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1.  In multiple sclerosis oligoclonal bands connect to peripheral B cell responses 
Annals of neurology  2014;75(2):266-276.
Objective
To determine to what extent oligoclonal band (OCB) specificities are clonally interrelated and to what degree they are associated with corresponding B-cell responses in the peripheral blood of multiple sclerosis (MS) patients.
Methods
Mass-spectrometric proteomic analysis of isoelectric focused (IEF) CSF IgG was used in combination with next-generation deep-immune repertoire sequencing of peripheral blood (PB) and CSF IgG heavy chain variable regions (IgG-VH) from MS patients.
Results
We find evidence for ongoing stimulation and maturation to antibody expressing B cells to occur primarily inside the CNS compartment. B cells participating in OCB production can also be identified in peripheral blood; these cells appear to migrate across the blood-brain barrier (BBB) and may also undergo further antigen-stimulation in the periphery. In individual patients different bands comprising OCB are clonally related.
Interpretation
Our data provide a high-resolution molecular analysis of OCB and strongly support the concept that OCB are not merely the terminal result of a targeted immune response in MS but represent a component of active B cell immunity that is dynamically supported on both sides of the blood-brain barrier.
doi:10.1002/ana.24088
PMCID: PMC3961546  PMID: 24375699
2.  Medial Temporal Lobe Function and Structure in Mild Cognitive Impairment 
Annals of neurology  2004;56(1):27-35.
Functional magnetic resonance imaging (fMRI) was used to study memory-associated activation of medial temporal lobe (MTL) regions in 32 nondemented elderly individuals with mild cognitive impairment (MCI). Subjects performed a visual encoding task during fMRI scanning and were tested for recognition of stimuli afterward. MTL regions of interest were identified from each individual’s structural MRI, and activation was quantified within each region. Greater extent of activation within the hippocampal formation and parahippocampal gyrus (PHG) was correlated with better memory performance. There was, however, a paradoxical relationship between extent of activation and clinical status at both baseline and follow-up evaluations. Subjects with greater clinical impairment, based on the Clinical Dementia Rating Sum of Boxes, recruited a larger extent of the right PHG during encoding, even after accounting for atrophy. Moreover, those who subsequently declined over the 2.5 years of clinical follow-up (44% of the subjects) activated a significantly greater extent of the right PHG during encoding, despite equivalent memory performance. We hypothesize that increased activation in MTL regions reflects a compensatory response to accumulating AD pathology and may serve as a marker for impending clinical decline.
doi:10.1002/ana.20163
PMCID: PMC4335689  PMID: 15236399
3.  Natural History of Denervation in SMA: Relation to Age, SMN2 Copy Number, and Function 
Annals of neurology  2005;57(5):704-712.
Denervation was assessed in 89 spinal muscular atrophy (SMA) 1, 2, and 3 subjects via motor unit number estimation (MUNE) and maximum compound motor action potential amplitude (CMAP) studies, and results correlated with SMN2 copy, age, and function. MUNE and maximum CMAP values were distinct among SMA subtypes (p < 0.05). Changes in MUNE and maximum CMAP values over time were dependent on age, SMA type, and SMN2 copy number. SMN2 copy number less than 3 correlated with lower MUNE and maximum CMAP values (p < 0.0001) and worse functional outcomes. As SMN2 copy number increases, so does functional status (p < 0.0001). Change in MUNE longitudinally over the time intervals examined in this study was not statistically significant for any SMA cohort. However, a decline in maximum CMAP over time was apparent in SMA2 subjects (p = 0.049). Age-dependent decline in MUNE and maximum CMAP was apparent in both SMA 1 (p < 0.0001) and SMA 2 (p < 0.0001) subjects, with age as an independent factor regardless of type. Maximum CMAP at the time of the initial assessment was most predictive of functional outcome (p < 0.0001). Prospective longitudinal studies in four prenatally diagnosed infants demonstrated significant progressive denervation in association with symptomatic onset or functional decline. These data highlight the potential value of such measures in increasing our understanding of pathophysiological factors involved in denervation in SMA.
doi:10.1002/ana.20473
PMCID: PMC4334582  PMID: 15852397
4.  Glucose Metabolism and Pancreatic Defects in Spinal Muscular Atrophy 
Annals of neurology  2012;72(2):256-268.
