Brain Volume Alterations Using 3tesla Magnetic Resonance and Neuropsychological Findings in Gaucher Disease Patients

Gaucher Disease (GD) is a metabolic inherited disorder, that classically includes three clinical phenotypes. Type I GD (GD1) is considered as a non-neuronopathic variant and can mimic a hematologic disease; type II (GD2) is classified as an acute neuropathic form and type III (GD3) is characterized by a slower and progressive neurological involvement. A protective role of N370S mutation against neurological impairment had been previously hypothesized in GD1, however, increasing data and our experience show that patients with GD1 may present clinical signs of parkinsonism, frequently combined with cognitive impairment and behavioral alterations. Structural brain changes associated with cognitive impairments in GD patients, especially type I, has not yet been reported. The prospective study, called SENOPRO_GAUCHER, has investigated in depth, the neurological status in GD patients using a multidisciplinary approach, including 3Tesla magnetic resonance (3T MR). This study was approved by our Ethics Committee. Baseline data on neurological, psychological, psychiatric, somatosensory, and motor evaluations, and electroencephalography (EEG) surprisingly revealed two, or more, clinical and/or instrumental, neurological signs in all GD1 patients, and a wide spectrum of neurological abnormalities in all GD3 patients. We, hereby, report the results of investigations on brain structure of GD patients, compared to that of healthy subjects, using 3T MR, in order to evaluate whether brain alterations can predict neurocognitive impairment in GD patients, especially GD1. Out of 22 GD patients, assessed for neuropsychological and psychiatric functions, 19 (17 GD1, 2 GD3, median age = 44, range 17 - 68 years) underwent 3T MR examination. Regarding genotyping, all but one of the 17 GD1 patients was N370S mutation heterozygous. A Voxel-based Morphometry (VBM) analysis was performed to investigate brain structure in the 19 GD patients and in 19 healthy subjects with no neurological or neuropsychiatric disease. The healthy subjects, matched to patients by age, sex, and education, in addition to 3T MR, underwent neuropsychological assessment using Mini-Mental State Exam and Mental Deterioration Battery, to evaluate cognitive functions and mental deterioration, respectively. Qualitative MR examinations revealed unspecific abnormalities in 7/17 GD1 patients. In particular, cortical and/or subcortical areas of gliosis (4 patients), vascular ectasia extended from the left frontal surface of the brain to the lateral ventricle (1 patient), dilation of perivascular spaces in the sub-cortical and nucleus-basal area (1 patient), diffuse suffering of the brain white matter due to a chronic ischemic vascular damage (1 patient) were found. Concurrently, results of the VBM analysis revealed that a set of regions, mostly located in the prefrontal and parieto-occipital cortex, were significantly reduced in GD patients, compared to healthy subjects. The most affected regions included the frontal eye fields, the dorso-lateral prefrontal cortex, the posterior cingulate cortex, and the V3B area. Psychological and psychiatric evaluations underlined anxiety, depression, and somatic concerns in 10/17 GD1 patients, combined with cognitive impairments in two of them. Moreover, 1/2 GD3 and 5/17 GD1 patients showed cognitive impairments in attention, language, short-term memory, and executive cognitive functions. The local brain volume reductions, detected in GD1 patients, were in line with the cognitive impairments resulted in the neuropsychological assessment. Cortical reductions of fronto-parietal regions, responsible for the planning and execution of movements, are compatible with impairments in executive functions (digit-span) and visuo-spatial memory, found in GD1 patients. Moreover, impairments in short-term memory observed in GD1 patients are compatible with cortical reductions in the frontal cortex found by the VBM analysis. In conclusion, focal brain differences found between GD1 patients and healthy subjects may predict and clarify the cognitive impairments and behavioural alterations observed in GD patients. The brain structural analyses, associated with neuropsychological assessment, suggest that a multidisciplinary approach is necessary in evaluating GD1 patients. Disclosures Giona: Sanofi Genzyme: Consultancy, Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Novartis: Consultancy.

