EEG abnormalities in patients with chronic neuronopathic Gaucher disease: A retrospective review.

The clinical phenotype of Gaucher disease type 3 (GD3), a neuronopathic lysosomal storage disorder, encompasses a wide array of neurological manifestations including neuro-ophthalmological findings, developmental delay, and seizures including progressive myoclonic epilepsy. Electroencephalography (EEG) is a widely available tool used to identify abnormalities in cerebral function, as well as epileptiform abnormalities indicating an increased risk of seizures. We characterized the EEG findings in GD3, reviewing 67 patients with 293 EEGs collected over nearly 50 years. Over 93% of patients had some form of EEG abnormality, most consisting of background slowing (90%), followed by interictal epileptiform discharges (IEDs) (54%), and photoparoxysmal responses (25%). The seven patients without background slowing were all under age 14 (mean 6.7 years). There was a history of seizures in 37% of this cohort; only 30% of these had IEDs on EEG. Conversely, only 56% of patients with IEDs had a history of seizures. These observed EEG abnormalities document an important aspect of the natural history of GD3 and could potentially assist in identifying neurological involvement in a patient with subtle clinical findings. Additionally, this comprehensive description of longitudinal EEG data provides essential baseline data for understanding central nervous system involvement in neuronopathic GD.

A characterization of Gaucher iPS-derived astrocytes: Potential implications for Parkinson's disease.

While astrocytes, the most abundant cells found in the brain, have many diverse functions, their role in the lysosomal storage disorder Gaucher disease (GD) has not been explored. GD, resulting from the inherited deficiency of the enzyme glucocerebrosidase and subsequent accumulation of glucosylceramide and its acylated derivative glucosylsphingosine, has both non-neuronopathic (GD1) and neuronopathic forms (GD2 and 3). Furthermore, mutations in GBA1, the gene mutated in GD, are an important risk factor for Parkinson's disease (PD). To elucidate the role of astrocytes in the disease pathogenesis, we generated iAstrocytes from induced pluripotent stem cells made from fibroblasts taken from controls and patients with GD1, with and without PD. We also made iAstrocytes from an infant with GD2, the most severe and progressive form, manifesting in infancy. Gaucher iAstrocytes appropriately showed deficient glucocerebrosidase activity and levels and substrate accumulation. These cells exhibited varying degrees of astrogliosis, Glial Fibrillary Acidic Protein (GFAP) up-regulation and cellular proliferation, depending on the level of residual glucocerebrosidase activity. Glutamte uptake assays demonstrated that the cells were functionally active, although the glutamine transporter EEAT2 was upregulated and EEAT1 downregulated in the GD2 samples. GD2 iAstrocytes were morphologically different, with severe cytoskeletal hypertrophy, overlapping of astrocyte processes, pronounced up-regulation of GFAP and S100ß, and significant astrocyte proliferation, recapitulating the neuropathology observed in patients with GD2. Although astrocytes do not express α-synuclein, when the iAstrocytes were co-cultured with dopaminergic neurons generated from the same iPSC lines, excessive α-synuclein released from neurons was endocytosed by astrocytes, translocating into lysosomes. Levels of aggregated α-synuclein increased significantly when cells were treated with monomeric or fibrillar α-synuclein. GD1-PD and GD2 iAstrocytes also exhibited impaired Cathepsin D activity, leading to further α-synuclein accumulation. Cytokine and chemokine profiling of the iAstrocytes demonstrated an inflammatory response. Thus, in patients with GBA1-associated parkinsonism, astrocytes appear to play a role in α-synuclein accumulation and processing, contributing to neuroinflammation.

Alleles with more than one mutation can complicate genotype/phenotype studies in Mendelian disorders: Lessons from Gaucher disease.

Autosomal resessive Mendelian disorders usually result from two inherited disease-causing mutations. However, this is not always the case. Focusing on Gaucher disease, which results from mutations in GBA1, we found that more comprehensive genotyping revealed important exceptions. For example, patients with uniparental disomy or new mutations do not inherit a mutation from each parent. Furthermore, we identified patients found to carry more than one GBA1 mutation on the same allele. It is essential to examine the entire GBA1 gene in order to establish an accurate genotype. Missing the second mutation can complicate genotype/phenotype studies and result in improper genetic counseling.

A New Glucocerebrosidase Chaperone Reduces α-Synuclein and Glycolipid Levels in iPSC-Derived Dopaminergic Neurons from Patients with Gaucher Disease and Parkinsonism.

