hispanic CCM1 patients (n=188) harboring the founder Q455X 'common Hispanic mutation' (CHM) in the KRIT1 gene were analyzed at baseline. Participants were enrolled between June 2010 and March 2014 either through the Brain Vascular Malformation Consortium (BVMC) study or through the Angioma Alliance organization. Clinical assessment and cerebral susceptibility-weighted magnetic resonance imaging were performed to determine ICH as well as total and large (≥5 mm in diameter) lesion counts. Samples were genotyped on the Affymetrix Axiom Genome-Wide LAT1 Human Array. We analyzed 830 variants in 56 inflammatory and immune response genes for association with ICH and residuals of log-transformed total or large lesion count adjusted for age at enrollment and gender. Variants were analyzed individually or grouped by sub-pathways or whole pathways. RESULTS: At baseline, 30.3% of CCM1-CHM subjects had ICH, with a mean ± standard deviation (SD) of 60.1±115.0 (range 0-713) for total lesions and 4.9±8.7 (range 0-104) for large lesions. The heritability estimates explained by all autosomal variants were 0.20 (SE=0.31), 0.81 (SE=0.17), and 0.48 (SE=0.19), for ICH, total lesion count, and large lesion count, respectively. TGFBR2 rs9823731 was significantly associated with ICH as well as with the total and large lesion counts (p≤0.017). Further, IL-4 rs9327638, CD14 rs778588, IL-6R rs114660934 and MSR1 rs62489577 were associated with two markers of disease severity. Finally, the whole pathway was associated with total lesion count (p=0.005) with TLR-4 rs10759930, CD14 rs778588, IL-6R rs114660934 and IGH rs57767447 mainly bearing this association. Eicosanoid signaling, extracellular pattern recognition, and immune response sub-pathways were also associated with the total lesion count. CONCLUSIONS: These results suggest that polymorphisms in inflammatory and immune response pathways contribute to variability in CCM1 disease severity and might be used as predictors of disease severity. In particular, TGFBR2 rs9823731 was associated with all three markers of CCM1 disease severity tested, suggesting that TGFBR2 might be a key participant in the mechanism underlying CCM1 disease severity and phenotype variability. However, further longitudinal studies in larger sample sizes are needed to confirm these findings.

Common variants on 9p21.3 are associated with brain arteriovenous malformations with accompanying arterial aneurysms.

OBJECTIVE: To investigate whether previously reported 9p21.3 single nucleotide polymorphisms (SNPs) are associated with risk of brain arteriovenous malformations (BAVM), which often have accompanying arterial aneurysms. Common variants in the 9p21.3 locus have been reported to be associated with multiple cardiovascular phenotypes, including coronary artery disease and intracranial aneurysms (rs10757278 and rs1333040). METHODS: We used data from 338 BAVM cases participating in the University of California, San Francisco (UCSF)-Kaiser Brain AVM Study Project and 504 healthy controls to evaluate genotypes for seven common SNPs (minor allele frequency>0.05) that were imputed using 1000 Genomes Phase 1 European data (R(2)>0.87). Association with BAVM was tested using logistic regression adjusting for age, sex and the top three principal components of ancestry. Subgroup analysis included 205 BAVM cases with aneurysm data: 74 BAVM with aneurysm versus 504 controls and 131 BAVM without aneurysm versus 504 controls. RESULTS: We observed suggestive association with BAVM and rs10757278-G (OR=1.23, 95% CI 0.99 to 1.53, p=0.064) and rs1333040-T (OR=1.27, 95% CI 1.01 to 1.58, p=0.04). For rs10757278-G, the association was stronger in BAVM cases with aneurysm (OR=1.52, 95% CI 1.03 to 2.22, p=0.032) than in BAVM without aneurysm (OR=0.98, 95% CI 0.72 to 1.34, p=0.91). Similar patterns of effects were observed for rs1333040 and for other SNPs in linkage disequilibrium (r(2)>0.8) with rs10757278. CONCLUSIONS: Common 9p21.3 variants showed similar effect sizes for association with BAVM as previously reported for aneurysmal disease. The association with BAVM appears to be explained by known associations with aneurysms, suggesting that BAVM-associated aneurysms share similar vascular pathology mechanisms with other aneurysm types.

Association of cardiovascular risk factors with disease severity in cerebral cavernous malformation type 1 subjects with the common Hispanic mutation.

