Malignant glioma in L-2-Hydroxyglutaric Aciduria: thorough molecular characterization of a case and literature review.

L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare neurometabolic disorder characterized by accumulation of L2-hydroxyglutarate (L-2-HG) due to mutations in the L2HGDH gene. L-2-HGA patients have a significantly increased lifetime risk of central nervous system (CNS) tumors. Here, we present a 16-year-old girl with L-2-HGA who developed a tumor in the right cerebral hemisphere, which was discovered after left-sided neurological deficits of the patient. Histologically, the tumor had a high-grade diffuse glioma phenotype. DNA sequencing revealed the inactivating homozygous germline L2HGDH mutation as well as inactivating mutations in TP53, BCOR and NF1. Genome-wide DNA-methylation analysis was unable to classify the tumor with high confidence. More detailed analysis revealed that this tumor clustered amongst IDH-wildtype gliomas by methylation profiling and did not show the glioma CpG island methylator phenotype (G-CIMP) in contrast to IDH-mutant diffuse gliomas with accumulated levels of D-2-HG, the stereoisomer of L-2-HD. These findings were against all our expectations given the inhibitory potential of 2-HG on DNA-demethylation enzymes. Our final integrated histomolecular diagnosis of the tumor was diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype. Due to rapid tumor progression the patient died nine months after initial diagnosis. In this manuscript, we provide extensive molecular characterization of the tumor as well as a literature review focusing on oncogenetic considerations of L-2-HGA-associated CNS tumors.

Region-specific and age-related differences in astrocytes in the human brain.

Astrocyte heterogeneity and its relation to aging in the normal human brain remain poorly understood. We here analyzed astrocytes in gray and white matter brain tissues obtained from donors ranging in age between the neonatal period to over 100 years. We show that astrocytes are differently distributed with higher density in the white matter. This regional difference in cellular density becomes less prominent with age. Additionally, we confirm the presence of morphologically distinct astrocytes, with gray matter astrocytes being morphologically more complex. Notably, gray matter astrocytes morphologically change with age, while white matter astrocytes remain relatively consistent in morphology. Using regional mass spectrometry-based proteomics, we did, however, identify astrocyte specific proteins with regional differences in abundance, reflecting variation in cellular density or expression level. Importantly, the expression of some astrocyte specific proteins region-dependently decreases with age. Taken together, we provide insights into region- and age-related differences in astrocytes in the human brain.

Progressive demyelinating polyneuropathy after hematopoietic cell transplantation in metachromatic leukodystrophy: a case series.

Metachromatic leukodystrophy (MLD) is a neuro-metabolic disorder due to arylsulfatase A deficiency, causing demyelination of the central and peripheral nervous system. Hematopoietic cell transplantation (HCT) can provide a symptomatic and survival benefit for pre-symptomatic and early symptomatic patients by stabilizing CNS disease. This case series, however, illustrates the occurrence of severely progressive polyneuropathy shortly after HCT in two patients with late-infantile, one with late-juvenile, and one with adult MLD, leading to the inability to walk or sit without support. The patients had demyelinating polyneuropathy before HCT, performed at the ages of 2 years in the first two patients and at 14 and 23 years in the other two patients. The myeloablative conditioning regimen consisted of busulfan, fludarabine and, in one case, rituximab, with anti-thymocyte globulin, cyclosporine, steroids, and/or mycophenolate mofetil for GvHD prophylaxis. Polyneuropathy after HCT progressed parallel with tapering immunosuppression and paralleled bouts of infection and graft-versus-host disease (GvHD). Differential diagnoses included MLD progression, neurological GvHD or another (auto)inflammatory cause. Laboratory, electroneurography and pathology investigations were inconclusive. In two patients, treatment with immunomodulatory drugs led to temporary improvement, but not sustained stabilization of polyneuropathy. One patient showed recovery to pre-HCT functioning, except for a Holmes-like tremor, for which a peripheral origin cannot be excluded. One patient showed marginal response to immunosuppressive treatment and died ten months after HCT due to respiratory failure. The extensive diagnostic and therapeutic attempts highlight the challenge of characterizing and treating progressive polyneuropathy in patients with MLD shortly after HCT. We advise to consider repeat electro-neurography and possibly peripheral nerve biopsy in such patients. Nerve conduction blocks, evidence of the presence of T lymphocytes and macrophages in the neuronal and surrounding nerve tissue, and beneficial effects of immunomodulatory drugs may indicate a partially (auto)immune-mediated pathology. Polyneuropathy may cause major residual disease burden after HCT. MLD patients with progressive polyneuropathy could potentially benefit from a more intensified immunomodulatory drug regime following HCT, especially at times of immune activation.

