CpG island hypermethylation of the neurofibromatosis type 2 (NF2) gene is rare in sporadic vestibular schwannomas

P. J. Kullar, D. M. Pearson, D. S. Malley, V. P. Collins and K. Ichimura (2010) Neuropathology and Applied Neurobiology 36, 505–514
CpG island hypermethylation of the neurofibromatosis type 2 (NF2) gene is rare in sporadic vestibular schwannomas Aims: Loss of both wild‐type copies of the neurofibromatosis type 2 (NF2) gene is found in both sporadic and neurofibromatosis type 2‐associated vestibular schwannomas (VS). Previous studies have identified a subset of VS with no loss or mutation of NF2. We hypothesized that methylation of NF2 resulting in gene silencing may play a role in such tumours. Methods: Forty sporadic VS were analysed by array comparative genomic hybridization using 1 Mb whole genome and chromosome 22 tile path arrays. The NF2 genes were sequenced and methylation of NF2 examined by pyrosequencing. Results: Monosomy 22 was the only recurrent change found. Twelve tumours had NF2 mutations. Eight tumours had complete loss of wild‐type NF2, four had one mutated and one wild‐type allele, 11 had only one wild‐type allele and 17 showed no abnormalities. Methylation analysis showed low‐level methylation in four tumours at a limited number of CpGs. No high‐level methylation was found. Conclusions: This study shows that a significant proportion of sporadic VS (>40%) have unmethylated wild‐type NF2 genes. This indicates that other mechanisms, yet to be identified, are operative in the oncogenesis of these VSs.     

Proton magnetic resonance spectroscopy of normal appearing white matter in familial and sporadic multiple sclerosis

Objective To assess metabolite levels in normal–appearing white matter in sporadic and familial multiple sclerosis (MS). Methods Using H–MRS and applying one voxel measure we assessed NAA/Cho,NAA/Cr and Cho/Cr ratios in 26 patients with sporadic and in 25 with familial MS and compared them with healthy subjects. Results In both MS groups NAA/Cho and NAA/Cr ratio were significantly lower than in healthy individuals whereas Cho/Cr ratio was higher in MS patients. In sporadic MS patients NAA/Cho and NAA/Cr ratios were lower, although not significantly, than in familial cases. The Cho/Cr ratio was similar in both MS groups. Conclusion These results suggest that subtle differences in H–MRS measures corresponding to axonal rather than to myelin pathology might occur in sporadic and familial MS.     

Free fatty acid patterns in normal and multiple sclerosis white matter

The relative compositions of free fatty acids (FFA) in white matter from six multiple sclerosis (MS) and seven control brains were determined by gas chromatography. The major components of all samples were oleic, stearic and palmitic acids, with lesser amounts of arachidonic acid, and a long chain fatty acid, identified as 24:4. MS samples had significantly less of the 24:4 fraction compared with controls, but no other differences were noted. The spectrum of FFA was similar in most respects to those reported for phosphatidylcholine and cholesterol esters. This supports current theories that a major pathway of lipid catabolism in the brain involves transfer of fatty acids from phosphatidylcholine to cholesterol.     

Gray matter integrity predicts white matter network reorganization in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease leading to gray matter atrophy and brain network reconfiguration as a response to increasing tissue damage. We evaluated whether white matter network reconfiguration appears subsequently to gray matter damage, or whether the gray matter degenerates following alterations in white matter networks. MRI data from 83 patients with clinically isolated syndrome and early relapsing–remitting MS were acquired at two time points with a follow‐up after 1 year. White matter network integrity was assessed based on probabilistic tractography performed on diffusion‐weighted data using graph theoretical analyses. We evaluated gray matter integrity by computing cortical thickness and deep gray matter volume in 94 regions at both time points. The thickness of middle temporal cortex and the volume of deep gray matter regions including thalamus, caudate, putamen, and brain stem showed significant atrophy between baseline and follow‐up. White matter network dynamics, as defined by modularity and distance measure changes over time, were predicted by deep gray matter volume of the atrophying anatomical structures. Initial white matter network properties, on the other hand, did not predict atrophy. Furthermore, gray matter integrity at baseline significantly predicted physical disability at 1‐year follow‐up. In a sub‐analysis, deep gray matter volume was significantly related to cognitive performance at baseline. Hence, we postulate that atrophy of deep gray matter structures drives the adaptation of white matter networks. Moreover, deep gray matter volumes are highly predictive for disability progression and cognitive performance.     

