Brain Microglial Activation Increased in Glucocerebrosidase (GBA) Mutation Carriers without Parkinson's disease.

BACKGROUND: Glucocerebrosidase gene mutations are a common genetic risk factor for Parkinson's disease. They exhibit incomplete penetrance. The objective of the present study was to measure microglial activation and dopamine integrity in glucocerebrosidase gene mutation carriers without Parkinson's disease compared to controls. METHODS: We performed PET scans on 9 glucocerebrosidase gene mutation carriers without Parkinson's disease and 29 age-matched controls. We measured microglial activation as 11 C-(R)-PK11195 binding potentials, and dopamine terminal integrity with 18 F-dopa influx constants. RESULTS: The 11 C-(R)-PK11195 binding potential was increased in the substantia nigra of glucocerebrosidase gene carriers compared with controls (Student t test; right, t = -4.45, P = 0.0001). Statistical parametric mapping also localized significantly increased 11 C-(R)-PK11195 binding potential in the occipital and temporal lobes, cerebellum, hippocampus, and mesencephalon. The degree of hyposmia correlated with nigral 11 C-(R)-PK11195 regional binding potentials (Spearman's rank, P = 0.0066). Mean striatal 18 F-dopa uptake was similar to healthy controls. CONCLUSIONS: In vivo 11 C-(R)-PK11195 PET imaging detects neuroinflammation in brain regions susceptible to Lewy pathology in glucocerebrosidase gene mutation carriers without Parkinson's. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells.

Gaucher disease is caused by mutations in the glucocerebrosidase gene, which encodes the lysosomal hydrolase glucosylceramidase. Patients with Gaucher disease and heterozygous glucocerebrosidase mutation carriers are at increased risk of developing Parkinson's disease. Indeed, glucocerebrosidase mutations are the most frequent risk factor for Parkinson's disease in the general population. Therefore there is an urgent need to understand the mechanisms by which glucocerebrosidase mutations predispose to neurodegeneration to facilitate development of novel treatments. To study this we generated fibroblast lines from skin biopsies of five patients with Gaucher disease and six heterozygous glucocerebrosidase mutation carriers with and without Parkinson's disease. Glucosylceramidase protein and enzyme activity levels were assayed. Oxidative stress was assayed by single cell imaging of dihydroethidium. Glucosylceramidase enzyme activity was significantly reduced in fibroblasts from patients with Gaucher disease (median 5% of controls, P = 0.0001) and heterozygous mutation carriers with (median 59% of controls, P = 0.001) and without (56% of controls, P = 0.001) Parkinson's disease compared with controls. Glucosylceramidase protein levels, assessed by western blot, were significantly reduced in fibroblasts from Gaucher disease (median glucosylceramidase levels 42% of control, P < 0.001) and heterozygous mutation carriers with (median 59% of control, P < 0.001) and without (median 68% of control, P < 0.001) Parkinson's disease. Single cell imaging of dihydroethidium demonstrated increased production of cytosolic reactive oxygen species in fibroblasts from patients with Gaucher disease (dihydroethidium oxidation rate increased by a median of 62% compared to controls, P < 0.001) and heterozygous mutation carriers with (dihydroethidium oxidation rate increased by a median of 68% compared with controls, P < 0.001) and without (dihydroethidium oxidation rate increased by a median of 70% compared with controls, P < 0.001) Parkinson's disease. We hypothesized that treatment with the molecular chaperone ambroxol hydrochloride would improve these biochemical abnormalities. Treatment with ambroxol hydrochloride increased glucosylceramidase activity in fibroblasts from healthy controls, Gaucher disease and heterozygous glucocerebrosidase mutation carriers with and without Parkinson's disease. This was associated with a significant reduction in dihydroethidium oxidation rate of ∼50% (P < 0.05) in fibroblasts from controls, Gaucher disease and heterozygous mutation carriers with and without Parkinson's disease. In conclusion, glucocerebrosidase mutations are associated with reductions in glucosylceramidase activity and evidence of oxidative stress. Ambroxol treatment significantly increases glucosylceramidase activity and reduces markers of oxidative stress in cells bearing glucocerebrosidase mutations. We propose that ambroxol hydrochloride should be further investigated as a potential treatment for Parkinson's disease.

