Skin α-Synuclein Seeding Activity in Patients with Type 1 Gaucher Disease

Abstract

Background

Patients with Type 1 Gaucher disease (GD1) have a significantly increased risk of developing PD.

Objectives

To evaluate skin α-synuclein (αSyn) seeding activity as a biomarker for GD1-related PD (GD1-PD).

Methods

This single-center study administered motor and cognitive examinations and questionnaires of non-motor symptoms to adult patients with GD1. Optional skin biopsy was performed for skin αSyn seeding amplification assay (αSyn SAA) using real-time quaking-induced conversion (RT-QuIC) assay.

Results

Forty-nine patients were enrolled, and thirty-six underwent skin biopsy. Two study participants had PD. Ten participants were αSyn SAA positive (27.8%), 7 (19.4%) were intermediate, and 19 (52.8%) were negative. Positive αSyn seeding activity was observed in the single GD1-PD case who consented to biopsy. αSyn SAA positivity was associated with older age (p = 0.043), although αSyn SAA positivity was more prevalent in GD1 patients than historic controls.

Conclusions

Longitudinal follow is required to determine if skin αSyn seeding activity can be an early biomarker for GD1-PD.

Introduction

Type 1 Gaucher disease (GD1), an autosomal recessive disorder caused by biallelic pathogenic variants in the beta-glucocerebrosidase gene (GBA1), is characterized by deficient activity of the enzyme glucocerebrosidase (GCase).¹ The resultant accumulation of the glycolipid glucocerebroside and its deacetylated lysolipid, glucosylsphingosine, in the lysosomes of the monocytes and macrophages leads to chronic inflammation and multisystemic manifestations including cytopenia, hepatosplenomegaly and skeletal disease.^1,2

It is well established that pathogenic variants in GBA1 are the most common genetic risk factors for Parkinson disease (PD).³ The incidence of PD in patients with GD1 is 6 to 17 times higher than the general population.^4,5 While PD risk is higher in GD1 patients than non-carriers, most GD1 patients will never develop PD, and which patients will develop PD remains unknown. PD patients with pathogenic variants in GBA1 present earlier, with more predominant non-motor symptoms, such as sleep-behavior disorders, autonomic symptoms, cognitive changes, and psychiatric disturbances, as well as more severe motor symptoms.^6–8

Spinal fluid studies demonstrate that α-synuclein seed amplification assay (αSyn SAA) is a sensitive and specific assay for PD, and proof-of-concept studies suggest that skin αSyn seeding activity, detected by αSyn SAA, may serve as a novel biomarker for diagnosis of PD and other synucleinopathies.^14–18 Here, we aimed to evaluate skin αSyn seeding activity as a biomarker for GD1-related PD (GD1-PD).

Subjects and Methods

All study procedures were approved by the Icahn School of Medicine at Mount Sinai Institutional Review Board (IRB 21–01385). Adult subjects ≥40 years old with a confirmed genetic and biochemical diagnosis of GD1 were enrolled with written informed consent. An optional skin biopsy was performed in patients who agreed. Medical records were reviewed to collect patient demographics, GD-related medical and treatment history, laboratory studies, and genotype. Study subjects were assessed with validated tools, including the MDS-Sponsored Revision of the UPDRS (MDS-UPDRS Parts I, II, and IV), Montreal Cognitive Assessment (MoCA), 1-Question REM Sleep Behavior Disorder Screen (RBDQ1), and SCales for Outcomes in PArkinson’s disease - AUTonomic Dysfunction (SCOPA-AUT). Participants underwent a neurological exam per the MDS-UPDRS Part III, which was video-recorded. This was analyzed by a movement disorder specialist who was blinded to the subjects’ medical history (beyond a diagnosis of GD), the skin αSyn SAA result and remainder of study assessments. Results of assessments were recorded in a REDCap database.

The “abnormal” category of PD screening was created a priori (before analyzing skin αSyn SAA results) based on the following criteria: 1) 7+ total score on the MDS-UPDRS Part 3 , or 2) 3–6 total score of Part 3 and at least one of the criteria—an abnormal score on MoCA (under 26), RBDQ1 (score=1), SCOPA-AUT (above 10), and/or tremor or freezing question from Part 2—was met.