Objective
Spinal muscular atrophy (SMA) is the number 1 genetic killer of young children. It is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. Although SMA is primarily a motor neuron disease, metabolism abnormalities such as metabolic acidosis, abnormal fatty acid metabolism, hyperlipidemia, and hyperglycemia have been reported in SMA patients. We thus initiated an in-depth analysis of glucose metabolism in SMA.
Methods
Glucose metabolism and pancreas development were investigated in the Smn2B/− intermediate SMA mouse model and type I SMA patients.
Results
Here, we demonstrate in an SMA mouse model a dramatic cell fate imbalance within pancreatic islets, with a predominance of glucagon-producing α cells at the expense of insulin-producing β cells. These SMA mice display fasting hyperglycemia, hyperglucagonemia, and glucose resistance. We demonstrate similar abnormalities in pancreatic islets from deceased children with the severe infantile form of SMA in association with supportive evidence of glucose intolerance in at least a subset of such children.
Interpretation
Our results indicate that defects in glucose metabolism may play an important contributory role in SMA pathogenesis.
doi:10.1002/ana.23582
PMCID: PMC4334584  PMID: 22926856
5.  Adult Polyglucosan Body Disease: Natural History and Key Magnetic Resonance Imaging Findings 
Annals of neurology  2012;72(3):433-441.
Objective
Adult polyglucosan body disease (APBD) is an autosomal recessive leukodystrophy characterized by neurogenic bladder, progressive spastic gait, and peripheral neuropathy. Polyglucosan bodies accumulate in the central and peripheral nervous systems and are often associated with glycogen branching enzyme (GBE) deficiency. To improve clinical diagnosis and enable future evaluation of therapeutic strategies, we conducted a multinational study of the natural history and imaging features of APBD.
Methods
We gathered clinical, biochemical, and molecular findings in 50 APBD patients with GBE deficiency from Israel, the United States, France, and the Netherlands. Brain and spine magnetic resonance images were reviewed in 44 patients.
Results
The most common clinical findings were neurogenic bladder (100%), spastic paraplegia with vibration loss (90%), and axonal neuropathy (90%). The median age was 51 years for the onset of neurogenic bladder symptoms, 63 years for wheelchair dependence, and 70 years for death. As the disease progressed, mild cognitive decline may have affected up to half of the patients. Neuroimaging showed hyperintense white matter abnormalities on T2 and fluid attenuated inversion recovery sequences predominantly in the periventricular regions, the posterior limb of the internal capsule, the external capsule, and the pyramidal tracts and medial lemniscus of the pons and medulla. Atrophy of the medulla and spine was universal. p.Y329S was the most common GBE1 mutation, present as a single heterozygous (28%) or homozygous (48%) mutation.
Interpretation
APBD with GBE deficiency, with occasional exceptions, is a clinically homogenous disorder that should be suspected in patients with adult onset leukodystrophy or spastic paraplegia with early onset of urinary symptoms and spinal atrophy.
doi:10.1002/ana.23598
PMCID: PMC4329926  PMID: 23034915
6.  Sex is a major determinant of neuronal dysfunction in Neurofibromatosis Type 1 
Annals of neurology  2014;75(2):309-316.
Objective
Children with Neurofibromatosis-1 (NF1) are at risk for developing numerous nervous system abnormalities, including cognitive problems and brain tumors (optic pathway glioma). Currently, there are few prognostic factors that predict clinical manifestations or outcomes in patients, even in families with an identical NF1 gene mutation. In this study, we leveraged Nf1 genetically-engineered mice (GEM) to define the potential role of sex as a clinically-relevant modifier of NF1-associated neuronal dysfunction.
Methods
De-identified clinical data were analyzed to determine the impact of sex on optic glioma-associated visual decline in children with NF1. In addition, Nf1 GEM were employed as experimental platforms to investigate sexually dimorphic differences in learning/memory, visual acuity, retinal ganglion cell (RGC) death, and Nf1 protein (neurofibromin)-regulated signaling pathway function (RAS activity, cyclic AMP and dopamine levels).
Results
Female patients with NF1-associated optic glioma were twice as likely to undergo brain MRI for visual symptoms and three times more likely to require treatment for visual decline than their male counterparts. As such, only female Nf1 GEM exhibited a decrement in optic glioma-associated visual acuity, shorter RGC axons, and attenuated cAMP levels. In contrast, only male Nf1 GEM showed spatial learning/memory deficits, increased RAS activity, and reduced dopamine levels.
