Background
Mutations in GBA cause Gaucher disease when biallelic and are strong risk factors for Parkinson's disease when heterozygous. GBA analysis is complicated by the nearby pseudogene. We aimed ...to design and validate a method for sequencing GBA using long reads.
Methods
We sequenced GBA on the Oxford Nanopore MinION as an 8.9 kb amplicon from 102 individuals, including patients with Parkinson's and Gaucher diseases. We used NanoOK for quality metrics, NGMLR to align data (after comparing with GraphMap), Nanopolish and Sniffles to call variants, and WhatsHap for phasing.
Results
We detected all known missense mutations in these samples, including the common p.N409S (N370S) and p.L483P (L444P) in multiple samples, and nine rarer ones, as well as a splicing and a truncating mutation, and intronic SNPs. We demonstrated the ability to phase mutations, confirm compound heterozygosity, and assign haplotypes. We also detected two known risk variants in some Parkinson's patients. Rare false positives were easily identified and filtered, with the Nanopolish quality score adjusted for the number of reads a very robust discriminator. In two individuals carrying a recombinant allele, we were able to detect and fully define it in one carrier, where it included a 55‐base pair deletion, but not in another one, suggesting a limitation of the PCR enrichment method. Missense mutations were detected at the correct zygosity, except for the case where the RecNciI one was missed.
Conclusion
The Oxford Nanopore MinION can detect missense mutations and an exonic deletion in this difficult gene, with the added advantages of phasing and intronic analysis. It can be used as an efficient research tool, but additional work is required to exclude all recombinants.
The GBA gene is important in Parkinson's and Gaucher disease, but difficult to sequence due to a highly homologous adjacent pseudogene. Here we present a novel method using long reads on the Oxford Nanopore MinION, which can detect missense mutations and an exonic deletion, with the added advantage of phasing and intronic analysis.
Microglia are heterogenous cells characterized by distinct populations each contributing to specific biological processes in the nervous system, including neuroprotection. To elucidate the impact of ...sex-specific microglia heterogenicity to the susceptibility of neuronal stress, we video-recorded with time-lapse microscopy the changes in shape and motility occurring in primary cells derived from mice of both sexes in response to pro-inflammatory or neurotoxic stimulations. With this morpho-functional analysis, we documented distinct microglia subpopulations eliciting sex-specific responses to stimulation: male microglia tended to have a more pro-inflammatory phenotype, while female microglia showed increased sensitivity to conduritol-B-epoxide (CBE), a small molecule inhibitor of glucocerebrosidase, the enzyme encoded by the GBA1 gene, mutations of which are the major risk factor for Parkinson's Disease (PD). Interestingly, glucocerebrosidase inhibition particularly impaired the ability of female microglia to enhance the Nrf2-dependent detoxification pathway in neurons, attenuating the sex differences observed in this neuroprotective function. This finding is consistent with the clinical impact of GBA1 mutations, in which the 1.5-2-fold reduced risk of developing idiopathic PD observed in female individuals is lost in the GBA1 carrier population, thus suggesting a sex-specific role for microglia in the etiopathogenesis of PD-GBA1.
Abstract
GBA
variants carriers are at increased risk of Parkinson’s disease (PD) and Lewy body dementia (LBD). The presence of pseudogene
GBAP1
predisposes to structural variants, complicating ...genetic analysis. We present two methods to resolve recombinant alleles and other variants in
GBA
: Gauchian, a tool for short-read, whole-genome sequencing data analysis, and Oxford Nanopore sequencing after PCR enrichment. Both methods were concordant for 42 samples carrying a range of recombinants and
GBAP1
-related mutations, and Gauchian outperformed the GATK Best Practices pipeline. Applying Gauchian to sequencing of over 10,000 individuals shows that copy number variants (CNVs) spanning
GBAP1
are relatively common in Africans. CNV frequencies in PD and LBD are similar to controls. Gains may coexist with other mutations in patients, and a modifying effect cannot be excluded. Gauchian detects more
GBA
variants in LBD than PD, especially severe ones. These findings highlight the importance of accurate
GBA
analysis in these patients.