UNLABELLED: Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT: Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.

Macrophage models of Gaucher disease for evaluating disease pathogenesis and candidate drugs.

Gaucher disease is caused by an inherited deficiency of glucocerebrosidase that manifests with storage of glycolipids in lysosomes, particularly in macrophages. Available cell lines modeling Gaucher disease do not demonstrate lysosomal storage of glycolipids; therefore, we set out to develop two macrophage models of Gaucher disease that exhibit appropriate substrate accumulation. We used these cellular models both to investigate altered macrophage biology in Gaucher disease and to evaluate candidate drugs for its treatment. We generated and characterized monocyte-derived macrophages from 20 patients carrying different Gaucher disease mutations. In addition, we created induced pluripotent stem cell (iPSC)-derived macrophages from five fibroblast lines taken from patients with type 1 or type 2 Gaucher disease. Macrophages derived from patient monocytes or iPSCs showed reduced glucocerebrosidase activity and increased storage of glucocerebroside and glucosylsphingosine in lysosomes. These macrophages showed efficient phagocytosis of bacteria but reduced production of intracellular reactive oxygen species and impaired chemotaxis. The disease phenotype was reversed with a noninhibitory small-molecule chaperone drug that enhanced glucocerebrosidase activity in the macrophages, reduced glycolipid storage, and normalized chemotaxis and production of reactive oxygen species. Macrophages differentiated from patient monocytes or patient-derived iPSCs provide cellular models that can be used to investigate disease pathogenesis and facilitate drug development.

A germline or de novo mutation in two families with Gaucher disease: implications for recessive disorders.

Gaucher disease (GD) is an autosomal recessive storage disorder that most commonly results from the inheritance of one identifiable mutant glucocerebrosidase (GBA1) allele from each parent. Here, we report two cases of type 2 GD resulting from the inheritance of one identifiable paternal mutant allele and one allele that likely resulted from a maternal germline mutation. Germline mutations or mosiacism are not generally associated with autosomal recessive disorders. The probands from the two unrelated families had the same maternal mutation, leu444pro, that we propose resulted from a de novo maternal germline mutation occurring at this known 'hotspot' for mutation. This first report of a germline mutation for a common point mutation leu444pro (c.1448 T>C;p.leu483pro) in GD has significant implications for molecular diagnostics and genetic counseling in recessive disorders.

Induced pluripotent stem cell model recapitulates pathologic hallmarks of Gaucher disease.

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid ß-glucocerebrosidase gene. To model GD, we generated human induced pluripotent stem cells (hiPSC), by reprogramming skin fibroblasts from patients with type 1 (N370S/N370S), type 2 (L444P/RecNciI), and type 3 (L444P/L444P) GD. Pluripotency was demonstrated by the ability of GD hiPSC to differentiate to all three germ layers and to form teratomas in vivo. GD hiPSC differentiated efficiently to the cell types most affected in GD, i.e., macrophages and neuronal cells. GD hiPSC-macrophages expressed macrophage-specific markers, were phagocytic, and were capable of releasing inflammatory mediators in response to LPS. Moreover, GD hiPSC-macrophages recapitulated the phenotypic hallmarks of the disease. They exhibited low glucocerebrosidase (GC) enzymatic activity and accumulated sphingolipids, and their lysosomal functions were severely compromised. GD hiPSC-macrophages had a defect in their ability to clear phagocytosed RBC, a phenotype of tissue-infiltrating GD macrophages. The kinetics of RBC clearance by types 1, 2, and 3 GD hiPSC-macrophages correlated with the severity of the mutations. Incubation with recombinant GC completely reversed the delay in RBC clearance from all three types of GD hiPSC-macrophages, indicating that their functional defects were indeed caused by GC deficiency. However, treatment of induced macrophages with the chaperone isofagomine restored phagocytosed RBC clearance only partially, regardless of genotype. These findings are consistent with the known clinical efficacies of recombinant GC and isofagomine. We conclude that cell types derived from GD hiPSC can effectively recapitulate pathologic hallmarks of the disease.

A mutation in SCARB2 is a modifier in Gaucher disease.

