Rachel Kleinloog

Lessons learned from whole exome sequencing in multiplex families affected by a complex genetic disorder, intracranial aneurysm

Genetic risk factors for intracranial aneurysm (IA) are not yet fully understood. Genomewide association studies have been successful at identifying common variants; however, the role of rare variation in IA susceptibility has not been fully explored. In this study, we report the use of whole exome sequencing (WES) in seven densely-affected families (45 individuals) recruited as part of the Familial Intracranial Aneurysm study. WES variants were prioritized by functional prediction, frequency, predicted pathogenicity, and segregation within families. Using these criteria, 68 variants in 68 genes were prioritized across the seven families. Of the genes that were expressed in IA tissue, one gene (TMEM132B) was differentially expressed in aneurysmal samples (n=44) as compared to control samples (n=16) (false discovery rate adjusted p-value=0.023). We demonstrate that sequencing of densely affected families permits exploration of the role of rare variants in a relatively common disease such as IA, although there are important study design considerations for applying sequencing to complex disorders. In this study, we explore methods of WES variant prioritization, including the incorporation of unaffected individuals, multipoint linkage analysis, biological pathway information, and transcriptome profiling. Further studies are needed to validate and characterize the set of variants and genes identified in this study.

Regional differences in incidence and patient characteristics of moyamoya disease: a systematic review

Background and purpose Moyamoya disease (MMD) is a rare cause of stroke, initially described in Japan. In other countries, incidences and presenting symptoms may differ from those in Japan. The literature on regional differences in incidence and patient characteristics of MMD was systematically reviewed. Methods Medline, EMBASE and CINAHL were searched for population based studies on MMD published between January 1969 and January 2011. From studies that met predefined inclusion criteria, information was extracted on incidence and patient characteristics. Incidences with corresponding 95% CIs if possible were calculated and descriptive statistics for patient characteristics were used. Results 8 studies were included: three from Japan, one each from Taiwan and China and three from the USA. Incidences per 100 000 patient years ranged in Japan from 0.35 to 0.94 (95% CI 0.69 to 1.19), in the USA from 0.05 (−0.04 to 0.12) in Iowa to 0.17 (−0.06 to 0.40) in Hawaii and were 0.41 (0.28 to 0.54) in Nanjing, China and 0.02 (0.003 to 0.04) in Taiwan. Female to male ratio ranged from 1.1 (0.9 to 1.5) in Nanjing to 2.8 (1.2 to 6.1) in Iowa. Proportions with intracerebral haemorrhage as the initial presentation were 56% in China, 52% in Taiwan, 29% in Hawaii, 21% in Japan and 10% in Iowa. Patients with childhood onset presented most often with ischaemia (>75%) in all regions. Conclusions MMD incidence was higher in Japan and China than in Taiwan and North America and presenting symptoms showed regional differences, which are thus far unexplained. Population based data on MMD in Europe are lacking.

Visualization of the aneurysm wall: a 7.0-tesla magnetic resonance imaging study.

BACKGROUND Risk prediction of rupture of intracranial aneurysms is poor and is based mainly on lumen characteristics. However, characteristics of the aneurysm wall may be more informative predictors. The limited resolution of currently available imaging techniques and the thin aneurysm wall make imaging of wall thickness challenging. OBJECTIVE To introduce a novel protocol for imaging wall thickness variation using ultra--high-resolution 7.0-Tesla (7.0-T) magnetic resonance imaging (MRI). METHODS We studied 33 unruptured intracranial aneurysms in 24 patients with a T1-weighted 3-dimensional magnetization-prepared inversion-recovery turbo-spin-echo whole-brain sequence with a resolution of 0.8 × 0.8 × 0.8 mm. We performed a validation study with a wedge phantom and with 2 aneurysm wall biopsies obtained during aneurysm treatment using ex vivo MRI and histological examination and correlating variations in MRI signal intensity with variations in actual thickness of the aneurysm wall. RESULTS In vivo, the aneurysm wall was visible in 28 of the 33 aneurysms. Variation in signal intensity was observed in all visible aneurysm walls. Ex vivo MRI showed variation in signal intensity across the wall of the biopsies, similar to that observed on the in vivo images. Signal intensity and actual thickness in both biopsies had a linear correlation, with Pearson correlation coefficients of 0.85 and 0.86. CONCLUSION Unruptured intracranial aneurysm wall and its variation in thickness can be visualized with 7.0-T MRI. Aneurysm wall thickness variation can now be further studied as a risk factor for rupture in prospective studies.