TUFM variants lead to white matter abnormalities mimicking multiple sclerosis

Background and purpose

Defects in the mitochondrial respiratory chain (MRC) can lead to combined MRC dysfunctions (COXPDs) with heterogenous genotypes and clinical features. We report a patient carrying heterozygous variants in the TUFM gene who presented with clinical features compatible with COXPD4 and radiological findings mimicking multiple sclerosis (MS).

Methods

A 37-year-old French Canadian woman was investigated for recent onset of gait and balance problems. Her previous medical history included recurrent episodes of hyperventilation associated with lactic acidosis during infections, asymptomatic Wolff–Parkinson–White syndrome, and nonprogressive sensorineural deafness.

Results

Neurological examinations revealed fine bilateral nystagmus, facial weakness, hypertonia, hyperreflexia, dysdiadochokinesia, dysmetria, and ataxic gait. Brain magnetic resonance imaging (MRI) showed multifocal white matter abnormalities in cerebral white matter as well as cerebellar hemispheres, brainstem, and middle cerebellar peduncles, some of which mimicked MS. Analysis of native-state oxidative phosphorylation showed a combined decrease in CI/CII, CIV/CII, and CVI/CII. Exome sequencing detected two heterozygous TUFM gene variants. Little clinical progression was noted over a 5-year follow-up. Brain MRI remained unchanged.

Conclusions

Our report broadens the phenotypic and radiological spectrum of TUFM-related disorders by adding milder, later onset forms to the previously known early onset, severe presentations. The presence of multifocal white matter abnormalities can be misinterpreted as due to acquired demyelinating diseases, and thus TUFM-related disorders should be added to the list of mitochondrial MS mimickers.     

Microglia in normal appearing white matter of multiple sclerosis are alerted but immunosuppressed

Little is known about the functional phenotype of microglia in normal appearing white matter (NAWM) of multiple sclerosis (MS), although it may hold valuable clues about mechanisms for lesion development. Therefore, we studied microglia from NAWM obtained post‐mortem from controls (n = 25) and MS patients (n = 21) for their phenotype ex vivo and their immune responsiveness in vitro, using a microglia isolation method that omits culture and adherence. By flow cytometry, microglia in MS NAWM displayed elevated CD45 levels and increased size and granularity but were distinct from autologous choroid plexus macrophages by absent or low expression of additional markers, in particular CD206. Flow cytometric analysis of microglia from NAWM of three controls and four MS patients showed alterations in levels of Fc‐gamma receptors in MS. In primary microglia from a bigger sample of subjects, analysis of Fc‐gamma receptors by quantitative PCR indicated a significant increase in mRNA levels of the inhibitory CD32b isoform in MS NAWM. Despite their changed activation status, microglia from MS NAWM were unresponsive to lipopolysaccharide in vitro. Notably, culture with dexamethasone led to an impaired induction of the inflammation‐limiting cytokine CCL18 in microglia from MS NAWM compared with those from control NAWM. Together, these data demonstrate that microglia in MS NAWM are in an alerted state, but display features of immunosuppression. Thus, the activation status of microglia in NAWM of MS patients likely reflects a response to ongoing neuroinflammation, which coincides with upregulation of immunoregulatory molecules to prevent full activation and damage to the vulnerable milieu. GLIA 2013;61:1848–1861     

The effect of hypointense white matter lesions on automated gray matter segmentation in multiple sclerosis

Previous imaging studies assessing the relationship between white matter (WM) damage and matter (GM) atrophy have raised the concern that Multiple Sclerosis (MS) WM lesions may affect measures of GM volume by inducing voxel misclassification during intensity‐based tissue segmentation. Here, we quantified this misclassification error in simulated and real MS brains using a lesion‐filling method. Using this method, we also corrected GM measures in patients before comparing them with controls in order to assess the impact of this lesion‐induced misclassification error in clinical studies. We found that higher WM lesion volumes artificially reduced total GM volumes. In patients, this effect was about 72% of that predicted by simulation. Misclassified voxels were located at the GM/WM border and could be distant from lesions. Volume of individual deep gray matter (DGM) structures generally decreased with higher lesion volumes, consistent with results from total GM. While preserving differences in GM volumes between patients and controls, lesion‐filling correction revealed more lateralised DGM shape changes in patients, which were not evident with the original images. Our results confirm that WM lesions can influence MRI measures of GM volume and shape in MS patients through their effect on intensity‐based GM segmentation. The greater effect of lesions at increasing levels of damage supports the use of lesion‐filling to correct for this problem and improve the interpretability of the results. Volumetric or morphometric imaging studies, where lesion amount and characteristics may vary between groups of patients or change over time, may especially benefit from this correction. Hum Brain Mapp, 2012. © 2011 Wiley Periodicals, Inc.     