Interpretation
Collectively, these observations establish sex as a major prognostic factor underlying neuronal dysfunction in NF1, and suggest that sex should be considered when interpreting future preclinical and clinical study results.
doi:10.1002/ana.24093
PMCID: PMC4172335  PMID: 24375753
neurofibromin; cyclic AMP; dopamine; Ras; NF1; learning; vision; central nervous system
7.  The Child Is Father to the Man: Developmental Roles for Proteins of Importance for Neurodegenerative Disease 
Annals of neurology  2010;67(2):151-158.
Although Alzheimer’s and Parkinson’s diseases predominately affect elderly adults, the proteins that play a role in the pathogenesis of these diseases are expressed throughout life. In fact, many of the proteins hypothesized to be important in the progression of neurodegeneration play direct or indirect roles in the development of the central nervous system. The systems affected by these proteins include neural stem cell fate decisions, neuronal differentiation, cellular migration, protection from oxidative stress, and programmed cell death. Insights into the developmental roles of these proteins may ultimately impact the understanding of neurodegenerative diseases and lead to the discovery of novel treatments.
doi:10.1002/ana.21841
PMCID: PMC4315179  PMID: 20225270
8.  Glucose and NADPH oxidase drive neuronal superoxide formation in stroke 
Annals of neurology  2008;64(6):654-663.
Objective
Hyperglycemia has been recognized for decades to be an exacerbating factor in ischemic stroke, but the mechanism of this effect remains unresolved. Here we evaluated superoxide production by neuronal NADPH oxidase as a link between glucose metabolism and neuronal death in ischemia-reperfusion.
Methods
Superoxide production was measured by the ethidium method in cultured neurons treated with oxygen-glucose deprivation and in mice treated with forebrain ischemia-reperfusion. The role of NADPH oxidase was examined using genetic disruption of its p47phox subunit and with the pharmacological inhibitor, apocynin.
Results
In neuron cultures, post-ischemic superoxide production and cell death were completely prevented by removing glucose from the medium, by inactivating NADPH oxidase, or by inhibiting the hexose monophosphate shunt which generates NADPH from glucose. In murine stroke, neuronal superoxide production and death were decreased by the glucose anti-metabolite, 2-deoxyglucose, and increased by high blood glucose concentrations. Inactivating NADPH oxidase with either apocynin or deletion of the p47phox subunit blocked neuronal superoxide production and negated the deleterious effects of hyperglycemia.
Interpretation
These findings identify glucose as the requisite electron donor for reperfusion-induced neuronal superoxide production and establish a previously unrecognized mechanism by which hyperglycemia can exacerbate ischemic brain injury.
doi:10.1002/ana.21511
PMCID: PMC4304737  PMID: 19107988
ischemia; reperfusion; superoxide; NADPH oxidase; hexose monophosphate shunt
9.  [No title available] 
PMCID: PMC4294275  PMID: 14681886
10.  Cerebellar Morphology in Tourette Syndrome and Obsessive-Compulsive Disorder 
Annals of neurology  2010;67(4):479-487.
Objective
Neuroanatomical and functional imaging studies have identified the cerebellum as an integral component of motor and language control. Few studies, however, have investigated the role of the cerebellum in Tourette syndrome (TS), a condition defined by the presence of semi-involuntary movements and sounds.
Methods
Magnetic resonance imaging was conducted in 163 persons with TS and 147 control participants. Multivariate linear regression models were used to explore effects on cerebellar surface morphology and underlying volumes for the main diagnosis effects of TS as well as comorbid obsessive-compulsive disorder (OCD) and attention-deficit/hyperactivity disorder. Additionally, the correlations of symptom severity with cerebellar morphology were also assessed.
Results
The TS group demonstrated reduced volumes of the cerebellar hemispheres bilaterally that derived primarily from reduced gray matter in crus I and lobules VI, VIIB, and VIIIA. These decreased regional volumes accompanied increasing tic symptom severity and motoric disinhibition as demonstrated by a finger tapping test. Males had reduced volumes of these same regions compared with females, irrespective of diagnosis. Comorbid OCD was associated with relative enlargement of these regions in proportion to the increasing severity of OCD symptoms.