The GBA1 gene encodes the lysosomal enzyme glucocerebrosidase (GCase), which is involved in sphingolipid metabolism. Biallelic variants in GBA1 cause Gaucher disease (GD), a lysosomal storage ...disorder characterised by loss of GCase activity and aberrant intracellular accumulation of GCase substrates. Carriers of GBA1 variants have an increased risk of developing Parkinson disease (PD), with odds ratio ranging from 2.2 to 30 according to variant severity. GBA1 variants which do not cause GD in homozygosis can also increase PD risk. Patients with PD carrying GBA1 variants show a more rapidly progressive phenotype compared to non-carriers, emphasising the need for disease modifying treatments targeting the GBA1 pathway.
Several mechanisms secondary to GCase dysfunction are potentially responsible for the pathological changes leading to PD. Misfolded GCase proteins induce endoplasmic reticulum stress and subsequent unfolded protein response and impair the autophagy-lysosomal pathway. This results in α-synuclein accumulation and spread, and promotes neurodegenerative changes. Preclinical evidence also shows that products of GCase activity can promote accumulation of α-synuclein, however there is no convincing evidence of substrate accumulation in GBA1-PD brains. Altered lipid homeostasis secondary to loss of GCase activity could also contribute to PD pathology.
Treatments that target the GBA1 pathway could reverse these pathological processes and halt/slow the progression of PD. These range from augmentation of GCase activity via GBA1 gene therapy, restoration of normal intracellular GCase trafficking via molecular chaperones, and substrate reduction therapy. This review discusses the pathways associated with GBA1-PD and related novel GBA1-targeted interventions for PD treatment.
Mutations of the glucocerebrosidase gene, GBA1 (OMIM 606463), are the most important risk factor for Parkinson disease (PD). In vitro and in vivo studies have reported that ambroxol increases ...β-glucocerebrosidase (GCase) enzyme activity and reduces α-synuclein levels. These observations support a potential role for ambroxol therapy in modifying a relevant pathogenetic pathway in PD.
To assess safety, tolerability, cerebrospinal fluid (CSF) penetration, and target engagement of ambroxol therapy with GCase in patients with PD with and without GBA1 mutations.
An escalating dose of oral ambroxol to 1.26 g per day.
This single-center open-label noncontrolled clinical trial was conducted between January 11, 2017, and April 25, 2018, at the Leonard Wolfson Experimental Neuroscience Centre, a dedicated clinical research facility and part of the University College London Queen Square Institute of Neurology in London, United Kingdom. Participants were recruited from established databases at the Royal Free London Hospital and National Hospital for Neurology and Neurosurgery in London. Twenty-four patients with moderate PD were evaluated for eligibility, and 23 entered the study. Of those, 18 patients completed the study; 1 patient was excluded (failed lumbar puncture), and 4 patients withdrew (predominantly lumbar puncture-related complications). All data analyses were performed from November 1 to December 14, 2018.
Primary outcomes at 186 days were the detection of ambroxol in the CSF and a change in CSF GCase activity.
Of the 18 participants (15 men 83.3%; mean SD age, 60.2 9.7 years) who completed the study, 17 (8 with GBA1 mutations and 9 without GBA1 mutations) were included in the primary analysis. Between days 0 and 186, a 156-ng/mL increase in the level of ambroxol in CSF (lower 95% confidence limit, 129 ng/mL; P < .001) was observed. The CSF GCase activity decreased by 19% (0.059 nmol/mL per hour; 95% CI, -0.115 to -0.002; P = .04). The ambroxol therapy was well tolerated, with no serious adverse events. An increase of 50 pg/mL (13%) in the CSF α-synuclein concentration (95% CI, 14-87; P = .01) and an increase of 88 ng/mol (35%) in the CSF GCase protein levels (95% CI, 40-137; P = .002) were observed. Mean (SD) scores on part 3 of the Movement Disorders Society Unified Parkinson Disease Rating Scale decreased (ie, improved) by 6.8 (7.1) points (95% CI, -10.4 to -3.1; P = .001). These changes were observed in patients with and without GBA1 mutations.
The study results suggest that ambroxol therapy was safe and well tolerated; CSF penetration and target engagement of ambroxol were achieved, and CSF α-synuclein levels were increased. Placebo-controlled clinical trials are needed to examine whether ambroxol therapy is associated with changes in the natural progression of PD.
ClinicalTrials.gov identifier: NCT02941822; EudraCT identifier: 2015-002571-24.
Abstract
The discovery of genes involved in familial as well as sporadic forms of Parkinson disease (PD) constitutes an important milestone in understanding this disorder's pathophysiology and ...potential treatment. Among these genes,
GBA1
is one of the most common and well‐studied, but it is still unclear how mutations in
GBA1
translate into an increased risk for developing PD. In this review, we provide an overview of the biochemical and structural relationship between
GBA1
and PD to help understand the recent advances in the development of PD therapies intended to target this pathway.