Genetic risk load according to the site of intracranial aneurysms

Objective: We investigated whether risk alleles of single nucleotide polymorphisms associated with intracranial aneurysm (IA) are enriched in patients with familial IA, IA located at the middle cerebral artery (MCA), or IA rupture at a younger age. Methods: In this case-only study, we calculated genetic risk scores (GRS) for 973 Dutch and 718 Finnish patients with IA by summing effect size–weighted risk allele counts of 7 single nucleotide polymorphisms associated with IAs previously identified through genome-wide association studies. We tested the GRS for association with presence of familial IA or IA at the MCA using logistic regression, and with age at time of IA rupture using linear regression. We also calculated odds ratios with 95% confidence intervals for the proportion of patients with each characteristic in the highest compared with the lowest GRS tertile. Results: GRS were higher in IA at the MCA in the Dutch (p = 2.5 × 10−4), Finnish (p = 0.039), and combined cohort (p = 4.9 × 10−5). GRS were not associated with familial IA in the Dutch (p = 0.34), Finnish (p = 0.45), and combined cohort (p = 0.98), or with age at time of IA rupture in the Dutch (p = 0.28), Finnish (p = 0.86), and combined cohort (p = 0.45). In the combined cohort, odds ratios were 0.89 (0.67–1.20) for familial IA, 1.03 (0.79–1.34) for lower age, and 1.54 (1.20–1.98) for MCA aneurysms. Conclusions: Our findings suggest that genetic risk factors have a larger role in the development of IA at the MCA than at other sites, and that genetic heterogeneity should be considered in future genetic studies.

RNA Sequencing Analysis of Intracranial Aneurysm Walls Reveals Involvement of Lysosomes and Immunoglobulins in Rupture

Background and Purpose— Analyzing genes involved in development and rupture of intracranial aneurysms can enhance knowledge about the pathogenesis of aneurysms, and identify new treatment strategies. We compared gene expression between ruptured and unruptured aneurysms and control intracranial arteries. Methods— We determined expression levels with RNA sequencing. Applying a multivariate negative binomial model, we identified genes that were differentially expressed between 44 aneurysms and 16 control arteries, and between 22 ruptured and 21 unruptured aneurysms. The differential expression of 8 relevant and highly significant genes was validated using digital polymerase chain reaction. Pathway analysis was used to identify enriched pathways. We also analyzed genes with an extreme pattern of differential expression: only expressed in 1 condition without any expression in the other. Results— We found 229 differentially expressed genes in aneurysms versus controls and 1489 in ruptured versus unruptured aneurysms. The differential expression of all 8 genes selected for digital polymerase chain reaction validation was confirmed. Extracellular matrix pathways were enriched in aneurysms versus controls, whereas pathways involved in immune response and the lysosome pathway were enriched in ruptured versus unruptured aneurysms. Immunoglobulin genes were expressed in aneurysms, but showed no expression in controls. Conclusions— For rupture of intracranial aneurysms, we identified the lysosome pathway as a new pathway and found further evidence for the role of the immune response. Our results also point toward a role for immunoglobulins in the pathogenesis of aneurysms. Immune-modifying drugs are, therefore, interesting candidate treatment strategies in the prevention of aneurysm development and rupture.

Thinner Regions of Intracranial Aneurysm Wall Correlate with Regions of Higher Wall Shear Stress: A 7T MRI Study

BACKGROUND AND PURPOSE: Both hemodynamics and aneurysm wall thickness are important parameters in aneurysm pathophysiology. Our aim was to develop a method for semi-quantitative wall thickness assessment on in vivo 7T MR images of intracranial aneurysms for studying the relation between apparent aneurysm wall thickness and wall shear stress. MATERIALS AND METHODS: Wall thickness was analyzed in 11 unruptured aneurysms in 9 patients who underwent 7T MR imaging with a TSE-based vessel wall sequence (0.8-mm isotropic resolution). A custom analysis program determined the in vivo aneurysm wall intensities, which were normalized to the signal of nearby brain tissue and were used as measures of apparent wall thickness. Spatial wall thickness variation was determined as the interquartile range in apparent wall thickness (the middle 50% of the apparent wall thickness range). Wall shear stress was determined by using phase-contrast MR imaging (0.5-mm isotropic resolution). We performed visual and statistical comparisons (Pearson correlation) to study the relation between wall thickness and wall shear stress. RESULTS: 3D colored apparent wall thickness maps of the aneurysms showed spatial apparent wall thickness variation, which ranged from 0.07 to 0.53, with a mean variation of 0.22 (a variation of 1.0 roughly means a wall thickness variation of 1 voxel [0.8 mm]). In all aneurysms, apparent wall thickness was inversely related to wall shear stress (mean correlation coefficient, −0.35; P < .05). CONCLUSIONS: A method was developed to measure the wall thickness semi-quantitatively, by using 7T MR imaging. An inverse correlation between wall shear stress and apparent wall thickness was determined. In future studies, this noninvasive method can be used to assess spatial wall thickness variation in relation to pathophysiologic processes such as aneurysm growth and rupture.