Interpretation
The cerebellum is involved in the pathogenesis of TS and tic-related OCD. Baseline gender differences in cerebellar morphology may in part account for the more prevalent expression of TS in males.
doi:10.1002/ana.21918
PMCID: PMC4286180  PMID: 20437583
11.  The α2B adrenergic receptor is mutant in cortical myoclonus and epilepsy 
Annals of neurology  2014;75(1):77-87.
Objective
Autosomal dominant cortical myoclonus and epilepsy (ADCME) is characterized by distal, fairly rhythmic myoclonus and epilepsy with variable severity. We have previously mapped the disease locus on chromosome 2p11.1-q12.2 by genome-wide linkage analysis. Additional pedigrees affected by similar forms of epilepsy have been associated to chromosome 8q, 5p and 3q, but none of the causing genes has been identified. We aim at identifying the mutant gene responsible for this epileptic form.
Methods
Genes included in the ADCME critical region were prioritized and directly sequenced. Co-immunoprecipitation, immunofluorescence and electrophysiology approaches on transfected human cells have been utilized for testing the functional significance of the identified mutation.
Results
Here we show that mutation in the α2-adrenergic receptor subtype B (α2B-AR) associates to ADCME by identifying a novel in-frame insertion/deletion in two Italian families. The mutation alters several conserved residues of the third intracellular (3i) loop, neither hampering the α2B-AR plasma membrane localization nor the arrestin-mediated internalization capacity, but altering the binding with the scaffolding protein spinophilin upon neurotransmitter activation. Spinophilin, in turn, regulates interaction of GPCRs with Regulators of G proteins Signaling proteins. Accordingly, the mutant α2B-AR increases the epinephrine-stimulated calcium signaling.
Interpretation
The identified mutation is responsible for ADCME, as the loss of α2B-AR/spinophilin interaction causes a gain of function effect. This work implicates for the first time the α-adrenergic system in human epilepsy and opens new ways for understanding the molecular pathway of epileptogenesis, widening the spectrum of possible therapeutic targets.
doi:10.1002/ana.24028
PMCID: PMC3932827  PMID: 24114805
12.  Glial Localization of Antiquitin: Implications for Pyridoxine-Dependent Epilepsy 
Annals of neurology  2014;75(1):22-32.
Objective
A high incidence of structural brain abnormalities has been reported in individuals with pyridoxine-dependent epilepsy (PDE). PDE is caused by mutations in ALDH7A1, also known as antiquitin. How antiquitin dysfunction leads to cerebral dysgenesis is unknown. In this study, we analyzed tissue from a child with PDE as well as control human and murine brain to determine the normal distribution of antiquitin, its distribution in PDE, and associated brain malformations.
Methods
Formalin-fixed human brain sections were subjected to histopathology and fluorescence immunohistochemistry studies. Frozen brain tissue was utilized for measurement of PDE-associated metabolites and Western blot analysis. Comparative studies of antiquitin distribution were performed in developing mouse brain sections.
Results
Histologic analysis of PDE cortex revealed areas of abnormal radial neuronal organization consistent with type Ia focal cortical dysplasia. Heterotopic neurons were identified in subcortical white matter, as was cortical astrogliosis, hippocampal sclerosis, and status marmoratus of the basal ganglia. Highly elevated levels of lysine metabolites were present in postmortem PDE cortex. In control human and developing mouse brain, antiquitin immunofluorescence was identified in radial glia, mature astrocytes, ependyma, and choroid plexus epithelium, but not in neurons. In PDE cortex, antiquitin immunofluorescence was greatly attenuated with evidence of perinuclear accumulation in astrocytes.
Interpretation
Antiquitin is expressed within glial cells in the brain, and its dysfunction in PDE is associated with neuronal migration abnormalities and other structural brain defects. These malformations persist despite postnatal pyridoxine supplementation and likely contribute to neurodevelopmental impairments.
doi:10.1002/ana.24027
PMCID: PMC3945410  PMID: 24122892
13.  Characterization of Atypical Language Activation Patterns in Focal Epilepsy 
Annals of neurology  2014;75(1):33-42.
Objective
Functional magnetic resonance imaging is sensitive to the variation in language network patterns. Large populations are needed to rigorously assess atypical patterns, which, even in neurological populations, are a minority.
Methods
We studied 220 patients with focal epilepsy and 118 healthy volunteers who performed an auditory description decision task. We compared a data-driven hierarchical clustering approach to the commonly used a priori laterality index (LI) threshold (LI < 0.20 as atypical) to classify language patterns within frontal and temporal regions of interest. We explored (n = 128) whether IQ varied with different language activation patterns.