Lysosomal integral membrane protein type 2 (LIMP-2) is responsible for proper sorting and lysosomal targeting of glucocerebrosidase, the enzyme deficient in Gaucher disease (GD). Mutations in the gene for LIMP-2, SCARB2, are implicated in inherited forms of myoclonic epilepsy, and myoclonic epilepsy is part of the phenotypic spectrum associated with GD. We investigated whether SCARB2 mutations impact the Gaucher phenotype focusing on patients with myoclonic epilepsy, including a pair of siblings with GD who were discordant for myoclonic seizures. Sequencing of SCARB2 genomic and cDNA identified a heterozygous, maternally inherited novel mutation, c.1412A>G (p.Glu471Gly), in the brother with GD and myoclonic epilepsy, absent from his sibling and controls. Glucocerebrosidase activity, Western blots, real-time PCR, and immunofluorescence studies demonstrated markedly decreased LIMP-2 and glucocerebrosidase in cells from the sibling with (p.Glu471Gly) LIMP-2, and diminished glucocerebrosidase in lysosomes. The cells secreted highly glycosylated enzyme and showed mistrafficking of glucocerebrosidase. Sequencing of SCARB2 in 13 other subjects with GD and myoclonic epilepsy and 40 controls failed to identify additional mutations. The study provides further evidence for the association of LIMP-2 and myoclonic epilepsy, explains the drastically different phenotypes encountered in the siblings, and demonstrates that LIMP-2 can serve as a modifier in GD.

Identification of recombinant alleles using quantitative real-time PCR implications for Gaucher disease.

Pseudogenes, resulting from duplications of functional genes, contribute to the functional complexity of their parental genes. The glucocerebrosidase gene (GBA), located in a gene-rich region on chromosome 1q 21, is mutated in Gaucher disease. The presence of contiguous, highly homologous pseudogenes for both GBA and metaxin 1 at this locus increases the likelihood of DNA rearrangement. We describe a facile method to identify and analyze recombinant alleles in patients with Gaucher disease. Genomic DNA from 20 patients with recombinant GBA alleles and five controls was evaluated to identify DNA rearrangements or copy number variation using six probes specific for either the GBA gene or pseudogene. Quantitative real-time PCR was performed on genomic DNA, and Southern blot analyses using HincII together with sequencing confirmed the real-time results. Both GBA fusions and duplications could be detected. Different sites of crossover were identified, and alleles resulting from gene conversion could be distinguished from reciprocal recombinant alleles. Quantitative real-time PCR is a sensitive and rapid method to detect fusions and duplications in patients with recombinant GBA alleles. This technique is more sensitive, faster, and cheaper than Southern blot analysis, and can be used in diagnostic laboratories, and to detect other recombinant alleles within the genome.

Evaluation of quinazoline analogues as glucocerebrosidase inhibitors with chaperone activity.

Gaucher disease is a lysosomal storage disorder (LSD) caused by deficiency in the enzyme glucocerebrosidase (GC). Small molecule chaperones of protein folding and translocation have been proposed as a promising therapeutic approach to this LSD. Most small molecule chaperones described in the literature contain an iminosugar scaffold. Here we present the discovery and evaluation of a new series of GC inhibitors with a quinazoline core. We demonstrate that this series can improve the translocation of GC to the lysosome in patient-derived cells. To optimize this chemical series, systematic synthetic modifications were performed and the SAR was evaluated and compared using three different readouts of compound activity: enzymatic inhibition, enzyme thermostabilization, and lysosomal translocation of GC.

Glucocerebrosidase is present in α-synuclein inclusions in Lewy body disorders.

Mutations in the gene encoding the lysosomal enzyme glucocerebrosidase, known to cause Gaucher disease (GD), are a risk factor for the development of Parkinson disease (PD) and related disorders. This association is based on the concurrence of parkinsonism and GD, the identification of glucocerebrosidase mutations in cohorts with PD from centers around the world, and neuropathologic findings. The contribution of glucocerebrosidase to the development of parkinsonian pathology was explored by studying seven brain samples from subjects carrying glucocerebrosidase mutations with pathologic diagnoses of PD and/or Lewy body dementia. Three individuals had GD and four were heterozygous for glucocerebrosidase mutations. All cases had no known family history of PD and the mean age of disease onset was 59 years (range 42-77). Immunofluorescence studies on brain tissue samples from patients with parkinsonism associated with glucocerebrosidase mutations showed that glucocerebrosidase was present in 32-90% of Lewy bodies (mean 75%), some ubiquitinated and others non-ubiquitinated. In samples from seven subjects without mutations, <10% of Lewy bodies were glucocerebrosidase positive (mean 4%). This data demonstrates that glucocerebrosidase can be an important component of α-synuclein-positive pathological inclusions. Unraveling the role of mutant glucocerebrosidase in the development of this pathology will further our understanding of the lysosomal pathways that likely contribute to the formation and/or clearance of these protein aggregates.