Fibroblasts from idiopathic Parkinson’s disease exhibit deficiency of lysosomal glucocerebrosidase activity associated with reduced levels of the trafficking receptor LIMP2

Lysosomal dysfunction is a central pathway associated with Parkinson’s disease (PD) pathogenesis. Haploinsufficiency of the lysosomal hydrolase GBA (encoding glucocerebrosidase (GCase)) is one of the largest genetic risk factors for developing PD. Deficiencies in the activity of the GCase enzyme have been observed in human tissues from both genetic (harboring mutations in the GBA gene) and idiopathic forms of the disease. To understand the mechanisms behind the deficits of lysosomal GCase enzyme activity in idiopathic PD, this study utilized a large cohort of fibroblast cells from control subjects and PD patients with and without mutations in the GBA gene (N370S mutation) (control, n = 15; idiopathic PD, n = 31; PD with GBA N370S mutation, n = 6). The current data demonstrates that idiopathic PD fibroblasts devoid of any mutations in the GBA gene also exhibit reduction in lysosomal GCase activity, similar to those with the GBA N370S mutation. This reduced GCase enzyme activity in idiopathic PD cells was accompanied by decreased expression of the GBA trafficking receptor, LIMP2, and increased ER retention of the GBA protein in these cells. Importantly, in idiopathic PD fibroblasts LIMP2 protein levels correlated significantly with GCase activity, which was not the case in control subjects or in genetic PD GBA N370S cells. In conclusion, idiopathic PD fibroblasts have decreased GCase activity primarily driven by altered LIMP2-mediated transport of GBA to lysosome and the reduced GCase activity exhibited by the genetic GBA N370S derived PD fibroblasts occurs through a different mechanism.

Multi-Parameter Neurological Study Based on Combined Conventional and Functional Assessments in Gaucher Disease Patients (SENOPRO_GAUCHER Study)

Gaucher Disease (GD), a metabolic inherited disorder, includes three clinical phenotypes. Type I GD (GD1), that can mimic a hematologic disease, has been classically considered as a non-neuropathic variant; type II (GD2) and type III (GD3) are classified as acute and chronic neuropathic forms, respectively. A protective role of N370S mutation against neurological impairment had been previously hypothesized in GD1, however, clinical signs of parkinsonism have recently been reported in this category of patients. The aim of our observational, monocentric, and prospective study, called SENOPRO_GAUCHER, was to evaluate in depth the neurological and neuropsychiatric aspects in GD1 patients, using clinical and instrumental investigations, in order to identify clinical and subclinical neurological manifestations, including cognitive impairment and behavioural alterations. Neurological assessments were scheduled for 22 GD patients (19 GD1 and 3 GD3) aged >12 years. Clinical evaluation, including Severity Scoring Tool Unified Parkinson's Disease Rating scale and Epworth Sleepiness scale; psychological tests and psychiatric evaluation, using a cognitive test battery and two psychiatric (BPRS) and psychological (CBA 2.0) questionnaires; somatosensory, motor and visual evoked potential, spectral domain optical coherence tomography (SD-OCT), standard electroretinogram; electroencephalography (EEG), and magnetic resonance (MR) 3Tesla, were planned at baseline, after 12 and 24 months. Results at the baseline evaluation of all 22 enrolled patients (9 males and 13 females; median age at the study: 44.5 years, range 17 - 68) are here reported. Regarding genotyping, all but one of the 19 GD1 pts was heterozygous for the N370S mutation. Parkinsonian motor signs were found in 10/22 patients (9 GD1): 7 pts (6 GD1) had isolated bradykinesia and 3 GD1 patients presented bradykinesia combined with rigidity. Abnormal saccadic movements were found in all GD3 pts and in one GD1. Excessive daytime sleepiness (EDS) was detected in 9/22 patients (8 GD1). EEG revealed focal, or diffuse slow waves in 6 patients (5 GD1). Significant cognitive impairments in attention, language, memory and executive functions were found in 4/19 GD1 and in 2/3 GD3 patients. Moreover, psychological and psychiatric evaluations underlined anxiety, depression and somatic concerns in 10/22 patients (9 GD1), combined with cognitive impairments in 3/12. Sixteen patients (13 GD1) showed slight or moderate sensorineural hearing loss (SNHL), and 4 of them had a cookie and reverse cookie bite morphology, typically associated with genetic SNHL. Ophthalmological evaluation revealed a degree of retinal blood vessel tortuosity in all but one patient, and superficial corneal dystrophies in 3 patients (2 GD1). An increased latency and moderate reduction in the amplitudes of optic nerve function were found in 13/22 patients (10 GD1). The SD-OCT examination showed impairment in retinal nerve fibre layers in 7 patients (6 GD1), combined with macular damage in 2 of them. Qualitative MR 3Tesla examinations revealed unspecific abnormalities in 7/19 (37%) GD1 patients; in particular, cortical and/or subcortical areas of gliosis (4 patients), vascular ectasia extended from the left frontal surface of the brain to the lateral ventricle (1 patient), dilation of perivascular spaces in the sub-cortical and nucleus-basal area (1 patient), diffuse suffering of the brain white matter due to a chronic ischemic vascular damage (1 patient). In summary, all GD3 patients showed a wide spectrum of neurological abnormalities, as expected. Surprisingly, however, all GD1 pts also presented at least two, or more, clinical and/or instrumental, neurological signs. The high prevalence of EDS in our population suggests a possible underlying sleep disorder which should be further investigated. The N370S mutation did not impact the likelihood of developing neurological abnormalities in our cohort of GD1 patients. In conclusion, our results have demonstrated that a multidisciplinary, and in-depth approach is necessary in evaluating GD1 patients. Disclosures Giona: Sanofi Genzyme: Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Novartis: Research Funding.