Results
The rate of atypical language among healthy volunteers (2.5%) and patients (24.5%) agreed with previous studies; however, we found 6 patterns of atypical language: a symmetrically bilateral, 2 unilaterally crossed, and 3 right dominant patterns. There was high agreement between classification methods, yet the cluster analysis revealed novel correlations with clinical features. Beyond the established association of left-handedness, early seizure onset, and vascular pathology with atypical language, cluster analysis identified an association of handedness with frontal lateralization, early seizure onset with temporal lateralization, and left hemisphere focus with a unilateral right pattern. Intelligence quotient was not significantly different among patterns.
Interpretation
Language dominance is a continuum; however, our results demonstrate meaningful thresholds in classifying laterality. Atypical language patterns are less frequent but more variable than typical language patterns, posing challenges for accurate presurgical planning. Language dominance should be assessed on a regional rather than hemispheric basis, and clinical characteristics should inform evaluation of atypical language dominance. Reorganization of language is not uniformly detrimental to language functioning.
doi:10.1002/ana.24015
PMCID: PMC4209919  PMID: 24038442
14.  Metallothioneins as Dynamic Markers for Brain Disease in Lysosomal Disorders 
Annals of neurology  2014;75(1):127-137.
Objective
To facilitate development of novel disease-modifying therapies for lysosomal storage disorder (LSDs) characterized by nervous system involvement such as metachromatic leukodystrophy (MLD), molecular markers for monitoring disease progression and therapeutic response are needed. To this end, we sought to identify blood transcripts associated with the progression of MLD.
Methods
Genome-wide expression analysis was performed in primary T lymphocytes of 24 patients with MLD compared to 24 age- and sex-matched healthy controls. Genes associated with MLD were identified, confirmed on a quantitative polymerase chain reaction platform, and replicated in an independent patient cohort. mRNA and protein expression of the prioritized gene family of metallothioneins was evaluated in postmortem patient brains and in mouse models representing 6 other LSDs. Metallothionein expression during disease progression and in response to specific treatment was evaluated in 1 of the tested LSD mouse models. Finally, a set of in vitro studies was planned to dissect the biological functions exerted by this class of molecules.
Results
Metallothionein genes were significantly overexpressed in T lymphocytes and brain of patients with MLD and generally marked nervous tissue damage in the LSDs here evaluated. Overexpression of metallothioneins correlated with measures of disease progression in mice and patients, whereas their levels decreased in mice upon therapeutic treatment. In vitro studies indicated that metallothionein expression is regulated in response to oxidative stress and inflammation, which are biochemical hallmarks of lysosomal storage diseases.
Interpretation
Metallothioneins are potential markers of neurologic disease processes and treatment response in LSDs.
doi:10.1002/ana.24053
PMCID: PMC4237725  PMID: 24242821
15.  Sex-specific control of CNS autoimmunity by p38 MAPK signaling in myeloid cells 
Annals of neurology  2014;75(1):50-66.
Objective
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS), characterized by a global increasing incidence driven by relapsing-remitting disease in females. p38 MAP kinase (MAPK) has been described as a key regulator of inflammatory responses in autoimmunity, but its role in the sexual dimorphism in MS or MS models remains unexplored.
Methods
Toward this end, we used experimental autoimmune encephalomyelitis (EAE), the principal animal model of MS, combined with pharmacologic and genetic inhibition of p38 MAPK activity and transcriptomic analyses.
Results
Pharmacologic inhibition of p38 MAPK selectively ameliorated EAE in female mice. Conditional deletion studies demonstrated that p38α signaling in macrophages/myeloid cells, but not T cells or dendritic cells, recapitulated this sexual dimorphism. Analysis of CNS inflammatory infiltrates showed that female, but not male mice lacking p38α in myeloid cells exhibited reduced immune cell activation compared with controls, while peripheral T cell priming was unaffected in both sexes. Transcriptomic analyses of myeloid cells revealed differences in p38α-controlled transcripts comprising female- and male-specific gene modules, with greater p38α dependence of pro-inflammatory gene expression in females.
