Quantitative dopamine transporter imaging assessment in Parkinson's disease patients carrying GBA gene mutations compared with idiopathic PD patients: A case-control study.

BACKGROUND: Genetic risk factors impact around 15% of Parkinson's disease (PD) patients and at least 23 variants have been identified including Glucocerebrosidase (GBA) gene variants. Using different clinical and instrumental qualitative-based data, various studies have been published on GBA-PD cohorts which suggested possible differences in dopaminergic nigrostriatal denervation pattern, particularly in caudate and putamen nuclei. METHODS: This retrospective study included two consecutive homogenous cohorts of GBA-PD and idiopathic (I-PD) patients. Each consecutive GBA-PD patient has been matched with a 1:1 pairing method with a consecutive I-PD subject according to age, age at disease onset, sex, Hoehn & Yahr (H&Y) staging scale and comorbidity level (CCI). Semiquantitative volumetric data by the DaTQUANTTM software integrated in the DaTSCAN exam performed at time of the diagnosis (SPECT imaging performed according to current guidelines of I-123 FPCIT SPECT imaging) were extrapolated. Bilateral specific binding ratios (SBR) at putamen and caudate levels were calculated, using the occipital lobes uptake. The Mann-Whitney test was performed to compare the two cohorts while the Spearman's test was used to find correlations between motor and volumetric data in each group. Bonferroni correction was used to account for multiple comparisons. RESULTS: Two cohorts of 25 patients each (GBA-PD and I-PD), were included. By comparing GBA-PD and I-PD patients, lower SBR values were found in the most affected anterior putamen and left caudate of the GBA-PD cohort. Furthermore, in the GBA-PD cohort the SBR of the most affected posterior putamen negatively correlated with the H&Y scale. However, none of these differences or correlations remained significant after Bonferroni correction for multiple comparisons. CONCLUSIONS: We observed differences in SBR values in GBA-PD patients compared with I-PD. However, these differences were no longer significant after Bonferroni multiple comparisons correction highlighting the need for larger, longitudinal studies.

A Comprehensive Assessment of Qualitative and Quantitative Prodromal Parkinsonian Features in Carriers of Gaucher Disease-Identifying Those at the Greatest Risk.

Carriers of GBA1 gene variants have a significant risk of developing Parkinson's disease (PD). A cohort study of GBA carriers between 40−75 years of age was initiated to study the presence of prodromal PD features. Participants underwent non-invasive tests to assess different domains of PD. Ninety-eight unrelated GBA carriers were enrolled (43 males) at a median age (range) of 51 (40−74) years; 71 carried the N370S variant (c.1226A > G) and 25 had a positive family history of PD. The Montreal Cognitive Assessment (MoCA) was the most frequently abnormal (23.7%, 95% CI 15.7−33.4%), followed by the ultrasound hyperechogenicity (22%, 95% CI 14−32%), Unified Parkinson's Disease Rating Scale part III (UPDRS-III) (17.2%, 95% CI 10.2−26.4%), smell assessment (12.4%, 95% CI 6.6−20.6%) and abnormalities in sleep questionnaires (11%, 95% CI 5.7−19.4%). Significant correlations were found between tests from different domains. To define the risk for PD, we assessed the bottom 10th percentile of each prodromal test, defining this level as "abnormal". Then we calculated the percentage of "abnormal" tests for each subject; the median (range) was 4.55 (0−43.5%). Twenty-two subjects had more than 15% "abnormal" tests. The limitations of the study included ascertainment bias of individuals with GBA-related PD in relatives, some incomplete data due to technical issues, and a lack of well-characterized normal value ranges in some tests. We plan to enroll additional participants and conduct longitudinal follow-up assessments to build a model for identifying individuals at risk for PD and investigate interventions aiming to delay the onset or perhaps to prevent full-blown PD.

Imaging and genetics in Parkinson's disease: assessment of the GBA1 mutation.

