A comparative blind study between skin biopsy and seed amplification assay to disclose pathological α-synuclein in RBD.

To compare the diagnostic accuracy of the immunofluorescence (IF) technique and aSyn-seed amplification assay (aSyn-SAA) of skin and cerebrospinal fluid (CSF) in disclosing pathological α-syn in idiopathic idiopathic REM sleep behavior disorder (iRBD) as early phase of a synucleinopathy. We prospectively recruited 41 patients with iRBD and 40 matched clinical controls including RBD associated with type 1 Narcolepsy (RBD-NT1, 21 patients), iatrogenic causes (2 pt) or OSAS (6 pt) and 11 patients with peripheral neuropathies. IF from samples taken by skin biopsy and aSyn-SAA from skin and CSF samples were analysed blinded to the clinical diagnosis. IF showed a good diagnostic accuracy (89%) that was lower in the case of skin and CSF-based aSyn-SAA (70% and 69%, respectively) because of a lower sensitivity and specificity. However, IF showed a significant agreement with CSF aSyn-SAA. In conclusion, our data may favor the use of skin biopsy and aSyn-SAA as diagnostic tools for a synucleinopathy in iRBD.

Consistent skin α-synuclein positivity in REM sleep behavior disorder - A two center two-to-four-year follow-up study.

OBJECTIVE/METHODS: Phosphorylated alpha-synuclein (p-syn) in dermal nerves of patients with isolated REM sleep behavior disorder (iRBD) is detectable by immunofluorescence-labeling. Skin-biopsy-p-syn-positivity was recently postulated to be a prodromal marker of Parkinson's disease (PD) or related synucleinopathies. Here, we provide two-to four-year clinical and skin biopsy follow-up data of 33 iRBD patients, whose skin biopsy findings at baseline were reported in 2017. RESULTS: Follow-up biopsies were available from 25 patients (18 positive at baseline) and showed consistent findings over time in 24 patients. One patient converted from skin-biopsy-negativity to -positivity. P-syn-positivity was observed in iRBD patients who still had a normal FP-CIT-SPECT two years later. Clinically, five of the 23 at baseline skin-biopsy-positive patients (21.7%) had converted to PD or dementia with Lewy bodies at follow-up, but none of the skin-biopsy-negative patients. CONCLUSIONS: Dermal p-syn in iRBD is most probably an early consistent marker of synucleinopathy and may support other indicators of conversion to manifest disease state.

Abnormal α-synuclein deposits in skin nerves: intra- and inter-laboratory reproducibility.

BACKGROUND AND PURPOSE: Visualization of phosphorylated α-synuclein at serine 129 (p-syn) in skin nerves is a promising test for the in vivo diagnosis of synucleinopathies. Here the aim was to establish the intra- and inter-laboratory reproducibility of measurement of intraneural p-syn immunoreactivity in two laboratories with major expertise (Würzburg and Bologna). METHODS: In total, 43 patients affected by Parkinson's disease (PD 21 patients), dementia with Lewy bodies (DLB 1), rapid eye movement sleep behaviour disorder (RBD 11), multiple system atrophy (MSA-P 4) and small fibre neuropathy (SFN 6) were enrolled. Skin biopsy was performed at the C7 paravertebral spine region and distal skin sites (thigh or leg). The analysis was standardized in both laboratories and carried out blinded on a single skin section double stained with antibodies to p-syn and the pan-axonal marker protein gene product 9.5. Fifty skin sections were randomly selected for the analysis: 25 from C7 and 25 from distal sites. Differently classified sections were re-evaluated to understand the reasons for the discrepancy. RESULTS: The intra-laboratory analysis showed an excellent reproducibility both in Würzburg (concordance of classification 100% of sections; K = 1; P < 0.001) and Bologna (96% of sections; K = 0.92; P < 0.001). Inter-laboratory analysis showed reproducibility in 45 sections (90%; K = 0.8; P < 0.001) and a different classification in five sections, which was mainly due to fragmented skin samples or weak fluorescent signals. CONCLUSIONS: Analysis of p-syn showed excellent inter- and intra-laboratory reproducibility supporting the reliability of this technique. The few ascertained discordances were important to further improve the standardization of this technique.