Participants who consented to the procedure underwent punch biopsy from the anterior forearm for skin αSyn SAA using the blinded real-time quaking-induced conversion (RT-QuIC) assay as previously described.¹⁴ The endpoint fluorescence readings were analyzed with GraphPad Prism version 10.0. Student’s t-test and one-way ANOVA were used for comparison between 2 or 3 groups, respectively. The cut-off value of 76241 RFU (at around 30% of the maximum of fluorescence) for endpoint fluorescence was established based on the Receiver Operating Characteristic (ROC) analysis of positive controls (PD patients) and negative controls (non-neurodegenerative individuals) from a historical study.¹⁹ In other analyses, samples exhibiting ThT fluorescence lower than this threshold were considered to be αSyn SAA negative, values between the threshold to 35% of the maximum (91,000 RFU) were considered to be αSyn SAA intermediate, while those exceeding 91,000 RFU of the fluorescence reading were considered to be αSyn SAA positive.

Descriptive statistics using mean (SD), median, and range for continuous variables, frequencies, and percentages for categorical variables were reported. The Goodman method was used to calculate confidence intervals (CI) for the proportions of αSyn SAA result categories. To identify variables associated with αSyn seeding activity, Kruskal-Wallis rank sum tests were used for continuous variables, and Fisher’s exact tests were used for categorical variables. All analyses were done in R version 4.3.1 (The R Foundation for Statistical Computer).

Results

Fifty-one GD-1 patients were enrolled in the study. Two were excluded from final analysis due to withdrawal from the study and failure to complete the majority of study procedures, respectively. Study participants (mean age 59.3 years, 57.1% male) included 2 with clinically confirmed PD. The majority of participants were p.N409S homozygotes (67.3%) and 44 were receiving treatment for GD1 (see Table 1). Most subjects in the cohort (71.6%) did not have abnormal PD screening results, with only 14% being abnormal.

Table 1:

Subject Characteristics

Subject Characteristics	Total (n=49)
Male sex, n (%)	28 (57.1)
Mean age, years	59.3
Confirmed diagnosis of PD, n (%)	2 (4.1)
Patients on Treatment, n (%)	44 (89.8)
Treatment type
Imiglucerase, n (%)	18 (40.9)
Velaglucerase alfa, n (%)	17 (38.6)
Eliglustat, n (%)	9 (20.5)
Genotype
N409S/N409S, n (%)	33 (67.3)
N409S/84GG, n (%)	9 (18.4)
N409S/V394L, n (%)	2 (4.1)
N409S/L483P, n (%)	1 (2.0)
N409S/IVS2+1, n (%)	1 (2.0)
N409S/R535H, n (%)	1 (2.0)
R502C/R502C, n (%)	1 (2.0)
R535H/IVS2+1, n (%)	1 (2.0)

Thirty-six subjects consented to a skin biopsy (73.5%). Results of the clinical assessments were similar between the entire cohort and the skin biopsy group (see supplemental materials, Table 3). Ten participants had positive skin αSyn seeding activity (27.8%; 95% CI: [13.7%, 48.2%]), 7 (19.4%; 95% CI: [8.2%, 39.4%]) had intermediate αSyn seeding activity, and 19 (52.8%; 95% CI: [33.5%, 71.2%]) had low αSyn seeding activity, thus were αSyn SAA negative. The single GD1-PD case who consented to the biopsy was αSyn SAA positive. Positive αSyn seeding activity was associated with older age (p = 0.043). Age at GD1 diagnosis, treatment status, mode of treatment, GD1 biomarkers, GBA1 genotype severity, RBDQ1, SCOPA-AUT score, or MOCA score did not significantly correlate with skin αSyn SAA results (see supplemental materials, Figures 1 and 2).

Of the participants with positive skin αSyn seeding activity, 4 (40%) had abnormal PD screening results (see criteria above) and 6 (60%) did not have abnormal screening results (see Table 2). Of those with intermediate αSyn seeding activity, 3 (42.9%) had abnormal PD screening results while 4 (57.1%) did not. Of those with low αSyn seeding activity, 5 (26.3%) had abnormal PD screening results, whereas 14 (73.7%) did not.