INTRODUCTION: Several genetic variants are associated with an increased risk for developing Parkinson's Disease (PD) and limited genotype/phenotype correlation. Specifically, mutations in GBA1, the gene coding for the lysosomal enzyme glucocerebrosidase, are associated with an earlier age of onset and faster disease progression. Given these phenotypic differences associated with GBA1 variants, we explored whether cortical thickness and other biomarkers of neurodegeneration differed in healthy controls and PD patients with and without GBA1 variants. METHODS: To understand how different GBA1 variants influence PD phenotype early in the disease, we retrieved neuroimaging and biospecimen data from the Parkinson's Progression Markers Initiative database. Using FreeSurfer, we compared T1-weighted MRI images from healthy controls (N = 47) to PD patients with heterozygous N370S (N = 21), heterozygous E326K (N = 18) or heterozygous T369M (N = 8) variants, and GBA1 non-mutation carriers (N = 47). RESULTS: Cortical thickness in PD patients differed from controls in the parietal cortex, with E365K, T369M variants, and GBA1 non-mutation carriers showing more cortical thinning than N370S variants. Patients with N370S variants had significantly higher serum neurofilament light levels among all groups. CONCLUSION: Our results demonstrate significant cortical thinning in PD patients independent of genotype in superior parietal and postcentral regions when compared to the controls. They highlight the impact of GBA1 variants on cortical thickness in the parietal cortex. Finally, they suggest that recently diagnosed PD patients with N370S variants have a higher cortical thickness and increased active neurodegeneration when compared to PD patients without GBA1 mutations and PD patients with E326K or T369M variants.

The remote assessment of parkinsonism supporting the ongoing development of interventions in Gaucher disease.

Mutations in GBA which are causative of Gaucher disease in their biallelic form, are the most common genetic risk factor for Parkinson's disease (PD). The diagnosis of PD relies upon clinically defined motor features which appear after irreversible neurodegeneration. Prodromal symptoms of PD may provide a means to predict latent pathology, years before the onset of motor features. Previous work has reported prodromal features of PD in GBA mutation carriers, however this has been insufficiently sensitive to identify those that will develop PD. The Remote Assessment of Parkinsonism Supporting Ongoing Development of Interventions in Gaucher Disease (RAPSODI GD) study assesses a large cohort of GBA mutation carriers, to aid development of procedures for earlier diagnosis of PD.

Lay abstract Changes in a gene called GBA cause a rare condition called Gaucher disease and are the most common genetic risk factor for Parkinson's disease (PD). To diagnose PD, patients must show symptoms of disordered movement which only occur after irreversible brain cell loss. Earlier symptoms may allow for the prediction of PD, years before movement symptoms occur. Previous work has reported earlier symptoms of PD occurring in people with GBA changes, however these studies have not been able to identify those at risk of developing PD. The Remote Assessment of Parkinsonism Supporting Ongoing Development of Interventions in Gaucher Disease (RAPSODI GD) study assesses a large group of people with GBA changes, to help develop a way to diagnose PD earlier.

Cardiopulmonary assessment of patients diagnosed with Gaucher's disease type I.

BACKGROUND: Understanding the basis of the phenotypic variation in Gaucher's disease (GD) has proven to be challenging for efficient treatment. The current study examined cardiopulmonary characteristics of patients with GD type 1. METHODS: Twenty Caucasian subjects (8/20 female) with diagnosed GD type I (GD-S) and 20 age- and sex-matched healthy controls (C), were assessed (mean age GD-S: 32.6 ± 13.1 vs. C: 36.2 ± 10.6, p > .05) before the initiation of treatment. Standard echocardiography at rest was used to assess left ventricular ejection fraction (LVEF) and pulmonary artery systolic pressure (PASP). Cardiopulmonary exercise testing (CPET) was performed on a recumbent ergometer using a ramp protocol. RESULTS: LVEF was similar in both groups (GD-S: 65.1 ± 5.2% vs. C: 65.2 ± 5.2%, p > .05), as well as PAPS (24.1 ± 4.2 mmHg vs. C: 25.5 ± 1.3 mmHg, p > .05). GD-S had lower weight (p < .05) and worse CPET responses compared to C, including peak values of heart rate, oxygen consumption, carbondioxide production (VCO2 ), end-tidal pressure of CO2 , and O2 pulse, as well as HR reserve after 3 min of recovery and the minute ventilation/VCO2  slope. CONCLUSIONS: Patients with GD type I have an abnormal CPET response compared to healthy controls likely due to the complex pathophysiologic process in GD that impacts multiple systems integral to the physiologic response to exercise.

Validation and assessment of preanalytical factors of a fluorometric in vitro assay for glucocerebrosidase activity in human cerebrospinal fluid.

