Micrornas as diagnostic biomarkers and therapeutic agents for small fiber neuropathy

Abstract

The present patent application describes miRNAs in the diagnosis of and as a therapy for small fiber neuropathy (SFN).

Description

[0001] "MicroRNAs as diagnostic biomarkers and therapeutic agents for small fiber neuropathy" DESCRIPTION

[0002] Small Fiber Neuropathy (SEN) is a multifactorial condition which affects the Aƃ and C fibers. The typical clinical presentation comprises symmetrical, length-dependent, and autonomic sensory symptoms, often accompanied by chronic neuropathic pain, which significantly interfere with the patient's daily life1.

[0003] Due to the complexity thereof and the lack of evidence on pathophysiological mechanisms, existing treatment strategies remain limited to symptoms, are inefficient and based on a trial and error approach.

[0004] MicroRNAs (miRNAs) represent potential therapeutic targets in complex diseases, due to the pleiotropic nature thereof and the ability to regulate multiple molecular pathways and mediate intercellular communication.

[0005] To date, preclinical and clinical studies have identified various miRNAs associated with axonal degeneration which influence axonal guidance signals and mediate local post-transcriptional and post-translational changes in the axonal microenvironment2'3; however, the role of each miRNA is strongly dependent on the microenvironment in which it is expressed, especially in multifactorial diseases.

[0006] The publication of Huang Chen et al, "Micro RNAs in autoimmune liver diseases: from diagnosis to potential therapeutic targets", BIOMEDICINE & PHARMACOTHERAPY, ELSEVIER, ER, vol. 130, (2020-08-09)) and international patent application WO 2016 / 144265 describe microRNA-26b-5p and microRNA-20a-5p.

[0007] Abstract

[0008] The inventors of the present patent application have surprisingly identified that specific miRNAs significantly correlate with the degree of degeneration of intraepidermal nerve fibers (IENF) under the pathophysiology of SFN.

[0009] Brief description of the drawings

[0010] Figure 1 shows the design of the study which led to the present invention.

[0011] Figure 2 shows the morphological and molecular profiles in the skin biopsy of SFN patients. (A) Model of cutaneous innervation in the lower limb in a healthy subject (HC) and in an idiopathic SFN patient. The skin biopsies were immunostained with antibodies against phosphoglycolate Phosphatase 9.5 (PGP9.5) and acquired with a bright field microscope. Patients with SFN show a reduction in IENF. (B) The unsupervised heatmap shows the differential miRNA expression profiles between SFN and HC patients, represented as ACq values.

[0012] Figure 3 shows that miR-26b-5p and miR-20a-5p miRNAs are downregulated in the epidermis of SFN patients. A) Box plot of miR- 26b-3p in the exploration and validation cohort showing downregulation in SFN with respect to HC. The comparisons were carried out by applying the Wilcoxon-Mann-Whitney test. The p-values adjusted for BH are shown in the graph; B) and C) Correlation of miR-26b-5p expression and with the delta IENFD values of the subjects recruited in exploration (B) and in the validation step (C). D) Correlation of miR26b-5p and miR20a-5p expression. E) Box plot of miR-20a-5p in the exploration and validation cohort showing downregulation in SEN with respect to HC. The comparisons are carried out by applying the Wilcoxon rank sum test. The unadjusted p-values are reported, as the miRNA did not survive the multiplex test correction analysis F) and G) Spearman's correlation of miR-20a-5p expression and delta IENFD values of subjects recruited in the exploration step (F) and validation step (G). Spearman's correlation coefficients and p-value values are shown in the graphs. H) The ROC analysis shows a significant discrimination capacity for SFN.

[0013] Figure 4 shows the Pearson correlation showing the functional relationships of miR-20a-5p and miR-26b-5p miRNAs with the gene targets thereof.

[0014] Figure 5 shows the in situ hybridization revealing that miR- 26b-5p is primarily expressed in the epidermis.