In vivo base editing of a pathogenic Eif2b5 variant improves vanishing white matter phenotypes in mice.

Vanishing white matter (VWM) is a fatal leukodystrophy caused by recessive mutations in subunits of the eukaryotic translation initiation factor 2B. Currently, there are no effective therapies for VWM. Here, we assessed the potential of adenine base editing to correct human pathogenic VWM variants in mouse models. Using adeno-associated viral vectors, we delivered intein-split adenine base editors into the cerebral ventricles of newborn VWM mice, resulting in 45.9% ± 5.9% correction of the Eif2b5R191H variant in the cortex. Treatment slightly increased mature astrocyte populations and partially recovered the integrated stress response (ISR) in female VWM animals. This led to notable improvements in bodyweight and grip strength in females; however, locomotor disabilities were not rescued. Further molecular analyses suggest that more precise editing (i.e., lower rates of bystander editing) as well as more efficient delivery of the base editors to deep brain regions and oligodendrocytes would have been required for a broader phenotypic rescue. Our study emphasizes the potential, but also identifies limitations, of current in vivo base-editing approaches for the treatment of VWM or other leukodystrophies.

Megalencephalic leukoencephalopathy with subcortical cysts: a variant update and review of the literature.

The leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC) is characterized by infantile-onset macrocephaly and chronic edema of the brain white matter. With delayed onset, patients typically experience motor problems, epilepsy and slow cognitive decline. No treatment is available. Classic MLC is caused by bi-allelic recessive pathogenic variants in MLC1 or GLIALCAM (also called HEPACAM). Heterozygous dominant pathogenic variants in GLIALCAM lead to remitting MLC, where patients show a similar phenotype in early life, followed by normalization of white matter edema and no clinical regression. Rare patients with heterozygous dominant variants in GPRC5B and classic MLC were recently described. In addition, two siblings with bi-allelic recessive variants in AQP4 and remitting MLC have been identified. The last systematic overview of variants linked to MLC dates back to 2006. We provide an updated overview of published and novel variants. We report on genetic variants from 508 patients with MLC as confirmed by MRI diagnosis (258 from our database and 250 extracted from 64 published reports). We describe 151 unique MLC1 variants, 29 GLIALCAM variants, 2 GPRC5B variants and 1 AQP4 variant observed in these MLC patients. We include experiments confirming pathogenicity for some variants, discuss particularly notable variants, and provide an overview of recent scientific and clinical insight in the pathophysiology of MLC.

Dominant CST3 variants cause adult onset leukodystrophy without amyloid angiopathy.

Leukodystrophies are rare genetic white matter disorders that have been regarded as mainly occurring in childhood. Recent years altered this perception, as a growing number of leukodystrophies was described to have an onset at adult ages. Still, many adult patients presenting with white matter changes remain without a specific molecular diagnosis. We describe a novel adult onset leukodystrophy in 16 patients from eight families carrying one of four different stop-gain or frameshift dominant variants in the CST3 gene. Clinical and radiological features differ markedly from the previously described Icelandic Cerebral Amyloid Angiopathy that was found in patients carrying p.Leu68Asn substitution in CST3. The clinical phenotype consists of recurrent episodes of hemiplegic migraine associated with transient unilateral focal deficits and slowly progressing motor symptoms and cognitive decline in mid-old adult ages. In addition, in some cases acute onset clinical deterioration led to a prolonged episode with reduced consciousness and even early death. Radiologically, pathognomonic changes are found at typical predilection sites involving the deep cerebral white matter sparing a periventricular and directly subcortical rim, the middle blade of corpus callosum, posterior limb of the internal capsule, middle cerebellar peduncles, cerebral peduncles, and specifically the globus pallidus. Histopathologic characterization in two autopsy cases did not reveal angiopathy, but instead micro- to macrocystic degeneration of the white matter. Astrocytes were activated at early stages and later on displayed severe degeneration and loss. In addition, despite loss of myelin, elevated numbers of partly apoptotic oligodendrocytes were observed. A structural comparison of the variants in CST3 suggests that specific truncations of Cystatin C result in an abnormal function, possibly by rendering the protein more prone to aggregation. Future studies are required to confirm the assumed effect on the protein and to determine pathophysiologic downstream events at the cellular level.