From genetic associations to functional studies in multiple sclerosis

Genetic screens steadily reveal more loci that show robust associations to complex human diseases, including multiple sclerosis (MS). Although some of the identified genetic variants are easily interpreted into a biological function, most of the genetic associations are frequently challenging to interpret. Underlying these difficulties is the fact that chip‐based assays typically detect single nucleotide polymorphisms (SNPs) representative of a stretch of DNA containing many genomic variants in linkage disequilibrium. Furthermore, a large proportion of the SNPs with strongest association to MS are located in regions of the DNA that do not directly code for proteins. Here we discuss challenges faced by MS researchers to follow up the large‐scale genetic screens that have been published over the past years in search of functional consequences of the identified MS‐associated SNPs. We discuss experimental design, tools and methods that may provide the much‐needed biological insights in both disease etiology and disease manifestations.     

Elevated protein carbonylation in the brain white matter and gray matter of patients with multiple sclerosis

Oxidative stress has been implicated in the pathophysiology of multiple sclerosis (MS). Increased levels of reactive oxygen species (ROS) derived from infiltrating macrophages and microglial cells have been shown to reduce the levels of endogenous antioxidants and to cause the oxidation of various substrates within the MS plaque. To determine whether oxidative damage takes place beyond visible MS plaques, the occurrence of total carbonyls (TCOs) and protein carbonyls (PCOs) in the normal-appearing white matter (NAWM) and gray matter (NAGM) of eight MS brains was assessed and compared with those of four control brains. The data show that most (7/8) of the MS-WM samples contain increased amounts of PCOs as determined by reaction with 2,4-dinitrophenylhydrazine and Western blot analysis. These samples also have high levels of glial fibrilary acidic protein (GFAP), suggesting that oxidative damage is related to the presence of small lesions. In contrast, we detected no evidence of protein thiolation (glutathionylation and cysteinylation) in the diseased tissue. To our surprise, MS-NAGM specimens with high GFAP content also showed three times the concentration of TCOs and PCOs as the controls. The increase in PCOs is likely to be a consequence of reduced levels of antioxidants, in that the concentration of nonprotein thiols in both MS-WM and -GM decreased by 30%. Overall, our data support the current view that both NAWM and -GM from MS brains contain considerable biochemical alterations. The involvement of GM in MS was also supported by the decrease in the levels of neurofilament light protein in all the specimens analyzed. To the best of our knowledge, this is the first study demonstrating the presence of increased protein carbonylation in post-mortem WM and GM tissue of MS patients.     

Abnormal ubiquitination of axons in normally myelinated white matter in multiple sclerosis brain

The hallmark of the lesions in multiple sclerosis (MS) is inflammatory demyelination with sparing of axons. Recent neuropathological and neuroradiological investigations show that structural changes of the axons occur, both in plaques and in the normal appearing white matter. A better understanding of the axonal damage in MS is important, since this may be responsible for permanent disability. We have investigated the immunoreactivity for ubiquitin, a sensitive method to detect axonal dystrophy and accumulation of abnormal proteins in pathological conditions of the nervous system, in the brains of six cases of MS (age range 39-66 years). Tissue blocks were fixed in formalin and embedded in paraffin. A panel of antibodies was used: anti-ubiquitin, anti-neurofilament (SMI-31 + SMI-32), anti-amyloid precursor protein and anti-PGP9.5. We focused our attention on chronic plaques, recognized by the absence of Luxol Fast Blue B-positive inclusions in macrophages. SMI-31 + SMI-32 showed the presence of a variable amount of axons within the plaques; the axonal network within the plaques was looser than outside. No ubiquitin reactivity was present in chronic plaques. In the normally myelinated white matter surrounding the plaques, a dense granular ubiquitin immunoreactivity was found both near and far from the plaque edge. No similar staining was found in control brains. Ubiquitination is the first step of a non-lysosomal degradation pathway of proteins. The present findings suggest a derangement of this proteolytic pathway in the axons outside the plaques, possibly as a consequence of chronic absence of myelin in the axonal segment inside the plaque. The spectrum of axonal changes in MS appears to be wider than expected and involves the apparently normal white matter.     