[0015] Object of the invention

[0016] In a first object, the present invention describes the use of miRNAs in the diagnosis of small fiber neuropathy (SFN).

[0017] In a second object, the miRNAs of the invention are described for medical use in the therapy of small fiber neuropathy (SFN).

[0018] In a third object, the use of the same miRNAs to identify compounds for the therapy of small fiber neuropathy is described.

[0019] Detailed description of the invention

[0020] In accordance with a first object, the present invention describes the use of miRNAs in the diagnosis of small fiber neuropathy (SFN).

[0021] In particular, said miRNAs are:

[0022]

[0023] In a first aspect of the invention, small fiber neuropathy is diagnosed when one or both of the above-indicated two miRNAs are identified and downregulated.

[0024] In a preferred aspect, small fiber neuropathy is diagnosed when miR-26b-5p is downregulated.

[0025] In a more preferred aspect, small fiber neuropathy is diagnosed when at least miR-26b-5p, and possibly also miR-20a-5p is identified and downregulated.

[0026] In particular, the concentration of said miRNAs is measured in an isolated sample represented by the epidermis of a patient suspected of suffering from said disease.

[0027] More in particular, the fact of being down-regulated is determined with reference to a healthy control (HC).

[0028] In a second object, the miRNAs of the invention are described for medical use in the therapy of small fiber neuropathy (SFN).

[0029] To this end, each of the two miRNAs described above can be used in therapy or they can be used together.

[0030] In a third object, the use of the same miRNAs to identify compounds for the therapy of small fiber neuropathy is described. Object of the study and design

[0031] In the present study, the molecular profile of the epidermis associated with severe degeneration of the density of intraepidermal nerve fibers (IENF) was identified. Skin biopsy samples were taken from SFN patients and healthy subjects (HC). MiRNA profiling was performed in two independent steps, exploration (N=13) and validation (N=20), which allowed identifying two miRNAs, closely related to each other, differentially expressed significantly in SFN patients. Gene expression profiling at the mRNA level and correlation analysis with miRNA expression levels was carried out to investigate the effect of the two candidate miRNAs on the target transcripts thereof.

[0032] In-situ hybridization was used to identify the cellular location of the miRNAs (Figure 5). Furthermore, the homogeneous selection of the cohort is a strong element for identifying specific miRNAs for the epidermis and characterizing small fiber neuropathy, which could represent an additional marker in the clinical trial.

[0033] Subject recruitment

[0034] The patients (n=18) were consecutively recruited from the SFN outpatient clinic at the Fondazione IRCCS Istituto Neurologico Carlo Besta (Milan, Italy). The healthy controls (HC, n=15) were recruited under the PAIN-Net project (grant agreement number 721841).

[0035] The inclusion criteria for idiopathic SFN are: presence of pain or sensory disturbances with both a length-dependent and diffuse distribution, reduced degeneration of intraepidermal nerve fiber density, no underlying clinical condition.

[0036] Skin biopsy samples were collected following the standard procedure for the diagnostic evaluation of intraepidermal nerve fiber density5from all enrolled subjects. All the subjects gave written informed consent to participate in the study. Figure 1 shows the study. In particular, skin biopsy samples from two study groups (small fiber neuropathy, SFN and healthy controls, HC) were analyzed in two independent steps; exploration and validation. Two miRNAs were downregulated in patients with small fiber neuropathies and with a synergistic action with each other in SFN patients with respect to HC. The remaining RNA samples (available for 13 SFNs and 10 HCs) were used for gene transcript profiling as miRNA targets and allowed identifying genes expressed in a differentiated manner (DEG) in the SFN patients with respect to the HC group. The experimental miRNA and mRNA data were then used to predict in silico biological processes, gene ontology (GO) terms, and potentially altered pathways in the skin microenvironment in the SFN disease. The functional miRNA-mRNA network in the SFN patients was then reconstructed using experimental and computational data.

