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Abstract Fabry disease (FD, OMIM 301500) is a rare X‐linked inherited lysosomal storage disorder associated with reduced activities of α‐galactosidase A (aGal, EC 3.2.1.22). The current standard of care for FD is based on enzyme replacement therapy (ERT), in which a recombinantly produced version of αGal is intravenously (iv) applied to Fabry patients in biweekly intervals. Though the iv application is clinically efficacious, periodical infusions are inconvenient, time‐ and resource‐consuming and they negatively impact the patients’ quality of life. Subcutaneous (sc) injection, in contrast, is an established route of administration for treatment of chronic conditions. It opens the beneficial option of self‐administration, thereby improving patients’ quality of life and at the same time reducing treatment costs. We have previously shown that Moss‐α‐Galactosidase (moss‐aGal), recombinantly produced in the moss Physcomitrium patens, is efficient in degrading accumulated Gb3 in target organs of murine model of FD and in the phase I clinical study, we obtained first efficacy evidence in human patients following single iv infusion. Here, we tested the efficacy of subcutaneous administration of moss‐aGal and compared it with the results observed following iv infusion in Fabry mice. The obtained findings demonstrate that subcutaneously applied moss‐aGal is correctly transported to target organs and efficacious in degrading Gb3 deposits there and thus suggest the possibility of using this route of administration for therapy of Fabry disease.

Abstract Background Neurodegeneration due to cerebral folate transport deficiency is a rare autosomal recessive disorder caused by biallelic pathogenic variants in FOLR1. Onset typically occurs in late infancy and is characterized by psychomotor regression, epilepsy, and a hypomyelinating leukodystrophy on magnetic resonance imaging. If left untreated, progressive neurodegeneration occurs. However, early treatment with folinic acid has been shown to stabilize or reverse neurological features. Approximately thirty patients have been described worldwide. Here, we report the first two cases with genetically proven cerebral folate transport deficiency from South-Eastern Europe, describe the effect of oral folinic acid therapy on clinical and neuroradiological features and review the literature. Results Two siblings presented in childhood with clinical and radiological findings consistent with a hypomyelinating leukodystrophy. Exome sequencing revealed a novel homozygous pathogenic variant in FOLR1 (c.465_466delinsTG; p.W156G), confirming the diagnosis of neurodegeneration due to cerebral folate transport deficiency. Folinic acid treatment was promptly initiated in both patients. The younger sibling was treated early in disease course at 2 years of age, and demonstrated complete recovery in clinical and MRI features. The older sibling, who was 8 years of age at the time of diagnosis and treatment, demonstrated partial but substantial improvements. Conclusion We present the first account in the literature that early treatment initiation with oral folinic acid alone can result in complete neurological recovery of both clinical and radiological abnormalities in neurodegeneration due to cerebral folate deficiency. Moreover, through the report of these patients along with review of the literature, we provide information about the natural history of the disease with comparison of treatment effects at different stages of disease progression. This report also reinforces the importance of universal access to genetic testing to ensure prompt diagnoses for treatable disorders.

Abstract Fabry disease is caused by a deficiency of α-galactosidase A (GLA) leading to the lysosomal accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids. Fabry patients experience significant damage to the heart, kidney, and blood vessels that can be fatal. Here we apply directed evolution to generate more stable GLA variants as potential next generation treatments for Fabry disease. GLAv05 and GLAv09 were identified after screening more than 12,000 GLA variants through 8 rounds of directed evolution. Both GLAv05 and GLAv09 exhibit increased stability at both lysosomal and blood pH, stability to serum, and elevated enzyme activity in treated Fabry fibroblasts (19-fold) and GLA–/– podocytes (10-fold). GLAv05 and GLAv09 show improved pharmacokinetics in mouse and non-human primates. In a Fabry mouse model, the optimized variants showed prolonged half-lives in serum and relevant tissues, and a decrease of accumulated Gb3 in heart and kidney. To explore the possibility of diminishing the immunogenic potential of rhGLA, amino acid residues in sequences predicted to bind MHC II were targeted in late rounds of GLAv09 directed evolution. An MHC II-associated peptide proteomics assay confirmed a reduction in displayed peptides for GLAv09. Collectively, our findings highlight the promise of using directed evolution to generate enzyme variants for more effective treatment of lysosomal storage diseases.