1H Magnetic resonance spectroscopy of normal appearing white matter in primary progressive multiple sclerosis

Recent magnetic resonance imaging (MRI) and pathological studies have indicated that axonal loss is a major contributor to disease progression in multiple sclerosis. ¹H magnetic resonance spectroscopy (MRS), through measurement of N-acetyl aspartate (NAA), a neuronal marker, provides a unique tool to investigate this. Patients with primary progressive multiple sclerosis have few lesions on conventional MRI, suggesting that changes in normal appearing white matter (NAWM), such as axonal loss, may be particularly relevant to disease progression in this group. To test this hypothesis NAWM was studied with MRS, measuring the concentration of N-acetyl derived groups (NA, the sum of NAA and N-acetyl aspartyl glutamate). Single-voxel MRS using a water-suppressed PRESS sequence was carried out in 24 patients with primary progressive multiple sclerosis and in 16 age-matched controls. Ratios of metabolite to creatine concentration (Cr) were calculated in all subjects, and absolute concentrations were measured in 18 patients and all controls. NA/Cr (median 1.40, range 0.86–1.91) was significantly lower in NAWM in patients than in controls (median 1.70, range 1.27–2.14; P = 0.006), as was the absolute concentration of NA (patients, median 6.90 mM, range 4.62–10.38 mM; controls, median 7.77 mM, range 6.60–9.71 mM; P = 0.032). There was no significant difference in the absolute concentration of creatine between the groups. This study supports the hypothesis that axonal loss occurs in NAWM in primary progressive multiple sclerosis and may well be a mechanism for disease progression in this group. Received: 22 January 1999 Received in revised form: 11 June 1999 Accepted: 16 June 1999     

Comparing MRI metrics to quantify white matter microstructural damage in multiple sclerosis

Quantifying white matter damage in vivo is becoming increasingly important for investigating the effects of neuroprotective and repair strategies in multiple sclerosis (MS). While various approaches are available, the relationship between MRI‐based metrics of white matter microstructure in the disease, that is, to what extent the metrics provide complementary versus redundant information, remains largely unexplored. We obtained four microstructural metrics from 123 MS patients: fractional anisotropy (FA), radial diffusivity (RD), myelin water fraction (MWF), and magnetisation transfer ratio (MTR). Coregistration of maps of these four indices allowed quantification of microstructural damage through voxel‐wise damage scores relative to healthy tissue, as assessed in a group of 27 controls. We considered three white matter tissue‐states, which were expected to vary in microstructural damage: normal appearing white matter (NAWM), T2‐weighted hyperintense lesional tissue without T1‐weighted hypointensity (T2L), and T1‐weighted hypointense lesional tissue with corresponding T2‐weighted hyperintensity (T1L). All MRI indices suggested significant damage in all three tissue‐states, the greatest damage being in T1L. The correlations between indices ranged from r = 0.18 to r = 0.87. MWF was most sensitive when differentiating T2L from NAWM, while MTR was most sensitive when differentiating T1L from NAWM and from T2L. Combining the four metrics into one, through a principal component analysis, did not yield a measure more sensitive to damage than any single measure. Our findings suggest that the metrics are (at least partially) correlated with each other, but sensitive to the different aspects of pathology. Leveraging these differences could be beneficial in clinical trials testing the effects of therapeutic interventions.     

Magnetic resonance studies of abnormalities in the normal appearing white matter and grey matter in multiple sclerosis

Abstract. Magnetic resonance (MR) techniques are of value in following the pathological process of multiple sclerosis in vivo. They are widely applied to monitor the disease natural history and its modification by treatment. However, serial studies of lesion measures have yielded generally disappointing correlations with the development of clinical disability. A potential explanation for this is the presence of abnormalities, beyond the visible lesions, in the normal appearing white matter (NAWM) and grey matter (NAGM). Quantitative structural MR techniques, including measures of magnetisation transfer, diffusion, relaxation times and spectroscopic metabolite concentrations, reveal that there are abnormalities in NAWM and NAGM. These are present from clinical onset and become more pronounced with clinical progression, increasing disability and increasing lesion load. Furthermore, functional MRI (fMRI) studies of motor and visual paradigms has identified a range of responses suggesting that cortical plasticity exists; such modified responses are seen in the earliest stages of disease and in the absence of visible lesions, but are more pronounced with disease progression and increasing lesion load and abnormality in the NAWM. Limited reproducibility and sensitivity to change can pose methodological limitations for MR studies of NAWM and NAGM, especially when follow up intervals are relatively short. Whilst existing quantitative MR measures from normal appearing tissues provide valuable information to understand the natural history and monitor treatment effects in MS, none of them fully or even predominantly accounts for the patients functional state nor can be relied on as a definitive surrogate measure of treatment effect. Better resolution of the abnormalities is needed especially in grey matter where pathological foci are known to be abundant. Studies correlating structural MR and fMRI parameters with measures of function in well defined anatomical pathways should further elucidate the pathogenic role of abnormality in the normal appearing tissues. In future, new imaging modalities are needed that provide a more specific measure of histopathological and cellular aspects of the disease process in vivo.     