[0037] Skin biopsy and assessment of intraepidermal nerve fiber density (IENFD).

[0038] For the evaluation of IENFD, skin biopsies 3 mm in diameter were obtained from all subjects in the distal part of the leg and processed according to the previously published protocol5. Briefly, the samples were fixed in a 2% paraformaldehyde-lysine-periodate solution for 24 hours at 4°C, cryoprotected overnight at 4°C, cut in series with a cryostat into 50 pm sections, and stored at -20°C until further processing. To assess IENFD, the 50 pm sections were immunostained with the antibody against protein gene product 9.5 (1: 500, Ultraclone). IENFD was quantified using a Zeiss brightfield microscope and calculated using the previously published standard5'6. Isolation of RNA from skin biopsy tissue The total RNA was isolated from the epidermis of two 50 pm skin biopsy sections of each subject, sectioned microscopically by dissection, using the TruXtract FFPE total RNA kit (Covaris, cat.no. PN520220) for the exploration cohort and PureLink™ FFPE Total RNA Isolation Kit (Invitrogen, cat.no. K1560-02) for the validation cohort, according to the manufacturer's instructions. RNA purity, concentrations and integrity were measured with the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) prior to the preparation of the miRNA array. All the RNA samples which achieved adequate purity ratios (A260 / A280 = 1.7-2.0) were used for the subsequent analysis.

[0039] Quantification

[0040] The quantification of miRNA expression was carried out using predesigned TaqMan™ Array Human MicroRNA A+B cards (Thermo Fisher) containing 754 miRNAs with pre-amplification, according to the manufacturer's instructions. Briefly, 15 ng of total RNA was reverse transcribed and then the cDNAs were pre-amplified. The preamplification products were diluted in 75 pl of 0.lx TE buffer, pH 8.0 and used for the RT-qPCR reaction. The PCR reaction mixture was prepared using 9 pl of the diluted pre-amplification product, 450 pl of TaqMan™ Fast Advanced Master Mix, and 441 pl of nuclease-free water. Each card tank was loaded with 100 pl of the PCR mixture and centrifuged. The RT-qPCR experiments were performed on ViiATM 7 Fast Real-Time PCR System (Thermo Fisher Scientific). The following cyclization protocol was used: enzymatic activation at 92°C for 10 min, followed by 40 cycles of denaturation at 95°C for 1 sec and annealing at 60°C for 20 sec. The reaction volume of each micro-well was 1 pl.

[0041] Gene expression quantification

[0042] Gene transcript expression was quantified by means of custom TaqMan™ microfluidic card arrays. 95 prioritized gene transcripts were selected for the involvement thereof in neuropathic pain-related pathways. Each custom array was used to analyze 4 subjects at a time. The RNA remaining after the miRNA analysis was reused, carrying out a new quality control. 23 subjects (13 SEN and 10 HC) for which we had enough sample were analyzed. Reverse transcription was carried out with the Invitrogen™ Superscript™ VILO™ cDNA synthesis kit (Thermo Fisher Scientific) in which 50 ng of total RNA was reverse transcribed into cDNA. The pre-amplification step was then carried out for all samples simultaneously. The pre-amplification PGR reaction was carried out under the following thermal cycle conditions: enzyme activation at 95°C for 10 minutes, followed by 14 cycles of denaturation at 95°C for 15 seconds and annealing at 60°C for 4 minutes. The enzyme was inactivated at 99°C for 10 min. The samples were then diluted in IX TE buffer (1:20 dilution factor) and the PGR mixture was prepared. The card tanks were filled with 100 pl of the prepared mixture and run on the ViiATM 7 Fast Real- Time PGR System (Thermo Fisher Scientific) with the following thermal protocol: enzyme activation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing at 60°C for 1 sec. The reaction volume of each micro-well was 1 pl.