Lysosomal dysfunction is an emerging feature in the pathology of Parkinson's disease and Dementia with Lewy bodies. Mutations in the GBA gene, encoding the enzyme Glucocerebrosidase (GCase), have been identified as a genetic risk factor for these synucleinopathies. As a result, there has been a growing interest in the involvement of GCase in these diseases. This GCase activity assay is based on the catalytic hydrolysis of 4-methylumbelliferyl ß-D-glucopyranoside that releases the highly fluorescent 4-methylumbelliferyl (4-MU). The final assay protocol was tested for the following parameters: Lower limit of quantification (LLOQ), precision, parallelism, linearity, spike recovery, number of freeze-thaw events, and sample handling stability. The GCase activity assay is within acceptable criteria for parallelism, precision and spike recovery. The LLOQ of this assay corresponds to an enzymatic activity of generating 0.26 pmol 4-MU/min/ml. The enzymatic activity was stable when samples were processed and frozen at - 80 °C within 4 h after the lumbar puncture procedure. Repetitive freeze-thaw events significantly decreased enzyme activity. We present the validation of an optimized in vitro GCase activity assay, based on commercially available components, to quantify its enzymatic activity in human cerebrospinal fluid and the assessment of preanalytical factors.

Parkinson's disease in Gaucher disease patients: what's changing in the counseling and management of patients and their relatives?

BACKGROUND: How to address the counseling of lifetime risk of developing Parkinson's disease in patients with Gaucher disease and their family members carrying a single variant of the GBA1 gene is not yet clearly defined. In addition, there is no set way of managing Gaucher disease patients, taking into account the possibility that they may show features of Parkinson's disease. METHODS: Starting from an overview on what has recently changed in our knowledge on this issue and grouping the experiences of healthcare providers of Gaucher disease patients, we outline a path of counseling and management of Parkinson's disease risk in Gaucher disease patients and their relatives. CONCLUSION: The approach proposed here will help healthcare providers to communicate Parkinson's disease risk to their patients and will reduce the possibility of patients receiving inaccurate information from inadequate sources. Furthermore, this resource will help to empower healthcare providers to identify early signs and/or symptoms of Parkinson's disease and decide when to refer these patients to the neurologist for appropriate specific therapy and follow-up.

A rapid and low-cost test for screening the most common Parkinson's disease-related GBA variants.

INTRODUCTION: Deleterious variants in the GBA gene confer a 2- to 20-fold increased risk of Parkinson's disease (PD) and are associated with a more severe disease course. The presence of a highly-similar pseudogene complicates genetic screening, both by Sanger and next-generation sequencing (NGS). Among PD-associated GBA variants, four missense substitutions (p.L444P, p.N370S, p.T369M, p.E326K) account for the majority of cases. Here, we aimed at developing an allele-specific PCR (AS-PCR) able to concomitantly detect the most common PD-related GBA variants. METHODS: A multiplex PCR assay was designed using allele-specific oligonucleotides that distinguish the gene from the pseudogene and can exclusively amplify the variant alleles. Primer sequences and molarity, and thermal profiles were empirically optimized. The assay was validated on 4016 DNAs extracted by standard salting-out and previously analyzed by whole-exome sequencing (WES) followed by Sanger validation. RESULTS: AS-PCRs performed on 4016 DNAs detected 103 variants; among them, 97 were true positives and 6 false positives. When comparing the results with the original WES data, for the "difficult" p.L444P, the number of false positives was 2/9 and 18/24 for multiplex-AS-PCR and WES, respectively. As we could have missed some p.L444P alleles by NGS, we verified the test performance on 50 DNAs from Sanger-validated p.L444P heterozygotes. All samples tested correctly. CONCLUSION: We set up and validated a rapid and inexpensive test for screening large cohorts of individuals for variants conferring a significant PD risk. This screening method is particularly interesting to identify patients who could benefit most from early access to personalized therapies.

Assessment of cellular cobalamin metabolism in Gaucher disease.