Longitudinal volumetric analysis of gray matter atrophy in metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder characterized by arylsulfatase A (ASA) deficiency, leading to sulfatide accumulation and myelin degeneration in the central nervous system. While primarily considered a white matter (WM) disease, gray matter (GM) is also affected in MLD, and hematopoietic stem cell transplantation (HSCT) may have limited effect on GM atrophy. We cross-sectionally and longitudinally studied GM volumes using volumetric MRI in a cohort of 36 (late-infantile, juvenile and adult type) MLD patients containing untreated and HSCT treated subjects. Cerebrum, cortical GM, (total) CSF, cerebellum, deep gray matter (DGM) (excluding thalamus) and thalamus volumes were analyzed. Longitudinal correlations with measures of cognitive and motor functioning were assessed. Cross-sectionally, juvenile and adult type patients (infantiles excluded based on limited numbers) were compared with controls at earliest scan, before possible treatment. Patients had lower cerebrum, cortical GM, DGM and thalamus volumes. Differences were most pronounced for adult type patients. Longitudinal analyses showed substantial and progressive atrophy of all regions and increase of CSF in untreated patients. Similar, albeit less pronounced, effects were seen in treated patients for cerebrum, cortical GM, CSF and thalamus volumes. Deterioration in motor performance (all patients) was related to atrophy, and increase of CSF, in all regions. Cognitive functioning (data available for treated patients) was related to cerebral, cortical GM and thalamus atrophy; and to CSF increase. Our findings illustrate the importance of recognizing GM pathology as a potentially substantial, clinically relevant part of MLD, apparently less amenable to treatment.

Lithium: effects in animal models of vanishing white matter are not promising.

Vanishing white matter (VWM) is a devastating autosomal recessive leukodystrophy, resulting in neurological deterioration and premature death, and without curative treatment. Pathogenic hypomorphic variants in subunits of the eukaryotic initiation factor 2B (eIF2B) cause VWM. eIF2B is required for regulating the integrated stress response (ISR), a physiological response to cellular stress. In patients' central nervous system, reduced eIF2B activity causes deregulation of the ISR. In VWM mouse models, the extent of ISR deregulation correlates with disease severity. One approach to restoring eIF2B activity is by inhibition of GSK3ß, a kinase that phosphorylates eIF2B and reduces its activity. Lithium, an inhibitor of GSK3ß, is thus expected to stimulate eIF2B activity and ameliorate VWM symptoms. The effects of lithium were tested in zebrafish and mouse VWM models. Lithium improved motor behavior in homozygous eif2b5 mutant zebrafish. In lithium-treated 2b4he2b5ho mutant mice, a paradoxical increase in some ISR transcripts was found. Furthermore, at the dosage tested, lithium induced significant polydipsia in both healthy controls and 2b4he2b5ho mutant mice and did not increase the expression of other markers of lithium efficacy. In conclusion, lithium is not a drug of choice for further development in VWM based on the limited or lack of efficacy and significant side-effect profile.

Adaptive behavior assessed by Vineland-3 as comprehensive outcome measure in vanishing white matter.

OBJECTIVES: Investigate the results and usability of the Vineland-3 as an outcome measure in vanishing white matter patients. METHODS: A cross-sectional investigation of the Vineland-3 based on interviews with caregivers, the Health Utilities Index, and the modified Rankin Scale in 64 vanishing white matter patients. RESULTS: Adaptive behavior measured with the Vineland-3 is impaired in the vast majority of vanishing white matter patients and significantly impacts daily life. Typically, the daily living skills domain is most severely affected and the socialization domain is the least affected. Based on the metric properties and the clinical relevance, the standard scores for the daily living skills domain and Adaptive Behavior Composite have the best properties to be used as an outcome measure. INTERPRETATION: The Vineland-3 appears to be a useful outcome measure to explore and quantify complex cognitive, behavioral, and psychiatric impairments affecting daily functioning in vanishing white matter. Further research should address the longitudinal evaluation of this tool and its additional value to standard neuropsychological and clinical examination.

Pridopidine subtly ameliorates motor skills in a mouse model for vanishing white matter.