[0043] Relative threshold method The "relative threshold" (Crt) method was used, proven by the manufacturer to be the most robust method for analyzing the data7. All the raw PCR data was imported in real-time into the DataConnect cloud and the automatic CRT threshold was applied in the design and analysis software (DA2) (Thermo Fisher Scientific, online version). Only miRNAs with good amplification quality (Ampscore > 1 and Cqconf > 0.8) were therefore included in the analysis.

[0044] NormFinder analysis

[0045] The accurate determination of relative miRNA levels requires normalization using a reference or endogenous control which ideally should be constant, stable, unregulated, and unaffected by experimental conditions. For this reason, it must be selected considering the quality and stability thereof in the sample of interest and in the groups studied. For the selection of the endogenous miRNA for both the exploration and validation step, the NormFinder software was used in both pools (A+B). In the exploration step, normalization was carried out with hsa-miR-200b-002251 for pool A and hsa-miR-1201-002781 for pool B, respectively. In the validation step, the miRNAs used as reference were hsa-miR-320- 002277 (poolA) and hsa-miR-1247-002893 (poolB). The non-human exogenous ath-miR159a acted as a negative control.

[0046] Relative expression analysis

[0047] The differential expression of the miRNAs was quantified as relative quantification (QR) by the 2-AAct8approach with the HC tissue used as reference group. AACt was calculated as the average of the ACt values for the miRNA of interest in the interest group - the average of the ACt values for the miRNAs of interest in the reference group. Thus, the fold change in the expression was calculated as 2_(AAcq). In-silico analysis of the target genes

[0048] To identify the genes which represent the potential targets of the differentially expressed miRNAs, an in-silico prediction analysis was carried out by fully retrieving the information from the miRTarBase, TargetScan and Tarbase resources. Furthermore, the ClueGO app (v2.5.8) of Cytoscape 3.9.19and DAVID10software were used to identify the pathways and enriched biological processes from lists of target genes or genes with known involvement in axon degeneration and maintenance. miRNAscope and immunofluorescence analysis

[0049] MicroRNA in situ hybridization (ISH) by miRNAscope™ LS Reagent Kit red (Advanced Cell Diagnostics, Hayward, CA, USA) was carried out to localize miR-26b-5p in skin biopsy tissue. The ISH was carried out following the kit guidelines. In short, on the first day, the adhesion of the tissues was carried out according to the manufacturer's instructions and the hydrophobic barrier was created. The slides were allowed to dry overnight and on the second day ISH was carried out using a human-specific miRNA probe (miR-26b-5p). The fluorescent probe was then incubated to identify the cell nuclei (DARI, Invitrogen, cat.no. D1306).

[0050] Statistical analysis

[0051] The associations between miRNA levels and clinical phenotype (SFN versus HC) were analyzed using the Wilcoxon-Mann-Whitney test. The FDR Benjamini-Hochberg method was applied as a correction for multiplex testing. The correlation analysis of the expression of the candidate miRNAs with that of the putative target genes was carried out by applying the Spearman correlation test considering coefficients h 0.5 or <-0.5 and p <0.05 as statistically significant. The Receiver Operating Characteristic (ROC) curve and area under the curve (AUC) were calculated to assess the ability of each miRNA to discriminate between SFN and HC patients in the validation step. To explore the diagnostic accuracy of combining multiple miRNAs, a combined ROC curve was calculated by multiple logistic regression analysis.

[0052] The statistical analysis was completed using the statistical programming language R, version 3.6 and STATA11 software.

[0053] Results

[0054] Exploration and validation of new candidate epidermis miRNAs associated with SFN

[0055] Study design and samples

[0056] To explore the molecular profile of the epidermis of SFN with respect to HC patients, an integrated analysis of miRNA and mRNA expression was carried out (Fig. 1). The study included an initial exploration cohort of 6 patients with idiopathic SFN and 7 HC analyzed with TaqMan Human MicroRNA cards containing 754 miRNAs. To further confirm the miRNA profile results, an independent validation cohort of 11 SFN and 8 HC patients applying the same methodology was included. Furthermore, an analysis of the mRNA expression of 95 gene targets, involved in the pathways related to neuropathic pain, was carried out in 13 SFN and 10 HC patients for whom biological material was still available. The demographic and clinical features of the study groups are presented in Table 1.