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of α-galactosidase A and subsequent accumulation of glycosphingolipids with terminal α-D-galactosyl residues. The molecular process through which this abnormal metabolism of glycosphingolipids causes multisystem dysfunction in Fabry disease is not fully understood. We sought to determine whether dysregulated DNA methylation plays a role in the development of this disease. In the present study, using isogenic cellular models derived from Fabry patient endothelial cells, we tested whether manipulation of α-galactosidase A activity and glycosphingolipid metabolism affects DNA methylation. Bisulfite pyrosequencing revealed that changes in α-galactosidase A activity were associated with significantly altered DNA methylation in the androgen receptor promoter, and this effect was highly CpG loci-specific. Methylation array studies showed that α-galactosidase A activity and glycosphingolipid levels were associated with differential methylation of numerous CpG sites throughout the genome. We identified 15 signaling pathways that may be susceptible to methylation alterations in Fabry disease. By incorporating RNA sequencing data, we identified 21 genes that have both differential mRNA expression and methylation. Upregulated expression of collagen type IV alpha 1 and alpha 2 genes correlated with decreased methylation of these two genes. Methionine levels were elevated in Fabry patient cells and Fabry mouse tissues, suggesting that a perturbed methionine cycle contributes to the observed dysregulated methylation patterns. In conclusion, this study provides evidence that α-galactosidase A deficiency and glycosphingolipid storage may affect DNA methylation homeostasis and highlights the importance of epigenetics in the pathogenesis of Fabry disease and, possibly, of other lysosomal storage disorders.

Fabry disease is caused by deficient activity of α-galactosidase A, an enzyme that hydrolyzes the terminal α-galactosyl moieties from glycolipids and glycoproteins, and subsequent accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), and galabiosylceramide. However, there is no known link between these compounds and disease severity. In this study, we compared Gb3 isoforms (various fatty acids) and lyso-Gb3 analogs (various sphingosine modifications) in two strains of Fabry disease mouse models: a pure C57BL/6 (B6) background or a B6/129 mixed background, with the latter exhibiting more prominent cardiac and renal hypertrophy and thermosensation deficits. Total Gb3 and lyso-Gb3 levels in the heart, kidney, and dorsal root ganglion (DRG) were similar in the two strains. However, levels of the C20-fatty acid isoform of Gb3 and particular lyso-Gb3 analogs (+18, +34) were significantly higher in Fabry-B6/129 heart tissue when compared with Fabry-B6. By contrast, there was no difference in Gb3 and lyso-Gb3 isoforms/analogs in the kidneys and DRG between the two strains. Furthermore, using immunohistochemistry, we found that Gb3 massively accumulated in DRG mechanoreceptors, a sensory neuron subpopulation with preserved function in Fabry disease. However, Gb3 accumulation was not observed in nonpeptidergic nociceptors, the disease-relevant subpopulation that has remarkably increased isolectin-B4 (the marker of nonpeptidergic nociceptors) binding and enlarged cell size. These findings suggest that specific species of Gb3 or lyso-Gb3 may play major roles in the pathogenesis of Fabry disease, and that Gb3 and lyso-Gb3 are not responsible for the pathology in all tissues or cell types.

Abstract Genetic white matter disorders have heterogeneous etiologies and overlapping clinical presentations. We performed a study of the diagnostic efficacy of genome sequencing in 41 unsolved cases with prior exome sequencing, resolving an additional 14 from an historical cohort (n = 191). Reanalysis in the context of novel disease‐associated genes and improved variant curation and annotation resolved 64% of cases. The remaining diagnoses were directly attributable to genome sequencing, including cases with small and large copy number variants (CNVs) and variants in deep intronic and technically difficult regions. Genome sequencing, in combination with other methodologies, achieved a diagnostic yield of 85% in this retrospective cohort.