Variation of Serum Lipid Profile during Enzyme Replacement Therapy, in Patients with Type 1 Gauchers Disease.

Enzyme Replacement Therapy (ERT) has dramatically changed the clinical course and prognosis of patients with Gaucher disease. We have assessed the extent to which the recently defined Goals of Therapy (Pastores et al, Semin Hematol 41(suppl 5):4–14) in 9 major fields (anemia, thrombocytopenia, hepatomegaly, splenomegaly, skeletal pathology,biomarkers, pulmonary manifestations, growth and functional health and well being) were achieved in our cohort of patients with type I Gaucher disease. Fifty-seven patients (27 females, 30 males, median age at diagnosis 30 years (range 2–77 years) and at start of ERT 39 years (range 5–79 years) were assessed. The median period of ERT was 94 months (range 24–155 months). Patients on ERT for less than 2 years were excluded. Twenty five specific goals are defined within the 9 fields. The median overall success rate was 20 of 25 goals or 80% per patient (range 12–25 goals or 60–100%). The success rate for 9 goals was 100% ( normalization of hemoglobin 1 year post-splenectomy, no major bleeding 1 year after ERT start, doubling of baseline platelet count in severely thrombocytopenic patients 2 years after ERT start, maintenance of platelet count in non-thrombocytopenic patients, prevention of bone crises, reversion of hepatopulmonary syndrome and dependence on oxygen, amelioration of pulmonary hypertension and prevention of rapid deterioration of pulmonary disease and sudden death). Higher success rates were achieved for the goals related to thrombocytopenia (97.9%) and pulmonary manifestations (97%) although in the latter case the applicability was low. Lower success rates were achieved for the goals related to bone manifestations (78.7%). There was no significant difference in the success rate between males and females, splenectomised and non-splenectomised patients, and between patients treated less or more than 6 years. However patients started on ERT before the 40th year of age exhibited significantly higher success rate as compared to patients started after the 40th year (p=0.047) and there was a significant inverse correlation between overall success rate and patient’s age at treatment start (r = − 0.437). Finally, the success rate was higher among double heterozygotes, carrying the N370S mutation, as compared to patients homozygous for the N370S mutation (89.2±9.6% vs. 80.2±12.9%, p=0.016).

The N370S (Asn370→Ser) mutation affects the capacity of glucosylceramidase to interact with anionic phospholipid-containing membranes and saposin C

The properties of the endolysosomal enzyme GCase (glucosylceramidase), carrying the most prevalent mutation observed in Gaucher patients, namely substitution of an asparagine residue with a serine at amino acid position 370 [N370S (Asn370→Ser) GCase], were investigated in the present study. We previously demonstrated that Sap (saposin) C, the physiological GCase activator, promotes the association of GCase with anionic phospholipid-containing membranes, reconstituting in this way the enzyme activity. In the present study, we show that, in the presence of Sap C and membranes containing high levels of anionic phospholipids, both normal and N370S GCases are able to associate with the lipid surface and to express their activity. Conversely, when the amount of anionic phospholipids in the membrane is reduced (∼20% of total lipids), Sap C is still able to promote binding and activation of the normal enzyme, but not of N370S GCase. The altered interaction of the mutated enzyme with anionic phospholipid-containing membranes and Sap C was further demonstrated in Gaucher fibroblasts by confocal microscopy, which revealed poor co-localization of N370S GCase with Sap C and lysobisphosphatidic acid, the most abundant anionic phospholipid in endolysosomes. Moreover, we found that N370S Gaucher fibroblasts accumulate endolysosomal free cholesterol, a lipid that might further interfere with the interaction of the enzyme with Sap C and lysobisphosphatidic acid-containing membranes. In summary, our results show that the N370S mutation primarily affects the interaction of GCase with its physiological activators, namely Sap C and anionic phospholipid-containing membranes. We thus propose that the poor contact between N370S GCase and its activators may be responsible for the low activity of the mutant enzyme in vivo.