Interpretation
Our findings demonstrate a key role for p38α in myeloid cells in CNS autoimmunity and uncover important molecular mechanisms underlying sex differences in disease pathogenesis. Taken together, our results suggest that the p38 MAPK signaling pathway represents a novel target for much needed disease modifying therapies for MS.
doi:10.1002/ana.24020
PMCID: PMC3945470  PMID: 24027119
16.  Anti-androgen Treatment for Spinal and Bulbar Muscular Atrophy 
Annals of neurology  2009;65(2):119-120.
doi:10.1002/ana.21633
PMCID: PMC4280995  PMID: 19259961
17.  Comparing PET imaging and CSF measurements of Aβ 
Annals of neurology  2013;74(6):826-836.
Objective
We examined agreement and disagreement between two biomarkers of Aβ deposition (amyloid PET and CSF Aβ1-42) in normal aging and dementia in a large multicenter study.
Methods
Concurrently acquired florbetapir-PET and CSF Aβ were measured in cognitively normal, mild cognitive impairment (MCI), and Alzheimer’s disease (AD) participants (N=374) from the Alzheimer’s Disease Neuroimaging Initiative (ADNI). We also compared Aβ measurements in a separate group with serial CSF measurements over 3.1 +/− 0.8 yrs that preceded a single florbetapir session. Additional biomarker and cognitive data allowed us to further examine profiles of discordant cases.
Results
Florbetapir and CSF Aβ were inversely correlated across all diagnostic groups, and dichotomous measurements were in agreement in 86% of subjects. Among subjects showing the most disagreement, the two discordant groups had different profiles: the florbetapir+/CSF Aβ− group was larger (N=13) and was made up of only normal and early MCI subjects; while the florbetapir−/CSF Aβ+ group was smaller (N=7), had poorer cognitive function and higher CSF tau, but no ApoE4 carriers. In the longitudinal sample, we observed both stable longitudinal CSF Aβ trajectories and those actively transitioning from normal to abnormal, but the final CSF Aβ measurements were in good agreement with florbetapir cortical retention.
Interpretation
CSF and amyloid-PET measurements of Aβ were consistent in the majority of subjects in the cross-sectional and longitudinal populations. Based on our analysis of discordant subjects, the available evidence did not show that CSF Aβ regularly becomes abnormal prior to fibrillar Aβ accumulation early in the course of disease.
doi:10.1002/ana.23908
PMCID: PMC3748164  PMID: 23536396
18.  Neuronal ER Stress in Axon Injury and Neurodegeneration 
Annals of neurology  2013;74(6):768-777.
Injuries to CNS axons result not only in Wallerian degeneration of the axon distal to the injury, but also in death or atrophy of the axotomized neurons, depending on injury location and neuron type. No method of permanently avoiding these changes has been found, despite extensive knowledge concerning mechanisms of secondary neuronal injury. The autonomous endoplasmic reticulum (ER) stress pathway in neurons has recently been implicated in retrograde neuronal degeneration. In addition to the emerging role of ER morphology in axon maintenance, we propose that ER stress is a common neuronal response to disturbances in axon integrity and a general mechanism for neurodegeneration. Thus manipulation of the ER stress pathway could have important therapeutic implications for neuroprotection.
doi:10.1002/ana.24005
PMCID: PMC3963272  PMID: 23955583
19.  Quantifying and Addressing Persistent Stroke Disparities in Hispanics 
Annals of neurology  2013;74(6):759-761.
doi:10.1002/ana.24025
PMCID: PMC4007247  PMID: 24114772
20.  SLC25A22 is a Novel Gene for Migrating Partial Seizures in Infancy 
Annals of neurology  2013;74(6):873-882.
Objective
To identify a genetic cause for migrating partial seizures in infancy (MPSI).
Methods
We characterized a consanguineous pedigree with MPSI and obtained DNA from affected and unaffected family members. We analyzed single nucleotide polymorphism (SNP) 500K data to identify regions with evidence for linkage. We performed whole exome sequencing and analyzed homozygous variants in regions of linkage to identify a candidate gene and performed functional studies of the candidate gene SLC25A22.
Results
In a consanguineous pedigree with two individuals with MPSI, we identified two regions of linkage, chromosome 4p16.1-p16.3 and chromosome 11p15.4-pter. Using whole exome sequencing, we identified 8 novel homozygous variants in genes in these regions. Only one variant, SLC25A22 c.G328C, results in a change of a highly conserved amino acid (p.G110R) and was not present in control samples. SLC25A22 encodes a glutamate transporter with strong expression in the developing brain. We show that the specific G110R mutation, located in a transmembraine domain of the protein, disrupts mitochondrial glutamate transport.