The effect of migalastat on long-term renal outcomes in enzyme replacement therapy (ERT)–naive and ERT-experienced patients with Fabry disease is not well defined. An integrated posthoc analysis of the phase 3 clinical trials and open-label extension studies was conducted to evaluate long-term changes in renal function in patients with Fabry disease and amenable GLA variants who were treated with migalastat for ≥2 years during these studies. The analysis included ERT-naive (n = 36 [23 females]; mean age 45 years; mean baseline estimated glomerular filtration rate (eGFR), 91.4 mL/min/mL/1.73 m2) and ERT-experienced (n = 42 [24 females]; mean age, 50 years; mean baseline eGFR, 89.2 mL/min/1.73m2) patients with amenable variants who received migalastat 123 mg every other day for ≥2 years. The annualized rate of change from baseline to last observation in estimated glomerular filtration rate using the Chronic Kidney Disease Epidemiology Collaboration equation (eGFRCKD-EPI) was calculated by both simple linear regression and a random coefficient model. In ERT-naive patients, mean annualized rates of change from baseline in eGFRCKD-EPI were − 1.6 mL/min/1.73 m2 overall and − 1.8 mL/min/1.73 m2 and − 1.4 mL/min/1.73 m2 in male and female patients, respectively, as estimated by simple linear regression. In ERT-experienced patients, mean annualized rates of change from baseline in eGFRCKD-EPI were − 1.6 mL/min/1.73 m2 overall and − 2.6 mL/min/1.73 m2 and − 0.8 mL/min/1.73 m2 in male and female patients, respectively. Mean annualized rate of change in eGFRCKD-EPI in ERT-naive patients with the classic phenotype (defined by white blood cell alpha galactosidase A [α-Gal A] activity of

Abstract Background Lysosomal storage diseases (LSD) are a large family of inherited disorders characterized by abnormal endolysosomal accumulation of cellular material due to catabolic enzyme and transporter deficiencies. Depending on the affected metabolic pathway, LSD manifest with somatic or central nervous system (CNS) signs and symptoms. Neuroinflammation is a hallmark feature of LSD with CNS involvement such as mucolipidosis type IV, but not of others like Fabry disease. Methods We investigated the properties of microglia from LSD with and without major CNS involvement in 2-month-old mucolipidosis type IV (Mcoln1 −/−) and Fabry disease (Gla y/−) mice, respectively, by using a combination of flow cytometric, RNA sequencing, biochemical, in vitro and immunofluorescence analyses. Results We characterized microglia activation and transcriptome from mucolipidosis type IV and Fabry disease mice to determine if impaired lysosomal function is sufficient to prime these brain-resident immune cells. Consistent with the neurological pathology observed in mucolipidosis type IV, Mcoln1 −/− microglia demonstrated an activation profile with a mixed neuroprotective/neurotoxic expression pattern similar to the one we previously observed in Niemann-Pick disease, type C1, another LSD with significant CNS involvement. In contrast, the Fabry disease microglia transcriptome revealed minimal alterations, consistent with the relative lack of CNS symptoms in this disease. The changes observed in Mcoln1 −/− microglia showed significant overlap with alterations previously reported for other common neuroinflammatory disorders including Alzheimer’s, Parkinson’s, and Huntington’s diseases. Indeed, our comparison of microglia transcriptomes from Alzheimer’s disease, amyotrophic lateral sclerosis, Niemann-Pick disease, type C1 and mucolipidosis type IV mouse models showed an enrichment in “disease-associated microglia” pattern among these diseases. Conclusions The similarities in microglial transcriptomes and features of neuroinflammation and microglial activation in rare monogenic disorders where the primary metabolic disturbance is known may provide novel insights into the immunopathogenesis of other more common neuroinflammatory disorders. Trial registration ClinicalTrials.gov, NCT01067742, registered on February 12, 2010

Lysosomal replacement enzymes are taken up by cell surface receptors that recognize glycans, the effects of different glycan features are unknown. Here the authors present a gene engineering screen in CHO cells that allows custom N-glycan-decorated enzymes with improved circulation time and organ distribution.