The leukodystrophy vanishing white matter (VWM) is characterized by chronic and episodic acute neurological deterioration. Curative treatment is presently unavailable. Pathogenic variants in the genes encoding eukaryotic initiation factor 2B (eIF2B) cause VWM and deregulate the integrated stress response (ISR). Previous studies in VWM mouse models showed that several ISR-targeting compounds ameliorate clinical and neuropathological disease hallmarks. It is unclear which ISR components are suitable therapeutic targets. In this study, effects of 4-phenylbutyric acid, tauroursodeoxycholic acid, or pridopidine (PDPD), with ISR targets upstream or downstream of eIF2B, were assessed in VWM mice. In addition, it was found that the composite ataxia score represented motor decline of VWM mice more accurately than the previously used neuroscore. 4-phenylbutyric acid and tauroursodeoxycholic acid did not improve VWM disease hallmarks, whereas PDPD had subtle beneficial effects on motor skills. PDPD alone does not suffice as treatment in VWM mice but may be considered for combination therapy. Also, treatments aimed at ISR components upstream of eIF2B do not improve chronic neurological deterioration; effects on acute episodic decline remain to be investigated.

Applicability of multiple quantitative magnetic resonance methods in genetic brain white matter disorders.

BACKGROUND AND PURPOSE: Magnetic resonance imaging (MRI) measures of tissue microstructure are important for monitoring brain white matter (WM) disorders like leukodystrophies and multiple sclerosis. They should be sensitive to underlying pathological changes. Three whole-brain isotropic quantitative methods were applied and compared within a cohort of controls and leukodystrophy patients: two novel myelin water imaging (MWI) techniques (multi-compartment relaxometry diffusion-informed MWI: MCR-DIMWI, and multi-echo T2 relaxation imaging with compressed sensing: METRICS) and neurite orientation dispersion and density imaging (NODDI). METHODS: For 9 patients with different leukodystrophies (age range 0.4-62.4 years) and 15 control subjects (2.3-61.3 years), T1-weighted MRI, fluid-attenuated inversion recovery, multi-echo gradient echo with variable flip angles, METRICS, and multi-shell diffusion-weighted imaging were acquired on 3 Tesla. MCR-DIMWI, METRICS, NODDI, and quality control measures were extracted to evaluate differences between patients and controls in WM and deep gray matter (GM) regions of interest (ROIs). Pearson correlations, effect size calculations, and multi-level analyses were performed. RESULTS: MCR-DIMWI and METRICS-derived myelin water fractions (MWFs) were lower and relaxation times were higher in patients than in controls. Effect sizes of MWF values and relaxation times were large for both techniques. Differences between patients and controls were more pronounced in WM ROIs than in deep GM. MCR-DIMWI-MWFs were more homogeneous within ROIs and more bilaterally symmetrical than METRICS-MWFs. The neurite density index was more sensitive in detecting differences between patients and controls than fractional anisotropy. Most measures obtained from MCR-DIMWI, METRICS, NODDI, and diffusion tensor imaging correlated strongly with each other. CONCLUSION: This proof-of-concept study shows that MCR-DIMWI, METRICS, and NODDI are sensitive techniques to detect changes in tissue microstructure in WM disorders.

Core protocol development for phase 2/3 clinical trials in the leukodystrophy vanishing white matter: a consensus statement by the VWM consortium and patient advocates.

BACKGROUND: The leukodystrophy "Vanishing White Matter" (VWM) is an orphan disease with neurological decline and high mortality. Currently, VWM has no approved treatments, but advances in understanding pathophysiology have led to identification of promising therapies. Several investigational medicinal products are either in or about to enter clinical trial phase. Clinical trials in VWM pose serious challenges, as VWM has an episodic disease course; disease phenotype is highly heterogeneous and predictable only for early onset; and study power is limited by the small patient numbers. To address these challenges and accelerate therapy delivery, the VWM Consortium, a group of academic clinicians with expertise in VWM, decided to develop a core protocol to function as a template for trials, to improve trial design and facilitate sharing of control data, while permitting flexibility regarding other trial details. Overall aims of the core protocol are to collect safety, tolerability, and efficacy data for treatment assessment and marketing authorization. METHODS: To develop the core protocol, the VWM Consortium designated a committee, including clinician members of the VWM Consortium, family and patient group advocates, and experts in statistics, clinical trial design and alliancing with industries. We drafted three age-specific protocols, to stratify into more homogeneous patient groups, of ages ≥ 18 years, ≥ 6 to < 18 years and < 6 years. We chose double-blind, randomized, placebo-controlled design for patients aged ≥ 6 years; and open-label non-randomized natural-history-controlled design for patients < 6 years. The protocol describes study populations, age-specific endpoints, inclusion and exclusion criteria, study schedules, sample size determinations, and statistical considerations. DISCUSSION: The core protocol provides a shared uniformity across trials, enables a pool of shared controls, and reduces the total number of patients necessary per trial, limiting the number of patients on placebo. All VWM clinical trials are suggested to adhere to the core protocol. Other trial components such as choice of primary outcome, pharmacokinetics, pharmacodynamics, and biomarkers are flexible and unconstrained by the core protocol. Each sponsor is responsible for their trial execution, while the control data are handled by a shared research organization. This core protocol benefits the efficiency of parallel and consecutive trials in VWM, and we hope accelerates time to availability of treatments for VWM. TRIAL REGISTRATION: NA. From a scientific and ethical perspective, it is strongly recommended that all interventional trials using this core protocol are registered in a clinical trial register.