[0057] The SFN and HC patients showed no significant differences in terms of sex (p = 0.59 exploration step, p = 0.55 validation step) and age (p = 1 exploration step, p = 0.20 validation step).

[0058] All the subjects underwent skin biopsy sampling used for the evaluation of IENFD, which was significantly reduced in the patients (Table 1 and Figure 2A). Only patients with a clear diagnosis of SFN were included in the study.

[0059]

[0060] ["Eta" = Age;

[0061] "Sesso" = Sex;

[0062] "Distribuzione" = Distribution;

[0063] "Allodinia meccanica" = Mechanical allodynia ; iperalgesia da puntura di spillo" = pinprick hyperalgesia;

[0064] "Dolore (sintomi)" = Pain (symptoms);

[0065] "Prurito (sintomi)" = Itch (symptoms);

[0066] "Evocato" = Evoked;

[0067] "Iperalgesia termica" = Thermal hyperalgesia;

[0068] "Disfunzione vasomotoria / sudomotoria (segni)" =

[0069] Vasomotor / sudomotor dysfunction (signs)";

[0070] "Profilo sensoriale" = Sensory profile;

[0071] "Perdita sensoriale (segni)" = Sensory loss (signs);

[0072] "Dolore profondo" = Deep pain;

[0073] "Freddo doloroso" = Painful cold;

[0074] "Bruciore dolorante" = Painful burning;

[0075] "Bruciante" = Burning;

[0076] "Pungente parossistico" = Paroxysmal stinging]

[0077] Table 1. Demographic and clinical features of the studied population. The patients SEN 1-SFN 6 were analyzed in the exploration step, while the patients SEN 7-SFN 18 were included in the validation step. IENFD, intraepidermal nerve fiber density; LD, length-dependent pain distribution; N-LD, non-length-dependent pain distribution; NRS, numeric pain rating scale; Sensory profile: TH, thermal hyperalgesia; MH, mechanical hyperalgesia; SL, sensory loss.

[0078] MiRNA profiling: exploration step

[0079] The microfluidic array contains 754 miRNAs which were evaluated in all the samples. After quality filtering, the expression data of 111 miRNAs present in more than 90% of the samples were analyzed by applying the relative expression method11. The altered levels of 20 miRNAs which survived multiplex correction were observed in the epidermis of 6 SEN patients with respect to 7 HC subjects, showing 13 down-regulated and 7 up-regulated miRNAs capable of strongly discriminating phenotypic groups (Figure 2B). MiRNA profiling: validation step

[0080] A validation study was then carried out applying the same profiling approach in the skin biopsy in an independent cohort of 12 SFN and 8 HC patients (Table 1 and Figure 1). It was confirmed that miR-26b- 5p is significantly differentially expressed (adjusted p-value BH = 3.28E-02, Foldchange = -2.55), confirming the same down-regulation pattern in SFN patients in both stages (Figure 3A).

[0081] To study the relationship between miR-26b-5p expression and innervation, Spearman's analysis was carried out which revealed a strong correlation between miR-26b-5p and denervation levels calculated as the difference between the measured IENFD and the cutoff value (deltalENFD), in both exploration (r=0.77, p=0.0053) and validation (r=0.63, p=0.0055) steps (Figure 3B-C).