Objectives The PRoposing Early Disease Indicators for Clinical Tracking in Fabry Disease (PREDICT-FD) initiative aimed to reach consensus among a panel of global experts on early indicators of disease progression that may justify FD-specific treatment initiation.Design and setting Anonymous feedback from panellists via online questionnaires was analysed using a modified Delphi consensus technique. Questionnaires and data were managed by an independent administrator directed by two non-voting cochairs. First, possible early indicators of renal, cardiac and central/peripheral nervous system (CNS/PNS) damage, and other disease and patient-reported indicators assessable in routine clinical practice were compiled by the cochairs and administrator from panellists’ free-text responses. Second, the panel scored indicators for importance (5-point scale: 1=not important; 5=extremely important); indicators scoring ≥3 among >75% of panellists were then rated for agreement (5-point scale: 1=strongly disagree; 5=strongly agree). Indicators awarded an agreement score ≥4 by >67% of panellists achieved consensus. Finally, any panel-proposed refinements to consensus indicator definitions were adopted if >75% of panellists agreed.Results A panel of 21 expert clinicians from 15 countries provided information from which 83 possible current indicators of damage (kidney, 15; cardiac, 15; CNS/PNS, 13; other, 16; patient reported, 24) were compiled. Of 45 indicators meeting the importance criteria, consensus was reached for 29 and consolidated as 27 indicators (kidney, 6; cardiac, 10; CNS/PNS, 2; other, 6; patient reported, 3) including: (kidney) elevated albumin:creatinine ratio, histological damage, microalbuminuria; (cardiac) markers of early systolic/diastolic dysfunction, elevated serum cardiac troponin; (CNS/PNS) neuropathic pain, gastrointestinal symptoms suggestive of gastrointestinal neuropathy; (other) pain in extremities/neuropathy, angiokeratoma; (patient-reported) febrile crises, progression of symptoms/signs. Panellists revised and approved proposed chronologies of when the consensus indicators manifest. The panel response rate was >95% at all stages.Conclusions PREDICT-FD captured global opinion regarding current clinical indicators that could prompt FD-specific treatment initiation earlier than is currently practised.

Abstract Background Fabry disease is frequently characterized by gastrointestinal symptoms, including diarrhea. Migalastat is an orally-administered small molecule approved to treat the symptoms of Fabry disease in patients with amenable mutations. Methods We evaluated minimal clinically important differences (MCID) in diarrhea based on the corresponding domain of the patient-reported Gastrointestinal Symptom Rating Scale (GSRS) in patients with Fabry disease and amenable mutations (N = 50) treated with migalastat 150 mg every other day or placebo during the phase 3 FACETS trial (NCT00925301). Results After 6 months, significantly more patients receiving migalastat versus placebo experienced improvement in diarrhea based on a MCID of 0.33 (43% vs 11%; p = .02), including the subset with baseline diarrhea (71% vs 20%; p = .02). A decline in kidney peritubular capillary globotriaosylceramide inclusions correlated with diarrhea improvement; patients with a reduction > 0.1 were 5.6 times more likely to have an improvement in diarrhea than those without (p = .031). Conclusions Migalastat was associated with a clinically meaningful improvement in diarrhea in patients with Fabry disease and amenable mutations. Reductions in kidney globotriaosylceramide may be a useful surrogate endpoint to predict clinical benefit with migalastat in patients with Fabry disease. Trial registration NCT00925301; June 19, 2009.