Reproducibility of native myocardial T1 mapping in the assessment of Fabry disease and its role in early detection of cardiac involvement by cardiovascular magnetic resonance.

BACKGROUND: Cardiovascular magnetic resonance (CMR) derived native myocardial T1 is decreased in patients with Fabry disease even before left ventricular hypertrophy (LVH) occurs and may be the first non-invasive measure of myocyte sphingolipid storage. The relationship of native T1 lowering prior to hypertrophy and other candidate early phenotype markers are unknown. Furthermore, the reproducibility of T1 mapping has never been assessed in Fabry disease. METHODS: Sixty-three patients, 34 (54%) female, mean age 48±15 years with confirmed (genotyped) Fabry disease underwent CMR, ECG and echocardiographic assessment. LVH was absent in 25 (40%) patients. Native T1 mapping was performed with both Modified Look-Locker Inversion recovery (MOLLI) sequences and a shortened version (ShMOLLI) at 1.5 Tesla. Twenty-one patients underwent a second scan within 24 hours to assess inter-study reproducibility. Results were compared with 63 healthy age and gender-matched volunteers. RESULTS: Mean native T1 in Fabry disease (LVH positive), (LVH negative) and healthy volunteers was 853±50 ms, 904±46 ms and 968±32 ms (for all p<0.0001) by ShMOLLI sequences. Native T1 showed high inter-study, intra-observer and inter-observer agreement with intra-class correlation coefficients (ICC) of 0.99, 0.98, 0.97 (ShMOLLI) and 0.98, 0.98, 0.98 (MOLLI). In Fabry disease LVH negative individuals, low native T1 was associated with reduced echocardiographic-based global longitudinal speckle tracking strain (-18±2% vs -22±2%, p=0.001) and early diastolic function impairment (E/E'=7 [6-8] vs 5 [5-6], p=0.028). CONCLUSION: Native T1 mapping in Fabry disease is a reproducible technique. T1 reduction prior to the onset of LVH is associated with early diastolic and systolic changes measured by echocardiography.

Novel pathogenic mutations in the glucocerebrosidase locus.

To determine the frequency of mutations responsible for Gaucher's disease, we systematically sequenced the GBA1 gene as part of a molecular characterization of 73 adult patients in the United Kingdom. Five hitherto unknown pathogenic variants were identified, one of which is a splice site change; the others are novel missense mutations. Given that GBA1 gene mutations are an important risk factor for the development of Parkinson's disease, we contend that a complete analysis and molecular characterization of both the known and novel GBA1 variants will be needed before the biochemical processes underlying this genetic association can be fully understood.

Hyposmia and cognitive impairment in Gaucher disease patients and carriers.

The objective of this study was to assess a cohort of Gaucher disease patients and their heterozygous carrier relatives for potential clinical signs of early neurodegeneration. Gaucher disease patients (n = 30), heterozygous glucocerebrosidase mutation carriers (n = 30), and mutation-negative controls matched by age, sex, and ethnicity (n = 30) were recruited. Assessment was done for olfactory function (University of Pennsylvania Smell Identification Test), cognitive function (Mini-Mental State Examination, Montreal Cognitive Assessment), rapid eye movement sleep disorder, autonomic symptoms, and parkinsonian motor signs (Unified Parkinson's Disease Rating Scale part III, Purdue pegboard). Olfactory function scores were significantly lower in Gaucher disease patients (P = .010) and heterozygous carriers (P < .001) than in controls. Cognitive assessment scores were significantly lower in Gaucher disease patients (P = .002) and carriers (P = .002) than in controls. Unified Parkinson's Disease Rating Scale motor subscale scores were significantly higher in Gaucher disease patients (P < .001) and heterozygotes (P = .0010) than in controls. There was no difference in scores for symptoms of rapid eye movement sleep disorder or autonomic dysfunction. Impairment of olfaction, cognition, and parkinsonian motor signs occurs more frequently in Gaucher disease patients and carriers than in controls, which may indicate the early stages of neurodegeneration.