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Since its first description in the XIV century, the understanding of Parkinson disease (PD) has advanced significantly. However, a considerable part of its pathogenesis remains elusive, and no ...disease modifying therapy has been successfully developed yet and partly because of this lack of knowledge. The genetic background of PD is diverse, accounting for both mendelian, familial and sporadic forms of the disease. One of the main contributors to the genetic risk of PD is the GBA gene, which encodes for a lysosomal enzyme and is also linked to Gaucher disease, an autosomal recessive storage disorder. GBA variants are relatively frequent in sporadic PD, making it a promising target for disease modifying therapies. However, the penetrance of GBA variants is low and mostly unexplained, with only a minority of GBA variants carriers developing PD. In addition, the study of GBA is complicated by difficulties in sequencing the gene, due to the presence of a highly homologous pseudogene (GBAP1) in close proximity to GBA. The aim of this PhD research is to study potential modifiers of risk of PD among GBA variant carriers. First, I collaborated in the development of RAPSODI, an online tool to assess and follow-up a cohort of GBA carriers, both with and without PD, and analysed the data produced, showing interesting differences between GBA carriers and non-carries. The main outcome of this preliminary assessment is that GBA carriers with PD have significantly worse motor and non-motor symptoms compared to GBA-negative PD patients. Further, I refined a method to sequence the GBA gene with Oxford Nanopore's MinION and developed a novel method for detecting reciprocal recombinants between the gene and pseudogene. By applying these tools, I was able to detect complex structural variants that might modify the risk of PD, as well as explore the role of intronic variants in GBA.
The discovery of genes involved in familial as well as sporadic forms of Parkinson disease (PD) constitutes an important milestone in understanding this disorder's pathophysiology and potential ...treatment. Among these genes, GBA1 is one of the most common and well‐studied, but it is still unclear how mutations in GBA1 translate into an increased risk for developing PD. In this review, we provide an overview of the biochemical and structural relationship between GBA1 and PD to help understand the recent advances in the development of PD therapies intended to target this pathway.
The discovery of genes involved in familial as well as sporadic forms of Parkinson disease (PD) constitutes an important milestone in understanding its pathopysiology and potential treatment. Among these genes, GBA1 is one of the most common and well‐studied, but it is still unclear how mutations in GBA1 can translate into an increased risk of developing PD. In this review, we analyzed the literature and tried to give a comprehensive overview of the biochemical and structural relationship between GBA1 and PD to better understand the recent advancements in the therapy of PD.
mutations are a frequent risk factor for Parkinson disease (PD). The aim of this study is to evaluate clinical features in a group of
mutation-positive individuals over a 6-year follow-up.
This is a ...longitudinal study on a cohort of
-positive carriers. We enrolled 31 patients with Gaucher disease type 1 (GD), 29
heterozygous carriers (Het
group) and 30 controls (HC) at baseline and followed them for 6 years. We assessed baseline motor and non-motor signs of PD in all subjects using clinical questionnaires and scales (reduced Unified Multiple System Atrophy Rating Scale (UMSARS), Montreal Cognitive assessment (MoCA), University of Pennsylvania Smell Identification Test (UPSIT), REM Sleep Behavior Disorder screening questionnaire (RBDsq), Movement Disorders Society Unified Parkinson's Disease Rating Scale motor subscale (MDS-UPDRS III) and Beck Depression Inventory (BDI). We repeated these at the 6-year follow-up alongside venous blood sampling for measurement of glucocerebrosidase enzymatic activity (GCase). We explored whether the GCase activity level was altered in leucocytes of these subjects and how it was related to development of PD.
We observed a significant worsening in UMSARS, RBDsq, MDS-UPDRS III and BDI scores at the 6-year follow-up compared with baseline in both the GD and Het
groups. Intergroup comparisons showed that GD subjects had significantly worse scores in UPSIT, UMSARS, MoCA and MDS-UPDRS III than HC, while Het
displayed worse outcomes in UPSIT and MDS-UPDRS III compared with HC. In
mutation-positive individuals (Het
and GD), an UPSIT score of 23 at baseline was correlated with worse outcome at 6 years in UPSIT, MoCA, MDS-UPDRS III and BDI.
In this 6-year-long longitudinal study,
mutation-positive subjects showed a worsening in motor and non-motor prodromal PD features.
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