ObjectivesTo evaluate the function of the oculomotor and vestibular systems and to correlate these findings with the clinical status of patients with Gaucher disease type 3 (GD3). The goal of this cross-sectional and longitudinal study was to find oculomotor biomarkers for future clinical trials.MethodsTwenty-six patients with GD3 were assessed for eligibility and 21 were able to perform at least one task. Horizontal and vertical reflexive saccades, smooth pursuit, gaze-holding, optokinetic nystagmus, and horizontal vestibulo-ocular reflex (VOR) were examined by video-oculography/video-head impulse test and compared concurrently with 33 healthy controls. The Scale for the Assessment and Rating of Ataxia (SARA), the modified Severity Scoring Tool (mSST), and Grooved Pegboard Test (GPT) were administered to assess overall neurological function. Eleven patients were also re-assessed after 1 year.ResultsNine out of 17 patients exhibited gaze-holding deficits. One patient had upbeat nystagmus. Three patients presented with bilateral abducens palsy in combination with central oculomotor disorders, suggesting a bilateral involvement of the abducens nucleus. Horizontal angular VOR gain was reduced in all patients (0.66 ± 0.37) compared with controls (1.1 ± 0.11, p

Genetic leukoencephalopathies (gLEs) are a group of heterogeneous disorders with white matter abnormalities affecting the central nervous system (CNS). The causative mutation in ~50% of gLEs is unknown. Using whole exome sequencing (WES), we identified homozygosity for a missense variant, VPS11: c.2536T>G (p.C846G), as the genetic cause of a leukoencephalopathy syndrome in five individuals from three unrelated Ashkenazi Jewish (AJ) families. All five patients exhibited highly concordant disease progression characterized by infantile onset leukoencephalopathy with brain white matter abnormalities, severe motor impairment, cortical blindness, intellectual disability, and seizures. The carrier frequency of the VPS11: c.2536T>G variant is 1:250 in the AJ population (n = 2,026). VPS11 protein is a core component of HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) protein complexes involved in membrane trafficking and fusion of the lysosomes and endosomes. The cysteine 846 resides in an evolutionarily conserved cysteine-rich RING-H2 domain in carboxyl terminal regions of VPS11 proteins. Our data shows that the C846G mutation causes aberrant ubiquitination and accelerated turnover of VPS11 protein as well as compromised VPS11-VPS18 complex assembly, suggesting a loss of function in the mutant protein. Reduced VPS11 expression leads to an impaired autophagic activity in human cells. Importantly, zebrafish harboring a vps11 mutation with truncated RING-H2 domain demonstrated a significant reduction in CNS myelination following extensive neuronal death in the hindbrain and midbrain. Thus, our study reveals a defect in VPS11 as the underlying etiology for an autosomal recessive leukoencephalopathy disorder associated with a dysfunctional autophagy-lysosome trafficking pathway.

The aim of our study was to measure globotriaosylceramide (Gb3) and lyso-Gb3 levels by tandem mass spectrometry in the urine and kidney in Fabry (gla knockout) mice and wild-type controls. We found that urine Gb3 of male and female Fabry mice was higher than wild-type mice of the same sex but also significantly higher in male mice compared with females of the same genotype. In kidney tissue, sex and genotype-dependent differences in Gb3 levels paralleled those in the urine. Isoforms C16, C22:1, and C24OHA were particularly higher in males compared with females in both wild-type and Fabry mice. Similarly, kidney lyso-Gb3 concentrations were significantly higher in 12-month-old male Fabry mice than in their homozygous female counterparts. However, lyso-Gb3 was undetectable in wild-type mice of both sexes. α-Galactosidase A activity and mRNA levels in kidney were significantly lower in male wild-type mice compared with female mice. This study shows the sex differences in kidney and urine Gb3 and kidney lyso-Gb3 levels in both wild-type and Fabry mice, and it suggests that these male-female differences should be taken into consideration when using murine models for Fabry disease.