Skin α-synuclein deposits differ in clinical variants of synucleinopathy: an in vivo study.

We aimed to characterize in vivo α-synuclein (α-syn) aggregates in skin nerves to ascertain: 1) the optimal marker to identify them; 2) possible differences between synucleinopathies that may justify the clinical variability. We studied multiple skin nerve α-syn deposits in 44 patients with synucleinopathy: 15 idiopathic Parkinson's disease (IPD), 12 dementia with Lewy Bodies (DLB), 5 pure autonomic failure (PAF) and 12 multiple system atrophy (MSA). Ten healthy subjects were used as controls. Antibodies against native α-syn, C-terminal α-syn epitopes such as phosphorylation at serine 129 (p-syn) and to conformation-specific for α-syn mature amyloid fibrils (syn-F1) were used. We found that p-syn showed the highest sensitivity and specificity in disclosing skin α-syn deposits. In MSA abnormal deposits were only found in somatic fibers mainly at distal sites differently from PAF, IPD and DLB displaying α-syn deposits in autonomic fibers mainly at proximal sites. PAF and DLB showed the highest p-syn load with a widespread involvement of autonomic skin nerve fibers. IN CONCLUSION: 1) p-syn in skin nerves was the optimal marker for the in vivo diagnosis of synucleinopathies; 2) the localization and load differences of aggregates may help to identify specific diagnostic traits and support a different pathogenesis among synucleinopathies.

Post-ganglionic autonomic neuropathy associated with anti-glutamic acid decarboxylase antibodies.

PURPOSE: Antibodies to glutamic acid decarboxylase (GAD-Abs) have been associated with several conditions, rarely involving the autonomic nervous system. Here, we describe two patients complaining of autonomic symptoms in whom a post-ganglionic autonomic neuropathy has been demonstrated in association with significantly elevated serum and CSF GAD-Abs levels. METHODS: Patients underwent nerve conduction studies, sympathetic skin response testing, evaluation of autonomic control of the cardiovascular system and skin biopsy. Also, serum screening to exclude predisposing causes of peripheral neuropathy was performed. Anti-GAD65 antibodies were evaluated in serum and CSF. RESULTS: GAD-Abs titer was increased in both serum and CSF in both patients. Sympathetic skin response was absent and skin biopsy revealed a non-length-dependent small-fiber neuropathy with sympathetic cholinergic and adrenergic post-ganglionic damage in both patients. Nerve conduction studies and evaluation of autonomic control of the cardiovascular system were normal in both patients. Both patients were treated with steroids with good, but partial, (patient 2) recovery of the autonomic dysfunctions. CONCLUSIONS: Although the pathophysiological mechanisms involved are not fully defined, GAD-abs positivity in serum and CSF should be searched in patients with autonomic neuropathy when no other acquired causes are evident. This positivity may help to clarify autoimmune etiology and, subsequently, to consider immunomodulatory treatment.

Skin biopsy and microneurography disclose selective noradrenergic dysfunction due to dopamine-ß-hydroxylase deficiency.

Skin biopsy and microneurography are autonomic tests directly evaluating adrenergic and cholinergic sympathetic fibers to identify selective deficiency of a specific peripheral sympathetic subdivision. We describe a patient with tomacular neuropathy due to a deletion of the PMP22 gene who complained of chronic orthostatic hypotension due to a dopamine-ß-hydroxylase deficiency confirmed by genetic analysis demonstrating two novel mutations in the DßH gene. To further characterize autonomic dysfunctions the proband underwent skin biopsy and microneurography. These tests disclosed a selective peripheral adrenergic dysfunction demonstrating the possibility to ascertain DßH deficiency. In conclusion, skin biopsy and microneurography may help to increase the diagnosis of this peculiar disorder particularly when routine autonomic nervous system tests show uncertain results.