Interpretation
We have shown that MPSI can be inherited and have identified a novel homozygous mutation in SLC25A22 in the affected individuals. Our data strongly suggest that SLC25A22 is responsible for MPSI, a severe condition with few known etiologies. We have demonstrated that a combination of linkage analysis and whole exome sequencing can be used for disease gene discovery. Finally, as SLC25A22 had been implicated in the distinct syndrome neonatal epilepsy with suppression bursts on EEG, we have expanded the phenotypic spectrum associated with SLC25A22.
PMCID: PMC4031329  PMID: 24596948
21.  Splicing biomarkers of disease severity in myotonic dystrophy 
Annals of neurology  2013;74(6):862-872.
Objective
To develop RNA splicing biomarkers of disease severity and therapeutic response in myotonic dystrophy type 1 (DM1) and type 2 (DM2).
Methods
In a discovery cohort we used microarrays to perform global analysis of alternative splicing in DM1 and DM2. The newly identified splicing changes were combined with previous data to create a panel of 50 putative splicing defects. In a validation cohort of 50 DM1 subjects we measured the strength of ankle dorsiflexion (ADF) and then obtained a needle biopsy of tibialis anterior (TA) to analyze splice events in muscle RNA. The specificity of DM-associated splicing defects was assessed in disease controls. The CTG expansion size in muscle tissue was determined by Southern blot. The reversibility of splicing defects was assessed in transgenic mice by using antisense oligonucleotides (ASOs) to reduce levels of toxic RNA.
Results
Forty-two splicing defects were confirmed in TA muscle in the validation cohort. Among these, 20 events showed graded changes that correlated with ADF weakness. Five other splice events were strongly affected in DM1 subjects with normal ADF strength. Comparison to disease controls and mouse models indicated that splicing changes were DM-specific, mainly attributable to MBNL1 sequestration, and reversible in mice by targeted knockdown of toxic RNA. Splicing defects and weakness were not correlated with CTG expansion size in muscle tissue.
Interpretation
Alternative splicing changes in skeletal muscle may serve as biomarkers of disease severity and therapeutic response in myotonic dystrophy.
doi:10.1002/ana.23992
PMCID: PMC4099006  PMID: 23929620
22.  In multiple sclerosis, oligoclonal bands connect to peripheral B-cell responses 
Annals of Neurology  2014;75(2):266-276.
Objective
To determine to what extent oligoclonal band (OCB) specificities are clonally interrelated and to what degree they are associated with corresponding B-cell responses in the peripheral blood (PB) of multiple sclerosis (MS) patients.
Methods
Mass-spectrometric proteomic analysis of isoelectric focused (IEF) cerebrospinal fluid (CSF) immunoglobulin G (IgG) was used in combination with next-generation deep-immune repertoire sequencing of PB and CSF IgG heavy chain variable regions from MS patients.
Results
We find evidence for ongoing stimulation and maturation to antibody-expressing B cells to occur primarily inside the central nervous system (CNS) compartment. B cells participating in OCB production can also be identified in PB; these cells appear to migrate across the blood–brain barrier and may also undergo further antigen stimulation in the periphery. In individual patients, different bands comprising OCBs are clonally related.
Interpretation
Our data provide a high-resolution molecular analysis of OCBs and strongly support the concept that OCBs are not merely the terminal result of a targeted immune response in MS but represent a component of active B cell immunity that is dynamically supported on both sides of the blood-brain barrier.
doi:10.1002/ana.24088
PMCID: PMC3961546  PMID: 24375699
23.  GRIN2B Mutations in West Syndrome and Intellectual Disability with Focal Epilepsy 
Annals of Neurology  2014;75(1):147-154.
Objective
To identify novel epilepsy genes using a panel approach and describe the functional consequences of mutations.
Methods
Using a panel approach, we screened 357 patients comprising a vast spectrum of epileptic disorders for defects in genes known to contribute to epilepsy and/or intellectual disability (ID). After detection of mutations in a novel epilepsy gene, we investigated functional effects in Xenopus laevis oocytes and screened a follow-up cohort.