False-positive results using a Gaucher diagnostic kit--RecTL and N370S.

The Pronto Gaucher kits and Pronto Gaucher RecTL Amplification Mix, marketed to identify mutations in the gene for glucocerebrosidase, are widely used for the diagnosis of Gaucher disease. Subjects genotyped using this kit have been reported with an allele including both the common N370S mutation and RecTL, a previously described Gaucher mutation arising from recombination between the glucocerebrosidase gene and pseudogene. Using direct sequencing and real-time PCR, we show that the RecTL, N370S allele is a false positive result, demonstrating possible pitfalls of diagnostic kits.

In silico and functional studies of the regulation of the glucocerebrosidase gene.

In Gaucher disease (GD), the inherited deficiency of glucocerebrosidase results in the accumulation of glucocerebroside within lysosomes. Although almost 300 mutations in the glucocerebrosidase gene (GBA) have been identified, the ability to predict phenotype from genotype is quite limited. In this study, we sought to examine potential GBA transcriptional regulatory elements for variants that contribute to phenotypic diversity. Specifically, we generated the genomic sequence for the orthologous genomic region ( approximately 39.4kb) encompassing GBA in eight non-human mammals. Computational comparisons of the resulting sequences, using human sequence as the reference, allowed the identification of multi-species conserved sequences (MCSs). Further analyses predicted the presence of two putative clusters of transcriptional regulatory elements upstream and downstream of GBA, containing five and three transcription factor-binding sites (TFBSs), respectively. A firefly luciferase (Fluc) reporter construct containing sequence flanking the GBA gene was used to test the functional consequences of altering these conserved sequences. The predicted TFBSs were individually altered by targeted mutagenesis, resulting in enhanced Fluc expression for one site and decreased expression for seven others sites. Gel-shift assays confirmed the loss of nuclear-protein binding for several of the mutated constructs. These identified conserved non-coding sequences flanking GBA could play a role in the transcriptional regulation of the gene contributing to the complexity underlying the phenotypic diversity seen in GD.

Glucocerebrosidase mutations in Chinese subjects from Taiwan with sporadic Parkinson disease.

BACKGROUND: An association between glucocerebrosidase, the enzyme deficient in Gaucher disease, and the synucleinopathies has been suggested both by the development of parkinsonism in Gaucher probands and carriers, as well as by the presence of mutations in the gene for glucocerebrosidase (GBA) in different series of subjects with synucleinopathies. In this study, an open access Parkinson repository was used to establish the incidence of GBA alterations in a different ethnic cohort with sporadic Parkinson disease (PD). METHODS: The glucocerebrosidase gene was sequenced in samples collected from 92 Chinese Parkinson disease patients from Taiwan along with 92 clinically screened controls, matched for age and ethnicity. FINDINGS: The frequency of GBA mutations among the Chinese PD probands was 4.3%, in contrast to 1.1% in Chinese controls. Mutant alleles identified included two known mutations, L444P and D409H, and two novel mutations, L174P and Q497R. INTERPRETATION: These results, ascertained in subjects from Taiwan collected in a standardized and clinically rigorous open access Parkinson disease repository and screened by direct sequencing of GBA, demonstrate that GBA mutations are also encountered in Chinese subjects with sporadic PD at a higher frequency than many other known PD genes. The study demonstrates that the association of GBA mutations with the development of parkinsonian pathology is not related to ethnic origin.

DNA targeting of rhinal cortex D2 receptor protein reversibly blocks learning of cues that predict reward.

When schedules of several operant trials must be successfully completed to obtain a reward, monkeys quickly learn to adjust their behavioral performance by using visual cues that signal how many trials have been completed and how many remain in the current schedule. Bilateral rhinal (perirhinal and entorhinal) cortex ablations irreversibly prevent this learning. Here, we apply a recombinant DNA technique to investigate the role of dopamine D2 receptor in rhinal cortex for this type of learning. Rhinal cortex was injected with a DNA construct that significantly decreased D2 receptor ligand binding and temporarily produced the same profound learning deficit seen after ablation. However, unlike after ablation, the D2 receptor-targeted, DNA-treated monkeys recovered cue-related learning after 11-19 weeks. Injecting a DNA construct that decreased N-methyl-d-aspartate but not D2 receptor ligand binding did not interfere with learning associations between the cues and the schedules. A second D2 receptor-targeted DNA treatment administered after either recovery from a first D2 receptor-targeted DNA treatment (one monkey), after N-methyl-d-aspartate receptor-targeted DNA treatment (two monkeys), or after a vector control treatment (one monkey) also induced a learning deficit of similar duration. These results suggest that the D2 receptor in primate rhinal cortex is essential for learning to relate the visual cues to the schedules. The specificity of the receptor manipulation reported here suggests that this approach could be generalized in this or other brain pathways to relate molecular mechanisms to cognitive functions.