Understanding and modifying Fabry disease: Rationale and design of a pivotal Phase 3 study and results from a patient-reported outcome validation study.

The use of available treatments for Fabry disease (FD) (including enzyme replacement therapy [ERT]) may be restricted by their limited symptom improvement and mode of administration. Lucerastat is currently being investigated in the MODIFY study as oral substrate reduction therapy for the treatment of FD. By reducing the net globotriaosylceramide (Gb3) load in tissues, lucerastat has disease-modifying potential to improve symptoms and delay disease progression. MODIFY is a multicenter, double-blind, randomized, placebo-controlled, parallel-group Phase 3 study (ClinicalTrial.gov: NCT03425539); here we present the rationale and design of this study. Eligible adults with a genetically confirmed diagnosis of FD and FD-specific neuropathic pain entered screening. Patients were randomized (2:1) to receive either oral lucerastat twice daily or placebo for 6 months; treatment allocation was stratified according to sex and ERT treatment status. The main objectives of MODIFY are to assess the effects of lucerastat on neuropathic pain, gastrointestinal (GI) symptoms, FD biomarkers, and determine its safety and tolerability. Neuropathic pain and GI symptoms are key features of FD that have a significant impact on quality of life. Despite various tools available to assess pain and GI symptoms, there are currently limited tools available to assess neuropathic and GI symptoms in FD, validated according to health authority guidelines. Based on FDA recommendations, we undertook a patient-reported outcome (PRO) validation study, using a novel eDiary-based PRO tool to assess the validity of evaluating neuropathic pain as a primary efficacy endpoint in MODIFY. Results from the PRO validation study are included. To date, MODIFY is the largest Phase 3 clinical study conducted in patients with FD. Enrollment to MODIFY is now complete, with 118 patients randomized. Results will be presented in a separate publication. Long-term effects of lucerastat are being assessed in the ongoing open-label extension study (NCT03737214).

Thirty-year clinical outcomes after haematopoietic stem cell transplantation in neuronopathic Gaucher disease.

BACKGROUND: Neuronopathic Gaucher Disease (nGD) describes the condition of a subgroup of patients with the Lysosomal Storage Disorder (LSD), Gaucher disease with involvement of the central nervous system (CNS) which results from inherited deficiency of ß-glucosylceramidase. Although systemic manifestations of disease are now corrected by augmentation with macrophage-targeted therapeutic enzyme (enzyme replacement therapy, ERT), neurological disease progresses unpredictably as a result of failure of therapeutic enzyme to cross the blood-brain barrier (BBB). Without therapy, the systemic and neurological effects of the disease progress and shorten life: investigators, principally in Sweden and the UK, pioneered bone marrow transplantation (BMT; Haematopoietic Stem Cell Transplantation HSCT) to supply healthy marrow-derived macrophages and other cells, to correct the peripheral disease. Here we report the first long-term follow-up (over 20 years in all cases) of nine patients in the UK and Sweden who underwent HSCT in the 1970s and 1980s. This retrospective, multicentre observational study was undertaken to determine whether there are neurological features of Gaucher disease that can be corrected by HSCT and the extent to which deterioration continues after the procedure. Since intravenous administration of ERT is approved for patients with the neuronopathic disease and ameliorates many of the important systemic manifestations but fails to correct the neurological features, we also consider the current therapeutic positioning of HSCT in this disorder. RESULTS: In the nine patients here reported, neurological disease continued to progress after transplantation, manifesting as seizures, cerebellar disease and abnormalities of tone and reflexes. CONCLUSIONS: Although neurological disease progressed in this cohort of patients, there may be a future role for HSCT in the treatment of nGD. The procedure has the unique advantage of providing a life-long source of normally functioning macrophages in the bone marrow, and possibly other sites, after a single administration. HSCT moreover, clearly ameliorates systemic disease and this may be advantageous-especially where sustained provision of high-cost ERT cannot be guaranteed. Given the remaining unmet needs of patients with neuronopathic Gaucher disease and the greatly improved safety profile of the transplant procedure, HSCT could be considered to provide permanent correction of systemic disease, including bone disease not ameliorated by ERT, when combined with emerging therapies directed at the neurological manifestations of disease; this could include ex-vivo gene therapy approaches.