BACKGROUND: Gaucher disease (GD) is a lysosomal disorder caused by biallelic pathogenic mutations in the GBA1 gene that encodes beta-glucosidase (GCase), and more rarely, by a deficiency in the GCase activator, saposin C. Clinically, GD manifests with heterogeneous multiorgan involvement mainly affecting hematological, hepatic and neurological axes. This disorder is divided into three types, based on the absence (type I) or presence and severity (types II and III) of involvement of the central nervous system. At the cellular level, deficiency of GBA1 disturbs lysosomal storage with buildup of glucocerebroside. The consequences of disturbed lysosomal metabolism on biochemical pathways that require lysosomal processing are unknown. Abnormal systemic markers of cobalamin (Cbl, B12) metabolism have been reported in patients with GD, suggesting impairments in lysosomal handling of Cbl or in its downstream utilization events. METHODS: Cultured skin fibroblasts from control humans (n = 3), from patients with GD types I (n = 1), II (n = 1) and III (n = 1) and an asymptomatic carrier of GD were examined for their GCase enzymatic activity and lysosomal compartment intactness. Control human and GD fibroblasts were cultured in growth medium with and without 500 nM hydroxocobalamin supplementation. Cellular cobalamin status was examined via determination of metabolomic markers in cell lysate (intracellular) and conditioned culture medium (extracellular). The presence of transcobalamin (TC) in whole cell lysates was examined by Western blot. RESULTS: Cultured skin fibroblasts from GD patients exhibited reduced GCase activity compared to healthy individuals and an asymptomatic carrier of GD, demonstrating a preserved disease phenotype in this cell type. The concentrations of total homocysteine (tHcy), methylmalonic acid (MMA), cysteine (Cys) and methionine (Met) in GD cells were comparable to control levels, except in one patient with GD III. The response of these metabolomic markers to supplementation with hydroxocobalamin (HOCbl) yielded variable results. The content of transcobalamin in whole cell lysates was comparable in control human and GD patients. CONCLUSIONS: Our results indicate that cobalamin transport and cellular processing pathways are overall protected from lysosomal storage damage in GD fibroblasts. Extending these studies to hepatocytes, macrophages and plasma will shed light on cell- and compartment-specific vitamin B12 metabolism in Gaucher disease.

Genome-wide assessment of Parkinson's disease in a Southern Spanish population.

Here, we set out to study the genetic architecture of Parkinson's disease (PD) through a Genome-Wide Association Study in a Southern Spanish population. About 240 PD cases and 192 controls were genotyped on the NeuroX array. We estimated genetic variation associated with PD risk and age at onset (AAO). Risk profile analyses for PD and AAO were performed using a weighted genetic risk score. Total heritability was estimated by genome-wide complex trait analysis. Rare variants were screened with single-variant and burden tests. We also screened for variation in known PD genes. Finally, we explored runs of homozygosity and structural genomic variations. We replicate PD association (uncorrected p-value < 0.05) at the following loci: ACMSD/TMEM163, MAPT, STK39, MIR4697, and SREBF/RAI1. Subjects in the highest genetic risk score quintile showed significantly increased risk of PD versus the lowest quintile (odds ratio = 3.6, p-value < 4e(-7)), but no significant difference in AAO. We found evidence of runs of homozygosity in 2 PD-associated regions: one intersecting the HLA-DQB1 gene in 6 patients and 1 control; and another intersecting the GBA-SYT11 gene in PD case. The GBA N370S and the LRRK2 G2019S variants were found in 8 and 7 cases, respectively, replicating previous work. A structural variant was found in 1 case in the PARK2 gene locus. This current work represents a comprehensive assessment at a genome-wide level characterizing a novel population in PD genetics.

A peptide-linked recombinant glucocerebrosidase for targeted neuronal delivery: Design, production, and assessment.

Although recombinant glucocerebrosidase (GCase) is the standard therapy for the inherited lysosomal storage disease Gaucher's disease (GD), enzyme replacement is not effective when the central nervous system is affected. We created a series of recombinant genes/proteins where GCase was linked to different membrane binding peptides including the Tat peptide, the rabies glycoprotein derived peptide (RDP), the binding domain from tetanus toxin (TTC), and a tetanus like peptide (Tet1). The majority of these proteins were well-expressed in a mammalian producer cell line (HEK 293F). Purified recombinant Tat-GCase and RDP-GCase showed similar GCase protein delivery to a neuronal cell line that genetically lacks the functional enzyme, and greater delivery than control GCase, Cerezyme (Genzyme). This initial result was unexpected based on observations of superior protein delivery to neurons with RDP as a vector. A recombinant protein where a fragment of the flexible hinge region from IgA (IgAh) was introduced between RDP and GCase showed substantially enhanced GCase neuronal delivery (2.5 times over Tat-GCase), suggesting that the original construct resulted in interference with the capacity of RDP to bind neuronal membranes. Extended treatment of these knockout neuronal cells with either Tat-GCase or RDP-IgAh-GCase resulted in an >90% reduction in the lipid substrate glucosylsphingosine, approaching normal levels. Further in vivo studies of RDP-IgAh-GCase as well as Tat-GCase are warranted to assess their potential as treatments for neuronopathic forms of GD. These peptide vectors are especially attractive as they have the potential to carry a protein across the blood-brain barrier, avoiding invasive direct brain delivery.