Phenotypic continuum in neuronopathic Gaucher disease: an intermediate phenotype between type 2 and type 3.

Neuronopathic Gaucher disease, classically divided into two types, can have a continuum of phenotypes, often defying categorization. Nine children had an intermediate phenotype characterized by a delayed age of onset but rapidly progressive neurological disease, including refractory seizures and oculomotor abnormalities. There was genotypic heterogeneity among these patients.

Myoclonic epilepsy in Gaucher disease: genotype-phenotype insights from a rare patient subgroup.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, presents with a wide spectrum of manifestations. Although Gaucher disease has been divided into three clinical types, patients with atypical presentations continue to be recognized. A careful phenotypic and genotypic assessment of patients with unusual symptoms may help define factors that modify phenotype in this disorder. One such example is a rare subgroup of patients with type 3 Gaucher disease who develop progressive myoclonic epilepsy. We evaluated 16 patients with myoclonic epilepsy, nine of whom were diagnosed by age 4 y with severe visceral involvement and myoclonus, and seven with a more chronic course, who were studied between ages 22 and 40. All of the patients had abnormal horizontal saccadic eye movements. Fourteen different genotypes were encountered, yet there were several shared alleles, including V394L (seen on two alleles), G377S (seen on three alleles), and L444P, N188S, and recombinant alleles (each found on four alleles). V394L, G377S, and N188S are mutations that have previously been associated with non-neuronopathic Gaucher disease. The spectrum of genotypes differed significantly from other patients with type 3 Gaucher disease, where genotypes L444P/L444P and R463C/null allele predominated. Northern blot studies revealed a normal glucocerebrosidase transcript, whereas Western studies showed that the patients studied lacked the processed 56 kD isoform of the enzyme, consistent with neuronopathic Gaucher disease. Brain autopsy samples from two patients demonstrated elevated levels of glucosylsphingosine, a toxic glycolipid, which could contribute to the development of myoclonus. Thus, although there were certain shared mutant alleles found in these patients, both the lack of a shared genotype and the variability in clinical presentations suggest that other modifiers must contribute to this rare phenotype.

Reciprocal and nonreciprocal recombination at the glucocerebrosidase gene region: implications for complexity in Gaucher disease.

Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme glucocerebrosidase. The glucocerebrosidase gene is located in a gene-rich region of 1q21 that contains six genes and two pseudogenes within 75 kb. The presence of contiguous, highly homologous pseudogenes for both glucocerebrosidase and metaxin at the locus increases the likelihood of DNA rearrangements in this region. These recombinations can complicate genotyping in patients with Gaucher disease and contribute to the difficulty in interpreting genotype-phenotype correlations in this disorder. In the present study, DNA samples from 240 patients with Gaucher disease were examined using several complementary approaches to identify and characterize recombinant alleles, including direct sequencing, long-template polymerase chain reaction, polymorphic microsatellite repeats, and Southern blots. Among the 480 alleles studied, 59 recombinant alleles were identified, including 34 gene conversions, 18 fusions, and 7 downstream duplications. Twenty-two percent of the patients evaluated had at least one recombinant allele. Twenty-six recombinant alleles were found among 310 alleles from patients with type 1 disease, 18 among 74 alleles from patients with type 2 disease, and 15 among 96 alleles from patients with type 3 disease. Several patients carried two recombinations or mutations on the same allele. Generally, alleles resulting from nonreciprocal recombination (gene conversion) could be distinguished from those arising by reciprocal recombination (crossover and exchange), and the length of the converted sequence was determined. Homozygosity for a recombinant allele was associated with early lethality. Ten different sites of crossover and a shared pentamer motif sequence (CACCA) that could be a hotspot for recombination were identified. These findings contribute to a better understanding of genotype-phenotype relationships in Gaucher disease and may provide insights into the mechanisms of DNA rearrangement in other disorders.