Results
We revealed de novo mutations in GRIN2B encoding the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor in 2 individuals with West syndrome and severe developmental delay as well as 1 individual with ID and focal epilepsy. The patient with ID and focal epilepsy had a missense mutation in the extracellular glutamate-binding domain (p.Arg540His), whereas both West syndrome patients carried missense mutations within the NR2B ion channel-forming re-entrant loop (p.Asn615Ile, p.Val618Gly). Subsequent screening of 47 patients with unexplained infantile spasms did not reveal additional de novo mutations, but detected a carrier of a novel inherited GRIN2B splice site variant in close proximity (c.2011-5_2011-4delTC). Mutations p.Asn615Ile and p.Val618Gly cause a significantly reduced Mg2+ block and higher Ca2+ permeability, leading to a dramatically increased Ca2+ influx, whereas p.Arg540His caused less severe disturbance of channel function, corresponding to the milder patient phenotype.
Interpretation
We identified GRIN2B gain-of-function mutations as a cause of West syndrome with severe developmental delay as well as of ID with childhood onset focal epilepsy. Severely disturbed channel function corresponded to severe clinical phenotypes, underlining the important role of facilitated NMDA receptor signaling in epileptogenesis.
doi:10.1002/ana.24073
PMCID: PMC4223934  PMID: 24272827
24.  White Matter Hyperintensities and Hypobaric Exposure 
Annals of neurology  2014;76(5):719-726.
Objective
Demonstrate that occupational exposure to nonhypoxic hypobaria is associated with subcortical white matter hyperintensities (WMHs) on fluid-attenuated inversion recovery magnetic resonance imaging (MRI).
Methods
Eighty-three altitude chamber personnel (PHY), 105 U-2 pilots (U2P), and 148 age- controlled and health-matched doctorate degree controls (DOC) underwent high-resolution MRI. Subcortical WMH burden was quantified as count and volume of subcortical WMH lesions after transformation of images to the Talairach atlas–based stereo-tactic frame.
Results
Subcortical WMHs were more prevalent in PHY (volume p = 0.011/count p = 0.019) and U2P (volume p<0.001/count p<0.001) when compared to DOC, whereas PHY were not significantly different than U2P.
Interpretation
This study provides strong evidence that nonhypoxic hypobaric exposure may induce subcortical WMHs in a young, healthy population lacking other risk factors for WMHs and adds this occupational exposure to other environmentally related potential causes of WMHs.
doi:10.1002/ana.24264
PMCID: PMC4219408  PMID: 25164539
25.  7T PHASE IMAGING OF ACUTE MS LESIONS: A NEW WINDOW INTO THE INFLAMMATORY PROCESS 
Annals of neurology  2013;74(5):10.1002/ana.23959.
Objectives
In multiple sclerosis (MS), accurate, in vivo characterization of dynamic inflammatory pathological changes occurring in newly forming lesions could have major implications for understanding disease pathogenesis and mechanisms of tissue destruction. Here, we investigated the potential of ultrahigh-field MRI (7T), particularly phase imaging combined with dynamic contrast enhancement, to provide new insights in acute MS lesions.
Methods
Sixteen active MS patients were studied at 7T. Noncontrast, high-resolution T2* magnitude and phase scans, T1 scans before/after gadolinium contrast injection, and dynamic contrast-enhanced (DCE) T1 scans were acquired. T2*/phase features and DCE pattern were determined for acute and chronic lesions. When possible, one-year follow-up 7T MRI was performed.
Results
Of 49 contrast-enhancing lesions, 44 could be analyzed. Centrifugal DCE lesions appeared isointense or hypointense on phase images, whereas centripetal DCE lesions showed thin, hypointense phase rims that clearly colocalized with the initial site of contrast enhancement. This pattern generally disappeared once enhancement resolved. On the other hand, in 43 chronic lesions also selected for the presence of hypointense phase rims, the findings were stable over time, and the rims were typically thicker and darker. These considerations suggest different underlying pathological processes in the two lesion types.
Interpretation
Ultrahigh-field MRI and, especially, phase contrast, are highly sensitive to tissue changes in acute MS lesions, which differ from the patterns seen in chronic lesions. In acute lesions, the hypointense phase rim reflects the expanding inflammatory edge and may directly correspond to inflammatory byproducts and sequelae of blood-brain barrier opening.
doi:10.1002/ana.23959
PMCID: PMC3812397  PMID: 23813441
inflammatory process; 7T phase imaging; multiple sclerosis

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