Risk Factors for Intracranial Aneurysm Rupture: A Systematic Review

BACKGROUND Intracranial aneurysm rupture prediction is poor, with only a few risk factors for rupture identified and used in clinical practice. OBJECTIVE To provide an overview of all the risk factors (including genetic, molecular, morphological, and hemodynamic factors) that have potential for use in clinical practice. METHODS We systematically searched PubMed and EMBASE and focused on factors that can be easily assessed in clinical practice, might be used for rupture prediction in clinical practice, and/or are potential targets for further research. Studies were categorized according to methodological quality, and a meta-analysis was performed, if possible. RESULTS We included 102 studies describing 144 risk factors that fulfilled predefined criteria. There was strong evidence for the morphological factors irregular shape (studied in 4 prospective cohort studies of high-quality, pooled odds ratio [OR] of 4.8 [95% confidence interval 2.7-8.7]), aspect ratio (pooled OR 10.2 [4.3-24.6]), size ratio, bottleneck factor, and height-to-width ratio to increase rupture risk. Moderate level of evidence was found for presence of contact with the perianeurysmal environment (pooled OR 3.5 [1.4-8.4]), unbalanced nature of this contact (pooled OR 17.8 [8.3-38.5]), volume-to-ostium ratio, and direction of the aneurysm dome (pooled OR 1.5 [1.2-1.9]). CONCLUSION Irregular aneurysm shape was identified as a risk factor with potential for use in clinical practice. The risk factors aspect ratio, size ratio, bottleneck factor, height-to-width ratio, contact with the perianeurysmal environment, volume-to-ostium ratio, and dome-direction should first be confirmed in multivariate analysis and incorporated in prediction models.

Comparative Ultrastructural and Stereological Analyses of Unruptured and Ruptured Saccular Intracranial Aneurysms

Insight into processes leading to rupture of intracranial aneurysms (IAs) may identify biomarkers for rupture or lead to management strategies reducing the risk of rupture. We characterized and quantified (ultra)structural differences between unruptured and ruptured aneurysmal walls. Six unruptured and 6 ruptured IA fundi were resected after microsurgical clipping and analyzed by correlative light microscopy for quantitative analysis (proportion of the vessel wall area) and transmission electron microscopy for qualitative ultrastructural analysis. Quantitative analysis revealed extensive internal elastic lamina (IEL) thickening in ruptured IA (36.3% ± 15%), while thin and fragmented IEL were common in unruptured IA (5.6% ± 7.1%). Macrophages were increased in ruptured IA (28.3 ± 24%) versus unruptured IA (2.7% ± 5.5%), as were leukocytes (12.85% ± 10% vs 0%). Vasa vasorum in ruptured but not in unruptured IA contained vast numbers of inflammatory cells and extravasation of these cells into the vessel wall. In conclusion, detection of thickened IEL, leaky vasa vasorum, and heavy inflammation as seen in ruptured IA in comparison to unruptured IA may identify aneurysms at risk of rupture, and management strategies preventing development of vasa vasorum or inflammation may reduce the risk of aneurysmal rupture.

Improved depiction of hemodynamics in intracranial aneurysms by 4D flow MRI at 7T compared to 3T

Background Intracranial aneurysms are life threatening conditions and occur in 3-6% of the population. The annual rupture rate is approximately 2% associated with significant morbidity and mortality. Hemodynamic information obtained by 4D flow MRI may contribute to the assessment of rupture risk of intracranial aneurysms. However, the small sizes of intracranial aneurysms require high spatial resolution to accurately capture complex intra-aneurysmal flow. Increasing spatial resolution, however, decreases signal-to-noise ratio (SNR) and thus velocity-to-noise ratio in flow images. Performing 4D flow at 7T has high potential to overcome low SNR limits and enable higher spatial resolution in 4D flow acquisitions. In this study 4D flow in intracranial aneurysms at 7T was compared with 4D flow at 3T.

Visualization of the Aneurysm Wall : A 7.0 Tesla MRI Study

BACKGROUND Risk prediction of rupture of intracranial aneurysms is poor and is based mainly on lumen characteristics. However, characteristics of the aneurysm wall may be more informative predictors. The limited resolution of currently available imaging techniques and the thin aneurysm wall make imaging of wall thickness challenging. OBJECTIVE To introduce a novel protocol for imaging wall thickness variation using ultra--high-resolution 7.0-Tesla (7.0-T) magnetic resonance imaging (MRI). METHODS We studied 33 unruptured intracranial aneurysms in 24 patients with a T1-weighted 3-dimensional magnetization-prepared inversion-recovery turbo-spin-echo whole-brain sequence with a resolution of 0.8 × 0.8 × 0.8 mm. We performed a validation study with a wedge phantom and with 2 aneurysm wall biopsies obtained during aneurysm treatment using ex vivo MRI and histological examination and correlating variations in MRI signal intensity with variations in actual thickness of the aneurysm wall. RESULTS In vivo, the aneurysm wall was visible in 28 of the 33 aneurysms. Variation in signal intensity was observed in all visible aneurysm walls. Ex vivo MRI showed variation in signal intensity across the wall of the biopsies, similar to that observed on the in vivo images. Signal intensity and actual thickness in both biopsies had a linear correlation, with Pearson correlation coefficients of 0.85 and 0.86. CONCLUSION Unruptured intracranial aneurysm wall and its variation in thickness can be visualized with 7.0-T MRI. Aneurysm wall thickness variation can now be further studied as a risk factor for rupture in prospective studies.