Potential biomarkers of osteonecrosis in Gaucher disease.

BACKGROUND: To investigate the relationship between chemokines and cytokines and osteonecrosis in Gaucher disease, we conducted multiplex assays in a cohort of 100 adult patients. METHODS: Mean age was 45 years (18-86); 92 Gaucher patients received imiglucerase (median duration 8 years (2-18)). Forty-three had experienced osteonecrosis (ON), and eight had ON despite enzyme therapy. Serum cytokines/chemokines were determined by fluorimetric bead arrays in samples from Gaucher patients and healthy volunteers (10 males and 10 females). Intra-assay and inter-assay coefficients of variation were 2%-9.8% and 5.6%-15%, respectively. RESULTS: VEGF and CCL5/RANTES did not differ between Gaucher and control samples. Concentrations of CCL3/MIP-1α, CCL4/MIP-1ß, CCL2/MCP-1, CXCL8/IL-8, IL-1ra and CCL18/PARC were elevated in Gaucher patients (p<0.05 for each). Median CCL4/MIP-1ß, CXCL8/IL-8, CCL5/RANTES and CCL18/PARC concentrations were greater in the 43 osteonecrosis patients (88.6 pg/mL, 30.5 pg/mL, 89.6 ng/mL and 434 ng/mL, respectively) compared with the 57 patients who had no evidence of osteonecrosis (medians of 59.4, 13.3, 62.7 and 283, respectively, p<0.05). Moreover, the eight patients with ON despite imiglucerase had median concentrations of CCL3/MIP-1α, CCL4/MIP-1ß, CXCL8/IL-8, CCL5/RANTES and CCL18/PARC (73.2, 120.9, 36.3 pg/mL, 105 and 767 ng/mL, respectively), which significantly exceeded the values in 84 patients now free of ON (52.3, 71.2, 16.5 pg/mL, 69.5 and 315 ng/mL, respectively, p<0.05). Treatment exposures were similar. CONCLUSION: Numerous serum cytokines are elevated in Gaucher disease. CCL18/PARC, CCL3/MIP-1α, CCL4/MIP-1ß, CCL5/RANTES and CXCL8/IL-8 are potential biomarkers of osteonecrosis and may allow prediction of this disabling complication.

Peripheral neuropathy in adult type 1 Gaucher disease: a 2-year prospective observational study.

Type 1 Gaucher disease is currently categorized as non-neuronopathic, although recent studies suggest peripheral neurological manifestations. We report prevalence and incidence data for peripheral neuropathy and associated conditions from a multinational, prospective, longitudinal, observational cohort study in patients with type 1 Gaucher disease, either untreated or receiving enzyme replacement therapy. The primary outcome parameters were the prevalence and incidence of polyneuropathy, evaluated by standardized assessments of neurological symptoms and signs, and electrophysiological studies. All diagnoses of polyneuropathy were adjudicated centrally. Secondary outcome parameters included the prevalence and incidence of mononeuropathy, other neurological or electrophysiological abnormalities not fulfilling the criteria for a mono- or polyneuropathy and general type 1 Gaucher disease symptoms. Furthermore, a literature search was performed to identify all studies reporting on prevalence and incidence of polyneuropathy in the general population. One hundred and three patients were enrolled [median (range) age: 42 (18-75) years; disease duration: 15 (0-56) years; 52% female]; 14 (13.6%) were untreated and 89 (86.4%) were on enzyme replacement therapy. At baseline, 11 patients [10.7%; 95% confidence interval (CI): 5.9-18.3] were diagnosed with sensory motor axonal polyneuropathy. Two (1.9%; 95% CI: 0.1-7.2) had a mononeuropathy of the ulnar nerve. The 2-year follow-up period revealed another six cases of polyneuropathy (2.9 per 100 person-years; 95% CI: 1.2-6.3). Patients with polyneuropathy were older than those without (P<0.001). Conditions possibly associated with polyneuropathy were identified in four patients only, being monoclonal gammopathy, vitamin B(1) deficiency, folic acid deficiency, type 2 diabetes mellitus, renal insufficiency, alcohol abuse and exposure to toxins related to profession. The 11 cases of polyneuropathy found at baseline were confirmed during follow-up. According to the literature, the prevalence of polyneuropathy in the general population was estimated between 0.09 and 1.3% and the incidence was estimated between 0.0046 and 0.015 per 100 person-years. Thus, we conclude that the prevalence and incidence of polyneuropathy in patients with type 1 Gaucher disease is increased compared with the general population.