[0082] Considering that miRNAs act in synergy, our research was extended to other miRNA candidates which did not survive the statistical correction, to highlight a possible relationship with the miR-26-5p candidate. Expression correlation analysis was carried out and a strong linear relationship was observed with miR-20a-5p (p-value= 4.7e-07, R=0.88) (Figure 3D). miR-20a-5p had a downregulation trend in SFN patients in both the exploration (HR= -1.48, unadjusted p- value = 4.80 E-02) and validation step (HR=-2.08, unadjusted p-value = 7.18 E-03) (Figure 3E) which also shows a strong correlation with delta IENFD in both the exploration (r=0.62, p=0.037) and validation steps (r=0.66, p=0.0031) (Figure 3F-G). miR-26b-5p and miR-20a-3p in the epidermis show strong discriminatory capacity for SFN To test the discriminatory capacity of two miRNA candidates for SFN, a ROC analysis was carried out and the area under the curve (AUG) calculated. miR-26b-5p reached an AUG of 96.4% with a sensitivity of 91.7% and a specificity of 85.7%, while miR-20a-5p showed an AUG value of 86.9% with sensitivity of 91.7% and specificity of 81.4% (Figure 3H).

[0083] Identification and validation of miRNA-targets

[0084] To further investigate the potential role of miR-26b-5p and miR-20a- 5p, in silico prediction analysis of target transcripts was carried out by querying three databases (miRTarBase, Tarbase, TargetScan). The identified genes were then used for the enrichment of the biological pathways and processes (BP). Significantly enriched terms (p <0.05), considered plausible for neuronal development and maintenance, neuronal microenvironment homeostasis, and neurotrophic factor signaling, were selected.

[0085] The gene expression analysis was carried out using a customized panel of pain-related genes. Significantly dysregulated expression of 8 genes was observed (Table 2A): four down-regulated (IKBKAP, MKNK2, PTGER3 and EDN1) and four up-regulated (MEF2C, SLC25A36, NTRK2 and PIK3CG).

[0086] Table 2A Gene p-value BH FoldChange(FCJ

[0087] IKBKAP %24E-05 3,78E-03 -2,19

[0088] MKNK2 M 7E-04 4,07E-03 -1,59

[0089] MEF2C 1J0E-04 4,07E-03 1,85

[0090] PTGER3 8,62E-04 l,55E-02 -1,73

[0091] SLC25A36 1,15E-O3 l,65E-02 1,70

[0092] NTRK2 4,12E-03 4,23E-02 2,32

[0093] PIK3CG 4,12E-03 4,23E-02 1,84

[0094] EDN1 5,18E-03 4,66E-02 -1,49

[0095] The gene expression analysis revealed altered epidermal expression for 8 genes.

[0096] Paired Pearson correlation was used to identify the functional relationships between miRNA and target mRNA expression. The correlation analysis was applied to each miRNA and to the putative target thereof, which was significantly altered in the SFN patients (Table 2A and 2B and Fig. 4). Our analysis showed correlations between the expression values of IKBKAP, miR-26b-5p (r=-0.75) and miR-20a-5p (r=-0.62), miR-26b-5p (r=0.75) and miR-20a-5p (r=0.67) significantly correlated with MEF2C, both miRNAs correlated with MKNK2, miR-20a-5p (r=-0.52), miR-26b-5p (r=-0.53); PIK3CG, miR-20a- 5p (r=0.53), miR-26b-5p (r=0.53) and SLC25A36, miR-20a-5p (r=0.48), miR-26b-5p (r=0.56) while miR-20a-5p expression correlated with

[0097] NTRK2 (r=0.59).

[0098] Table 2B

[0099] Gene annotation analysis

[0100] To identify the biological terms associated with the dysregulated genes, gene annotation analysis was carried out in silico. The dysregulated genes which survived the Benjamini-Hochberg correction were imported into the software and the most plausible terms were selected among the associated terms (pathways and biological processes), considering the tissue analyzed.