Fabry disease is an X-linked disorder caused by mutations in the GLA gene encoding for α-galactosidase A (AGA, EC 3.2.1.22). Measurement of AGA enzyme activity using cell homogenates can easily identify men with Fabry disease, but in women, the degree of X-inactivation in the tested tissue may produce activities in homogenates that are indistinguishable from normal. Monti et al. developed a series of lissamine rhodamine-labeled glycosphingolipid substrates that can be used to measure clearance of these lipids in intact cells (1). We report here that one of these substrates, lissamine rhodamine ceramide trihexoside (LR-CTH), can be used as a probe for functional activity of AGA in intact fibroblasts, endothelial cells, and T-lymphocytes from patients with Fabry disease. By utilizing standard detection techniques, such as microscopic imaging, fluorescence microplate spectrophotometry, and flow cytometry, cells with impaired AGA activity can easily be distinguished from wild-type (WT) cells, and these two cell types can be isolated into separate populations using fluorescence-activated cell sorting (FACS). The assay we report here can be adapted to evaluate new therapies by high-throughput screening, can aid in the study of AGA activity in living cells, and can assist in the diagnosis of women with the Fabry trait.

Gaucher disease, a recessive inherited metabolic disorder caused by defects in the gene encoding glucosylceramidase (GlcCerase), can be divided into three subtypes according to the appearance of symptoms associated with central nervous system involvement. We now identify a protein, glycoprotein non-metastatic B (GPNMB), that acts as an authentic marker of brain pathology in neurological forms of Gaucher disease. Using three independent techniques, including quantitative global proteomic analysis of cerebrospinal fluid (CSF) in samples from Gaucher disease patients that display neurological symptoms, we demonstrate a correlation between the severity of symptoms and GPNMB levels. Moreover, GPNMB levels in the CSF correlate with disease severity in a mouse model of Gaucher disease. GPNMB was also elevated in brain samples from patients with type 2 and 3 Gaucher disease. Our data suggest that GPNMB can be used as a marker to quantify neuropathology in Gaucher disease patients and as a marker of treatment efficacy once suitable treatments towards the neurological symptoms of Gaucher disease become available.

Gaucher disease is an inherited metabolic disorder caused by defective activity of the lysosomal enzyme, glucocerebrosidase, resulting in accumulation of the lipids, glucosylceramide (GlcCer), and glucosylsphingosine (GlcSph). Little is known about the mechanism leading from lipid accumulation to disease, particularly in the acute and subacute neuronopathic forms of Gaucher disease, types 2 and 3, respectively. Recent work from our laboratory has shown, in animal models, that GlcCer enhances agonist-induced calcium release from intracellular stores via the ryanodine receptor, which results in neuronal cell death. We now test whether calcium release is altered in human brain tissue obtained post-mortem from Gaucher disease patients. Agonist-induced calcium release via the ryanodine receptor was significantly enhanced (P < 0.05) in brain microsomes from the acute neuronopathic form of Gaucher disease (type 2) (43 ± 6% of the calcium in microsomes) compared to the subacute (type 3) (27 ± 3%) and the non-neuronopathic (type 1) (28 ± 6%) forms, and controls (18 ± 3%), and correlated with levels of GlcCer accumulation. These findings suggest that defective calcium homeostasis may be a mechanism responsible for neuropathophysiology in acute neuronopathic Gaucher disease, and may potentially offer new therapeutic approaches for disease management.

Krabbe disease is a devastating neurodegenerative lysosomal storage disorder caused by a deficiency of β-galactocerebrosidase (GALC). Gene therapy is a promising therapeutic approach for Krabbe disease. As the human brain is large and it is difficult to achieve global gene transduction, the efficacy of cross-correction is a critical determinant of the outcome of gene therapy for this disease. We investigated whether HIV Tat protein transduction domain (PTD) can improve the cross-correction of GALC. Tat-PTD significantly increased (~6-fold) cross-correction of GALC through enhanced secretion and uptake in a cell-culture model system. The effects of Tat-PTD were gene and flanking amino acids dependent. Tat-fusion increased the secretion of α-galactosidase A (α-gal A), but this did not improve its cross-correction. Tat-fusion did not change either secretion or uptake of β-glucocerebrosidase (GC). Tat-PTD increased GALC protein synthesis, abolished reactivity of GC to the 8E4 antibody, and likely reduced mannose phosphorylation in all these lysosomal enzymes. This study demonstrated that Tat-PTD can be useful for increasing cross-correction efficiency of lysosomal enzymes. However, Tat-PTD is not a mere adhesive motif but possesses a variety of biological functions. Therefore, the potential beneficial effect of Tat-PTD should be assessed individually on each lysosomal enzyme.