Gross Motor Function in Pediatric Onset TUBB4A-Related Leukodystrophy: GMFM-88 Performance and Validation of GMFC-MLD in TUBB4A.

TUBB4A pathogenic variants are associated with a spectrum of neurologic impairments including movement disorders and leukodystrophy. With the development of targeted therapies, there is an urgent unmet need for validated tools to measure mobility impairment. Our aim is to explore gross motor function in a pediatric-onset TUBB4A-related leukodystrophy cohort with existing gross motor outcome tools. Gross Motor Function Measure-88 (GMFM-88), Gross Motor Function Classification System (GMFCS-ER), and Gross Motor Function Classification-Metachromatic Leukodystrophy (GMFC-MLD) were selected through face validity. Subjects with a confirmed clinical and molecular diagnosis of TUBB4A-related leukodystrophy were enrolled. Participants' sex, age, genotype, and age at disease onset were collected, together with GMFM-88 and concurrent GMFCS-ER and GMFC-MLD. Performances on each measure were compared. GMFM-88 floor effect was defined as total score below 20%. A total of 35 subjects participated. Median performance by GMFM-88 was 16.24% (range 0-97.31), with 42.9% (n = 15) of individuals performing above the floor. GMFM-88 Dimension A (Lying and Rolling) was the best-performing dimension in the GMFM-88 (n = 29 above the floor). All levels of the Classification Scales were represented, with the exception of the GMFC-MLD level 0. Evaluation by GMFM-88 was strongly correlated with the Classification Scales (Spearman correlations: GMFCS-ER:GMFM-88 r = 0.90; GMFC-MLD:GMFM-88 r = 0.88; GMFCS-ER:GMFC-MLD: r = 0.92). Despite overall observation of a floor effect, the GMFM-88 is able to accurately capture the performance of individuals with attenuated phenotypes. GMFM-88 Dimension A shows no floor effect. GMFC-MLD shows a strong correlation with GMFCS-ER and GMFM-88, supporting its use as an age-independent functional score in TUBB4A-related leukodystrophy.

Regional vulnerability of brain white matter in vanishing white matter.

Vanishing white matter (VWM) is a leukodystrophy that primarily manifests in young children. In this disease, the brain white matter is differentially affected in a predictable pattern with telencephalic brain areas being most severely affected, while others remain allegedly completely spared. Using high-resolution mass spectrometry-based proteomics, we investigated the proteome patterns of the white matter in the severely affected frontal lobe and normal appearing pons in VWM and control cases to identify molecular bases underlying regional vulnerability. By comparing VWM patients to controls, we identified disease-specific proteome patterns. We showed substantial changes in both the VWM frontal and pons white matter at the protein level. Side-by-side comparison of brain region-specific proteome patterns further revealed regional differences. We found that different cell types were affected in the VWM frontal white matter than in the pons. Gene ontology and pathway analyses identified involvement of region specific biological processes, of which pathways involved in cellular respiratory metabolism were overarching features. In the VWM frontal white matter, proteins involved in glycolysis/gluconeogenesis and metabolism of various amino acids were decreased compared to controls. By contrast, in the VWM pons white matter, we found a decrease in proteins involved in oxidative phosphorylation. Taken together, our data show that brain regions are affected in parallel in VWM, but to different degrees. We found region-specific involvement of different cell types and discovered that cellular respiratory metabolism is likely to be differentially affected across white matter regions in VWM. These region-specific changes help explain regional vulnerability to pathology in VWM.

Quantitative MRI in leukodystrophies.