Proton magnetic resonance spectroscopy of normal appearing white matter in asymptomatic relatives of multiple sclerosis patients

Proton magnetic resonance spectroscopy of normal appearing white matter (NAWM) was performed in 25 first-degree relatives of patients with multiple sclerosis (MS). In relatives of MS patients, insignificant trends in the NAA/Cho ratio (to be lower) and in the Cho/Cr ratio (to be higher) were found when compared with healthy controls. These results demonstrated that there are only minimal, insignificant changes in commonly identified metabolite concentrations in NAWM of relatives of MS patients when compared with healthy subjects.     

Grey matter pathology in multiple sclerosis

Although multiple sclerosis (MS) has been considered a white matter disease, MS lesions are known to occur in grey matter. Recent immunohistochemical studies have demonstrated extensive grey matter demyelination in chronic MS. The most common lesion type consists of purely cortical lesions extending inward from the surface of the brain, this lesion subgroup is grossly underestimated by standard histochemical myelin staining methods. Some MS patients have subpial demyelination in all cortical areas of the brain; this pattern has been termed 'general cortical subpial demyelination'. Extensive cortical demyelination is associated with the progressive phases of disease, as less cortical demyelination has been detected in relapsing-remitting MS. The pathology of grey matter lesions differs from that of white matter lesions; grey matter lesions are less inflammatory, with less macrophage and lymphocyte infiltration. In purely cortical lesions there is no significant increase in lymphocytes compared with non-demyelinated adjacent cortical areas in MS patients or cerebral cortex in control patients. Significant axonal transection and neuronal loss have been demonstrated in grey matter MS lesions. Current magnetic resonance imaging (MRI) methods are not sensitive for purely cortical MS lesions. The clinical significance of cortical MS lesions may not be characterised until more sensitive MRI methods are developed.     

Differential white and gray matter damage in highly active multiple sclerosis: A prospective cohort study

We analyze the differential brain volume changes in highly active multiple sclerosis (HAMS) vs. non-HAMS patients during the disease onset.MethodsHAMS was defined as: a) patients with 1 relapse in the previous year and at least 1 T1 gadolinium-enhancing lesion or 9 or more T2 lesions while on therapy with other disease modifying treatment (DMD); or b) patients with 2 or more relapses in the previous year, whether on DMD or not. High-resolution T1 weighted MRI scans were acquired at onset and every 12 months for 2 years. Lesion load and brain volume measurements were determined. At onset, gray matter volume (GMV) and white matter volume (WMV) tissue volumes were calculated using the SIENAX. Longitudinal changes were estimated by using SIENA to calculate the percentage of brain volume loss. Differences between volumes per group at onset and at the end of the follow up were established.Results64 patients, mean age 38.4 years, 35 (57%) women were included. A total of 14 (21%) were classified as HAMS. At onset, HAMS patients showed lower GMV and WMV volume compared with non-HAMS patients (p = 0.003 and p = 0.01, respectively). During the follow up, HAMS patients showed a higher decrease in GM volume compared with non-HAMS patients (-0.61 vs. – 0.77, p < 0.001) independent from new lesion as well as relapse rate activity during follow up.ConclusionHAMS increased rates of GMV atrophy over 24 months compared to non-HAMS patients independent from relapse rate and new T2 lesions.     

Pathological abnormalities in the normal-appearing white matter in multiple sclerosis

In established cases of multiple sclerosis (MS), the normal-appearing white matter (NAWM), as defined for magnetic resonance imaging (MRI), is abnormal in the majority of cases. The clinical significance of these NAWM abnormalities is the subject of debate, but there is strong correlation with degree and progression of disability. New lesions form in NAWM before blood-brain barrier breakdown, as evidenced by gadolinium enhancement. The pathological basis of these neuroimaging abnormalities is largely unknown. Definitive pathological studies on the NAWM are few and are often based on small numbers of samples and of cases. Despite a variety of MS NAWM pathological studies, major research questions, of importance to our understanding of basic pathogenetic mechanisms and consequent rational therapies, remain unanswered. These relate to the frequency and extent of oligodendrocyte/myelin and axonal abnormalities in MS NAWM, and to the cellular basis of very early MS lesions detected by neuroimaging. In a pilot study of MS NAWM, microglial activation was demonstrated in 9 of 10 MS cases. We are currently testing the hypothesis that microglial activation, as defined by altered phenotype and HLA-DR positivity, will act as a marker for oligodendrocyte/myelin and axonal pathology in MS NAWM.