Leukodystrophies (LD) are rare inherited disorders that primarily affect the white matter (WM) of the central nervous system. The large heterogeneity of LD results from the diversity of the genetically determined defects that interfere with glial cells functions. Astrocytes have been identified as the primary target of LD with cystic myelin breakdown including those related to mutations in the ubiquitous translation initiation factor eIF2B. EIF2B is involved in global protein synthesis and its regulation under normal and stress conditions. Little is known about how eIF2B mutations have a major effect on WM. We performed a transcriptomic analysis using fibroblasts of 10 eIF2B-mutated patients with a severe phenotype and 10 age matched patients with other types of LD in comparison to control fibroblasts. ANOVA was used to identify genes that were statistically significantly differentially expressed at basal state and after ER-stress. The pattern of differentially expressed genes between basal state and ER-stress did not differ significantly among each of the three conditions. However, 70 genes were specifically differentially expressed in eIF2B-mutated fibroblasts whatever the stress conditions tested compared to controls, 96% being under-expressed. Most of these genes were involved in mRNA regulation and mitochondrial metabolism. The 13 most representative genes, including genes belonging to the Heterogeneous Nuclear Ribonucleoprotein (HNRNP) family, described as regulators of splicing events and stability of mRNA, were dysregulated during the development of eIF2B-mutated brains. HNRNPH1, F and C mRNA were over-expressed in foetus but under-expressed in children and adult brains. The abnormal regulation of HNRNP expression in the brain of eIF2B-mutated patients was concomitant with splicing dysregulation of the main genes involved in glial maturation such as PLP1 for oligodendrocytes and GFAP in astrocytes. These findings demonstrate a developmental deregulation of splicing events in glial cells that is related to abnormal production of HNRNP, in eIF2B-mutated brains.

BACKGROUND: Primary or secondary abnormalities of glycosylation have been reported in various brain diseases. Decreased asialotransferrin to sialotransferrin ratio in cerebrospinal fluid (CSF) is a diagnostic marker of leukodystrophies related to mutations of genes encoding translation initiation factor, EIF2B. We investigated the CSF glycome of eIF2B-mutated patients and age-matched normal individuals in order to further characterize the glycosylation defect for possible use as a biomarker. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a differential N-glycan analysis using MALDI-TOF/MS of permethylated N-glycans in CSF and plasma of controls and eIF2B-mutated patients. We found in control CSF that tri-antennary/bisecting and high mannose structures were highly represented in samples obtained between 1 to 5 years of age, whereas fucosylated, sialylated structures were predominant at later age. In CSF, but not in plasma, of eIF2B-mutated patient samples, we found increased relative intensity of bi-antennary structures and decreased tri-antennary/bisecting structures in N-glycan profiles. Four of these structures appeared to be biomarker candidates of glycomic profiles of eIF2B-related disorders. CONCLUSION: Our results suggest a dynamic development of normal CSF N-glycan profiles from high mannose type structures to complex sialylated structures that could be correlated with postnatal brain maturation. CSF N-glycome analysis shows relevant quantitative changes associated with eIF2B related disorders. This approach could be applied to other neurological disorders involving developmental gliogenesis/synaptogenesis abnormalities.