Leukodystrophies constitute a large and heterogeneous group of genetic diseases primarily affecting the white matter of the central nervous system. Different disorders target different white matter structural components. Leukodystrophies are most often progressive and fatal. In recent years, novel therapies are emerging and for an increasing number of leukodystrophies trials are being developed. Objective and quantitative metrics are needed to serve as outcome measures in trials. Quantitative MRI yields information on microstructural properties, such as myelin or axonal content and condition, and on the chemical composition of white matter, in a noninvasive fashion. By providing information on white matter microstructural involvement, quantitative MRI may contribute to the evaluation and monitoring of leukodystrophies. Many distinct MR techniques are available at different stages of development. While some are already clinically applicable, others are less far developed and have only or mainly been applied in healthy subjects. In this review, we explore the background, current status, potential and challenges of available quantitative MR techniques in the context of leukodystrophies.

Focal lesions following intracerebral gene therapy for mucopolysaccharidosis IIIA.

OBJECTIVE: Mucopolysaccharidosis type IIIA (MPSIIIA) caused by recessive SGSH variants results in sulfamidase deficiency, leading to neurocognitive decline and death. No disease-modifying therapy is available. The AAVance gene therapy trial investigates AAVrh.10 overexpressing human sulfamidase (LYS-SAF302) delivered by intracerebral injection in children with MPSIIIA. Post-treatment MRI monitoring revealed lesions around injection sites. Investigations were initiated in one patient to determine the cause. METHODS: Clinical and MRI details were reviewed. Stereotactic needle biopsies of a lesion were performed; blood and CSF were sampled. All samples were used for viral studies. Immunohistochemistry, electron microscopy, and transcriptome analysis were performed on brain tissue of the patient and various controls. RESULTS: MRI revealed focal lesions around injection sites with onset from 3 months after therapy, progression until 7 months post therapy with subsequent stabilization and some regression. The patient had transient slight neurological signs and is following near-normal development. No evidence of viral or immunological/inflammatory cause was found. Immunohistochemistry showed immature oligodendrocytes and astrocytes, oligodendrocyte apoptosis, strong intracellular and extracellular sulfamidase expression and hardly detectable intracellular or extracellular heparan sulfate. No activation of the unfolded protein response was found. INTERPRETATION: Results suggest that intracerebral gene therapy with local sulfamidase overexpression leads to dysfunction of transduced cells close to injection sites, with extracellular spilling of lysosomal enzymes. This alters extracellular matrix composition, depletes heparan sulfate, impairs astrocyte and oligodendrocyte function, and causes cystic white matter degeneration at the site of highest gene expression. The AAVance trial results will reveal the potential benefit-risk ratio of this therapy.

Aquaporin-4 and GPRC5B: old and new players in controlling brain oedema.

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema.

Neurodegenerative disease after hematopoietic stem cell transplantation in metachromatic leukodystrophy.

OBJECTIVE: Metachromatic leukodystrophy is a lysosomal storage disease caused by deficient arylsulfatase A. It is characterized by progressive demyelination and thus mainly affects the white matter. Hematopoietic stem cell transplantation may stabilize and improve white matter damage, yet some patients deteriorate despite successfully treated leukodystrophy. We hypothesized that post-treatment decline in metachromatic leukodystrophy might be caused by gray matter pathology. METHODS: Three metachromatic leukodystrophy patients treated with hematopoietic stem cell transplantation with a progressive clinical course despite stable white matter pathology were clinically and radiologically analyzed. Longitudinal volumetric MRI was used to quantify atrophy. We also examined histopathology in three other patients deceased after treatment and compared them with six untreated patients. RESULTS: The three clinically progressive patients developed cognitive and motor deterioration after transplantation, despite stable mild white matter abnormalities on MRI. Volumetric MRI identified cerebral and thalamus atrophy in these patients, and cerebellar atrophy in two. Histopathology showed that in brain tissue of transplanted patients, arylsulfatase A expressing macrophages were clearly present in the white matter, but absent in the cortex. Arylsulfatase A expression within patient thalamic neurons was lower than in controls, the same was found in transplanted patients. INTERPRETATION: Neurological deterioration may occur after hematopoietic stem cell transplantation in metachromatic leukodystrophy despite successfully treated leukodystrophy. MRI shows gray matter atrophy, and histological data demonstrate absence of donor cells in gray matter structures. These findings point to a clinically relevant gray matter component of metachromatic leukodystrophy, which does not seem sufficiently affected by transplantation.