Eye movement biomarkers allow for the definition of phenotypes in Gaucher Disease.

BACKGROUND: Neurological forms of Gaucher disease, the inherited disorder of ß-Glucosylceramidase caused by bi-allelic variants in GBA1, is a progressive disorder which lacks a disease-modifying therapy. Systemic manifestations of disease are effectively treated with enzyme replacement therapy, however, molecules which cross the blood-brain barrier are still under investigation. Clinical trials of such therapeutics require robust, reproducible clinical endpoints to demonstrate efficacy and clear phenotypic definitions to identify suitable patients for inclusion in trials. The single consistent clinical feature in all patients with neuronopathic disease is the presence of a supranuclear saccadic gaze palsy, in the presence of Gaucher disease this finding serves as diagnostic of 'type 3' Gaucher disease. METHODS: We undertook a study to evaluate saccadic eye movements in Gaucher patients and to assess the role of the EyeSeeCam in measuring saccades. The EyeSeeCam is a video-oculography device which was used to run a protocol of saccade measures. We studied 39 patients with non-neurological Gaucher disease (type 1), 21 patients with type 3 (neurological) disease and a series of 35 healthy controls. Mean saccade parameters were compared across disease subgroups. RESULTS: We confirmed the saccadic abnormality in patients with type 3 Gaucher disease and identified an unexpected subgroup of patients with type 1 Gaucher disease who demonstrated significant saccade parameter abnormalities. These patients also showed subtle neurological findings and shared a GBA1 variant. CONCLUSIONS: This striking novel finding of a potentially attenuated type 3 Gaucher phenotype associated with a specific GBA1 variant and detectable saccadic abnormality prompts review of current disease classification. Further, this finding highlights the broad spectrum of neuronopathic Gaucher phenotypes relevant when designing inclusion criteria for clinical trials.

Quantitative Myocardial Perfusion in Fabry Disease.

BACKGROUND: Fabry disease (FD) is an X-linked lysosomal storage disease resulting in tissue accumulation of sphingolipids. Key myocardial processes that lead to adverse outcomes in FD include storage, hypertrophy, inflammation, and fibrosis. These are quantifiable by multiparametric cardiovascular magnetic resonance. Recent developments in cardiovascular magnetic resonance perfusion mapping allow rapid in-line perfusion quantification permitting broader clinical application, including the assessment of microvascular dysfunction. We hypothesized that microvascular dysfunction in FD would be associated with storage, fibrosis, and edema. METHODS: A prospective, observational study of 44 FD patients (49 years, 43% male, 24 [55%] with left ventricular hypertrophy [LVH]) and 27 healthy controls with multiparametric cardiovascular magnetic resonance including vasodilator stress perfusion mapping. Myocardial blood flow (MBF) was measured and its associations with other processes investigated. RESULTS: Compared with LVH- FD, LVH+ FD had higher left ventricular ejection fraction (73% versus 68%), more late gadolinium enhancement (85% versus 15%), and a lower stress MBF (1.76 versus 2.36 mL/g per minute). The reduction in stress MBF was more pronounced in the subendocardium than subepicardium. LVH- FD had lower stress MBF than controls (2.36 versus 3.00 mL/g per minute; P=0.002). Across all FD, late gadolinium enhancement and low native T1 were independently associated with reduced stress MBF. On a per-segment basis, stress MBF was independently associated with wall thickness, T2, extracellular volume fraction, and late gadolinium enhancement. CONCLUSIONS: FD patients have reduced perfusion, particularly in the subendocardium with greater reductions with LVH, storage, edema, and scar. Perfusion is reduced even without LVH suggesting it is an early disease marker.