BACKGROUND: Cerebral cavernous malformations (CCM) are enlarged vascular lesions affecting 0.1-0.5% of the population worldwide and causing hemorrhagic strokes, seizures, and neurological deficits. Familial CCM type 1 (CCM1) is an autosomal dominant disease caused by mutations in the Krev Interaction Trapped 1 (KRIT1/CCM1) gene, and is characterized by multiple brain lesions whose number and size increase with age. The number of lesions varies widely for unknown reasons, even among carriers of similar ages with the same mutation. The purpose of this study was to investigate whether cardiovascular (CV) risk factors influence potential markers of familial CCM1 disease severity, such as lesion count and history of intracerebral hemorrhage. METHODS: We analyzed baseline data from 185 Hispanic subjects, enrolled in the Brain Vascular Malformation Consortium study between June 2010 and March 2013. All subjects were carriers of the founder Q455X 'Common Hispanic Mutation' (CHM) in the KRIT1 gene, and had a clinical diagnosis of CCM or had an affected first- or second-degree relative with CCM. We performed a cross-sectional study, collecting detailed clinical information of CCM1-CHM subjects and cerebral susceptibility-weighted magnetic resonance imaging to assess lesion count. Linear or logistic regression analysis of log-lesion count or history of intracerebral hemorrhage and CV risk factors (age, gender, obesity, diabetes, hypertension, hyperlipidemia and smoking status) and related quantitative traits (body mass index, glycosylated hemoglobin levels, blood pressure, lipids levels and pack-years of cigarette smoking) was performed accommodating familial clustering. RESULTS: CCM1-CHM subjects were mainly female (63.8%) and symptomatic at presentation (63.2%). Lesion count was highly variable (mean ± SD: 57.7 ± 110.6; range: 0-713); 90% of CCM1-CHM subjects had multiple lesions at enrollment. Age (p < 0.001) was positively correlated with lesion count and male gender (p = 0.035) was associated with a greater number of lesions. Obesity (p = 0.001) and higher body mass index (p = 0.002) were associated with fewer lesions. No association with hypertension was detected, however, systolic blood pressure (p = 0.002) was associated with fewer lesions. No significant association with lesion count was observed for diabetes, hyperlipidemia, smoking status or for related quantitative traits. History of intracerebral hemorrhage was not significantly associated with any CV risk factors, however, we found borderline associations of hemorrhage with obesity (p = 0.062), systolic blood pressure (p = 0.083) and pack-years of cigarette smoking (p = 0.055). After correction for multiple testing, age and obesity remained significantly associated with lesion count in CCM1-CHM subjects. CONCLUSIONS: These results suggest that several CV risk factors explain some of the variability in lesion count in Hispanic CCM1-CHM subjects. Although age, gender, obesity, body mass index and systolic blood pressure may influence familial CCM1 disease severity, further longitudinal studies in larger sample sizes are essential to confirm these findings.

Severity score for hereditary hemorrhagic telangiectasia.

BACKGROUND: A disease severity score in hereditary hemorrhagic telangiectasia (HHT) would be a useful tool for assessing burden of disease and for designing clinical trials. Here, we propose the first known HHT severity score, the HHT-score. METHODS: Demographics and disease characteristics were collected for the first 525 HHT patients recruited to the HHT Project of the Brain Vascular Malformation Consortium (BVMC). HHT-score was calculated based on presence of: organ arteriovenous malformations (maximum 3 points); chronic bleeding (maximum 2 points); and severe organ involvement (maximum 2 points). Points were summed and patients categorized as having mild (0-2), moderate (3-4) or severe (5-7) disease. The occurrence of "any adverse outcome" was evaluated for association with HHT-score categories. RESULTS: The frequency of "any adverse outcome" was significantly different across the three groups (49.6% in mild, 65.8% in moderate and 89.5% in severe, p<0.001). Adjusting for age and gender, the risk of "any adverse outcome" was higher in the moderate (OR=1.84, 95% CI: 1.15-2.95, p=0.011) and severe groups (OR=9.16, 95% CI: 1.99-42.09, p=0.004) compared to the mild. CONCLUSIONS: We have taken the first steps toward creating a global measure of disease severity in HHT. While the initial results are promising, further validation of the HHT-score is still required.