[0101] In situ hybridization of miR-26b

[0102] Our miRNA profiling provided a novel molecule which could carry out a regulatory role in the development of SFN; to explore its cellular location, the miRNAscope in situ hybridization assay, known as a highly sensitive technology for miRNA detection, was carried out. With this experiment, we demonstrate that miR-26b-5p is highly concentrated in the human epidermis (Fig. 5). From the above, the advantages of the present invention will be immediately apparent.

[0103] In particular, the role of miR-26b-5p and miR-20a-5p in discriminating patients with Small Fiber Neuropathy from healthy subjects was confirmed, with a predictive capacity which, in the ROC analysis, showed diagnostic accuracy with AUG 96.4% and 86.9%, respectively.

[0104] The strong correlation observed between innervation density (delta IENFD) and miR-26b-5p or miR-20a-5p expression supports the hypothesis of miRNA involvement in intraepidermal fiber maintenance, indicating that the significant down-regulation of the two miRNAs is in line with decreased cutaneous innervation.

[0105] The role of these two miRNAs also provides an important basis for the development of prevention strategies and drug therapy.

[0106] References

[0107] 1. Devigili, G., Cazzato, D. & Lauria, G. Clinical diagnosis and management of small fiber neuropathy: an update on best practice. Expert Rev Neurother 20, 967-980 (2020). https: / / doi .org:10.1080 / 14737175.2020.1794825

[0108] 2. Wang, B. & Bao, L. Axonal microRNAs: localization, function and regulatory mechanism during axon development. J Mol Cell Biol 9, 82- 90 (2017). https: / / doi.org:10.1093 / jmcb / mjw050

[0109] 3. Maimon, R. et al. miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non-Cell-Autonomous Mechanism in ALS. J Neurosci 38, 5478-5494 (2018). https: / / doi .org:10.1523 / JNEUROSCI.3037-17.2018

[0110] 4. Chakraborty, C., Sharma, A. R., Sharma, G. & Lee, S. S. Therapeutic advances of miRNAs: A preclinical and clinical update. J Adv Res 28, 127-138 (2021). https: / / doi.org:10.1016 / j.jare.2020.08.012

[0111] 5. Lauria, G. et al. Intraepidermal nerve fiber density at the distal leg: a worldwide normative reference study. J Peripher Nerv Syst 15, 202-207 (2010). https : / / doi.org:10.1111 / j.1529- 8027.2010.00271.x

[0112] 6. Lauria, G. & Devigili, G. Skin biopsy as a diagnostic tool in peripheral neuropathy. Nat Clin Pract Neurol 3, 546-557 (2007). https: / / doi.org:10.1038 / ncpneuro0 630

[0113] 7. Biosystems, A.

[0114] 8. Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3, 1101-1108 (2008). https: / / doi.org:10.1038 / nprot.2008.73 9. Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13, 2498-2504 (2003). https: / / doi.org:10.1101 / gr.1239303

[0115] 10. Huang, d. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4, 44-57 (2009). https: / / doi.org:10.1038 / nprot.2008.211

[0116] 11. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) Method. Methods 25, 402-408 (2001). https: / / doi.org:10.1006 / meth.2001.1262

Claims

CLAIMS1. Use of miRNAs in the diagnosis of small fiber neuropathy(SFN) in a patient, wherein said miRNAs are one or both of:

2. Use of miRNAs in the diagnosis of small fiber neuropathy (SFN) in a patient according to the preceding claim, wherein one or both of said miRNAs are downregulated with reference to a healthy control.

3. Use of miRNAs in the diagnosis of small fiber neuropathy (SFN) in a patient according to the preceding claim 1 or 2, wherein the concentration of said miRNAs is measured in an isolated epidermis sample of said patient.

4. MiRNAs for medical use in the therapy of small fiber neuropathy (SFN) in a patient, wherein said miRNAs are one or both of:

5. Use of miRNAs for identifying therapeutic compounds for the therapy of small fiber neuropathy (SFN) in a patient, wherein said miRNAs are one or both of:

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