The pattern of cerebral dopamine (DA) abnormalities in Huntington disease (HD) is complex, as reflected by the variable clinical benefit of both DA antagonists and agonists in treating HD symptoms. In addition, little is known about serotonin metabolism despite the early occurrence of anxiety and depression in HD. Post-mortem enzymatic changes are likely to interfere with the in vivo profile of biogenic amines. Hence, in order to reliably characterize the regional and chronological profile of brain neurotransmitters in a HD mouse model, we used a microwave fixation system that preserves in vivo concentrations of dopaminergic and serotoninergic amines. DA was decreased in the striatum of R6/2 mice at 8 and 12 weeks of age while DA metabolites, 3-methoxytyramine and homovanillic acid, were already significantly reduced in 4-week-old motorically asymptomatic R6/2 mice. In the striatum, hippocampus and frontal cortex of 4, 8 and 12-week-old R6/2 mice, serotonin and its metabolite 5-hydroxyindoleacetic acid were significantly decreased in association with a decreased turnover of serotonin. In addition, automated high-resolution behavioural analyses displayed stress-like behaviours such as jumping and grooming and altered spatial learning in R6/2 mice at age 4 and 6 weeks respectively. Therefore, we describe the earliest alterations of DA and serotonin metabolism in a HD murine model. Our findings likely underpin the neuropsychological symptoms at time of disease onset in HD.

Our objective was to characterize the saccadic eye movements in patients with type 3 Gaucher disease (chronic neuronopathic) in relationship to neurological and neurophysiological abnormalities. For approximately 4 years, we prospectively followed a cohort of 15 patients with Gaucher type 3, ages 8-28 years, by measuring saccadic eye movements using the scleral search coil method. We found that patients with type 3 Gaucher disease had a significantly higher regression slope of duration vs amplitude and peak duration vs amplitude compared to healthy controls for both horizontal and vertical saccades. Saccadic latency was significantly increased for horizontal saccades only. Downward saccades were more affected than upward saccades. Saccade abnormalities increased over time in some patients reflecting the slowly progressive nature of the disease. Phase plane plots showed individually characteristic patterns of abnormal saccade trajectories. Oculo-manual dexterity scores on the Purdue Pegboard test were low in virtually all patients, even in those with normal cognitive function. Vertical saccade peak duration vs amplitude slope significantly correlated with IQ and with the performance on the Purdue Pegboard but not with the brainstem and somatosensory evoked potentials. We conclude that, in patients with Gaucher disease type 3, saccadic eye movements and oculo-manual dexterity are representative neurological functions for longitudinal studies and can probably be used as endpoints for therapeutic clinical trials.ClinicalTrials.gov NCT00001289.

BACKGROUND:In recent years, the phenotypes of leukodystrophies linked to mutations in the eukaryotic initiation factor 2B genes have been extended, classically called CACH/VWM (Childhood ataxia with cntral hypomyélination/vanishing white matter disorder). The large clinical spectrum observed from the more severe antenatal forms responsible for fetal death to milder adult forms with an onset after 16 years old and restricted to slow cognitive impairment have lead to the concept of eIF2B-related disorders. The typical MRI pattern with a diffuse CSF-like aspect of the cerebral white matter can lack particularly in the adult forms whereas an increasing number of patients with clinical and MRI criteria for CACH/VWM disease but without eIF2B mutations are found. Then we propose the use of biochemical markers to help in this difficult diagnosis. The biochemical diagnosis of eIF2B-related disorder is difficult as no marker, except the recently described asialotransferrin/transferrin ratio measured in cerebrospinal fluid, has been proposed and validated until now. Decreased eIF2B GEF activity has been previously reported in lymphoblastoid cell lines from 30 eIF2B-mutated patients. Our objective was to evaluate further the utility of this marker and to validate eIF2B GEF activity in a larger cohort as a specific diagnostic test for eIF2B-related disorders. METHODOLOGY/PRINCIPAL FINDINGS:We performed eIF2B GEF activity assays in cells from 63 patients presenting with different clinical forms and eIF2B mutations in comparison to controls but also to patients with defined leukodystrophies or CACH/VWM-like diseases without eIF2B mutations. We found a significant decrease of GEF activity in cells from eIF2B-mutated patients with 100% specificity and 89% sensitivity when the activity threshold was set at < or =77.5%. CONCLUSION:These results validate the measurement of eIF2B GEF activity in patients' transformed-lymphocytes as an important tool for the diagnosis of eIF2B-related disorders.