Table 2:

Composite PD Screening Result^* vs. Skin αSyn SAA Result in Biopsy Patients

Skin αSyn SAA Result	Not Abnormal (n=24)	Abnormal (n=12)	Overall (n=36)
Positive, n (%)	6 (25)	4 (33)	10 (28)
Intermediate, n (%)	4 (17)	3 (25)	7 (19)
Negative, n (%)	14 (58)	5 (42)	19 (53)

^*Analysis of MDS-UPDRS Part 3 alongside RBDQ1, MoCA, SCOPA-AUT, and response to tremor or freezing questions from the MDS-UPDRS Part 2 (see methods above).

Discussion

There is an urgent need to develop biomarkers to identify patients at risk for PD. Skin αSyn SAA has been proposed as a highly sensitive and specific diagnostic test for PD,¹⁴ with the presence of αSyn seeding activity being detected even in the prodromal stages of disease.^17,18 In this study, we aimed to evaluate GD1 patients for motor and non-motor signs of PD and correlate our findings with skin αSyn SAA positivity. While similar evaluations have been performed in GBA1 heterozygotes,^16,20 this study is the first to perform comprehensive clinical assessments and skin αSyn SAA in GD1 patients.

The majority of GD1 patients in our cohort did not have abnormal clinical assessments when analyzed as a composite score which included results of the MDS-UPDRS Part 3, MoCA, RBDQ1, SCOPA-AUT, and the tremor or freezing question from the MDS-UPDRS Part 2. There was a statistically significant association between age and αSyn SAA positivity. Given this correlation with age, it is conceivable that the weakly positive cases, or those falling into the intermediate category, could represent the early stages of PD. Therefore, it stands to reason that there is value in establishing a group for these cases, situated between “positive” and “negative.” No significant correlation was identified between skin αSyn SAA results and GD1 disease history or composite results of the clinical assessments performed. As expected, the baseline prevalence of positive skin αSyn seeding activity among GD1 patients in our cohort was higher than historical controls (12.5%), but lower than PD patients previously reported (88.24%) (see supplemental materials, Figure 3).²¹ This observation must be interpreted with caution, as it is possible that the pathological mechanism leading to GD1-PD differs from that of sporadic PD and thus impacts baseline skin αSyn seeding activity.

While the mechanism to explain the correlation between GD1 and PD continues to evolve, it has been demonstrated that deficient GCase activity results in increased α-synuclein aggregation, and in a bidirectional loop, α-synuclein aggregation further suppresses GCase activity.^9–11,13 This relationship has been documented in sporadic PD as well, with decreased GCase activity in the substantia nigra and cerebellum of the brains of patients with PD and without pathogenic variants in GBA1.^22,23 Moreover, GCase activity in neurons has been shown to decline with age in healthy adults, thus lowering the threshold to develop PD.^13,24 Thus, the association between positive skin αSyn seeding activity and age is not unexpected. While the prevalence of skin αSyn SAA positivity among our cohort is higher than the incidence of GD1-PD, it is possible that there is a unique threshold at which clinical symptoms of GD1-PD occur.

Our cohort was relatively small, with diverse GBA genotypes (though almost all participants had at least one copy of the N409S mutation), and only two had a clinical diagnosis of PD. Other genetic risk factors for PD and family history were not captured. Study procedures took place at the peak of the COVID-19 pandemic. Therefore, participants were wearing masks intermittently throughout their exams, limiting the remote expert’s assessment of facial expression. Although olfactory dysfunction is a common prodromal feature of PD and GD1-PD, olfaction was not assessed in this study, as hyposmia and anosmia were known features of COVID-19 infection.^7,25–27 Neurological assessments were recorded on video for review remotely, but as a result, some exam components (ie, assessing cogwheel rigidity), were not included in the MDS-UPDRS Part 3 score. Skin biopsy was collected from the anterior forearm, which may have impacted results. Previous studies have included samples obtained from the posterior occipital region of the scalp and cervical spine (C7, in particular), and shown higher αSyn SAA sensitivity.^15,18 However in order to maximize recruitment and ease of sample attainment, a more peripheral site was chosen.

It remains unknown if the GD1 patients with positive skin αSyn seeding activity will advance to clinical PD. Therefore, longitudinal follow-up of this cohort is required. Until more is known about the utility of this or other biomarkers, physicians should proactively screen their GD1 patients for PD with thorough clinical assessments and refer to movement disorder specialists as necessary. With potential disease modifying therapies in clinical development,²⁸ early identification of patients is key to improving therapeutic outcomes.