An assessment of the frequency of mutations in the GBA and VPS35 genes in Hungarian patients with sporadic Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, with cases of either familial or sporadic origin. Several polymorphisms in a number of genes have been proved to have an important role in the development of PD. Particular attention has recently been paid to genes of the glucocerebrosidase (GBA) and the vacuolar protein sorting-associated protein 35 (VPS35). In this study, the three most common mutations (L444P, N370S and R120W) of the GBA gene and the D620N mutation of the VPS35 gene were examined in 124 Hungarian patients diagnosed with sporadic PD (SPD) and 122 control subjects. The frequency of the L444P mutation of the GBA gene proved to be higher in the PD patients (2.4%) than in the controls (0%), although the difference was not statistically significant. All the patients who carried the mutant allele were in the early-onset PD (EOPD) group. However, neither the R120W nor the N370S variant of the GBA gene nor D620N mutation of the VPS35 gene were detected among the PD cases or the controls. Even though these results suggest that the studied mutations are quite rare in SPD patients, the most frequent L444P mutation of the GBA gene may be associated with the development of EOPD in the Hungarian population.

A plant-derived recombinant human glucocerebrosidase enzyme--a preclinical and phase I investigation.

UNLABELLED: Gaucher disease is a progressive lysosomal storage disorder caused by the deficiency of glucocerebrosidase leading to the dysfunction in multiple organ systems. Intravenous enzyme replacement is the accepted standard of treatment. In the current report, we evaluate the safety and pharmacokinetics of a novel human recombinant glucocerebrosidase enzyme expressed in transformed plant cells (prGCD), administered to primates and human subjects. Short term (28 days) and long term (9 months) repeated injections with a standard dose of 60 Units/kg and a high dose of 300 Units/kg were administered to monkeys (n = 4/sex/dose). Neither clinical drug-related adverse effects nor neutralizing antibodies were detected in the animals. In a phase I clinical trial, six healthy volunteers were treated by intravenous infusions with escalating single doses of prGCD. Doses of up to 60 Units/kg were administered at weekly intervals. prGCD infusions were very well tolerated. Anti-prGCD antibodies were not detected. The pharmacokinetic profile of the prGCD revealed a prolonged half-life compared to imiglucerase, the commercial enzyme that is manufactured in a costly mammalian cell system. These studies demonstrate the safety and lack of immunogenicity of prGCD. Following these encouraging results, a pivotal phase III clinical trial for prGCD was FDA approved and is currently ongoing. TRIAL REGISTRATION: ClinicalTrials.gov NCT00258778.

The underrecognized progressive nature of N370S Gaucher disease and assessment of cancer risk in 403 patients.

Mutations in GBA1 gene that encodes lysosomal glucocerebrosidase result in Type 1 Gaucher Disease (GD), the commonest lysosomal storage disorder; the most prevalent disease mutation is N370S. We investigated the heterogeneity and natural course of N370S GD in 403 patients. Demographic, clinical, and genetic characteristics of GD at presentation were examined in a cross-sectional study. In addition, the relative risk (RR) of cancer in patients compared with age-, sex-, and ethnic-group adjusted national rates of cancer was determined. Of the 403 patients, 54% of patients were homozygous (N370S/N370S) and 46% were compound heterozygous for the N370S mutation (N370S/other). The majority of N370S/N370S patients displayed a phenotype characterized by late onset, predominantly skeletal disease, whereas the majority of N370S/other patients displayed early onset, predominantly visceral/hematologic disease, P < 0.0001. There was a striking increase in lifetime risk of multiple myeloma in the entire cohort (RR 25, 95% CI 9.17-54.40), mostly confined to N370S homozygous patients. The risk of other hematologic malignancies (RR 3.45, 95% CI 1.49-6.79), and overall cancer risk (RR 1.80, 95% CI 1.32-2.40) was increased. Homozygous N370S GD leads to adult-onset progressive skeletal disease with relative sparing of the viscera, a strikingly high risk of multiple myeloma, and an increased risk of other cancers. High incidence of gammopathy suggests an important role of the adaptive immune system in the development of GD. Adult patients with GD should be monitored for skeletal disease and cancers including multiple myeloma.