DOI: 10.5061/dryad.hqbzkh1st
title: Data from: Schwann cell transcript biomarkers for hereditary neuropathy skin biopsies

description: Charcot-Marie-Tooth (CMT) disease is most commonly caused by duplication
 of a chromosomal segment surrounding Peripheral Myelin Protein 22, or the
 PMP22 gene, which is classified as CMT1A. Several candidate therapies
 reduce Pmp22 mRNA levels in CMT1A rodent models, but the development of
 biomarkers for clinical trials in CMT1A is a challenge, given its slow
 progression and difficulty in obtaining nerve samples. Quantitative PCR
 measurements of PMP22 mRNA in dermal nerves were performed using skin
 biopsies in human clinical trials for CMT1A, but this approach did not
 show increased PMP22 mRNA in CMT1A patients compared to controls. One
 complicating factor is the variable amounts of Schwann cells (SCs) in the
 skin. The objective of the study was to develop a novel method for precise
 evaluation of PMP22 levels in skin biopsies that can discriminate CMT1A
 patients from controls. We have developed methods to normalize PMP22
 transcript levels to SC-specific genes that are not altered by CMT1A
 status. Several CMT1A-associated genes were assembled into a custom
 Nanostring panel to enable precise transcript measurements that can be
 normalized to variable SC content. The digital expression data from
 Nanostring analysis showed reproducible elevation of PMP22 levels in CMT1A
 versus control skin biopsies, particularly after normalization to
 SC-specific genes. This platform should be useful in clinical trials for
 CMT1A as a biomarker of target engagement that can be used to optimize
 dosing, and the same normalization framework is applicable to other types
 of CMT. 
description: Two 2-mm punch skin biopsies were performed 9cm proximal to the
 ulnar crease and placed in RNAlater (Thermo Fisher Scientific, Waltham,
 MA) before freezing, as previously described 12, 13, 24-26. Total RNA isolated from the samples was prepared for RNA-seq using NuGEN Ovation library prep kits. <em>Nanostring Analysis.</em> The custom 54-gene Codeset was designed by Nanostring to maximize detection of most transcript variants, except the probes targeted to exons 1A and 1B of the <em>PMP22</em> gene. SC-specific expression was also assessed using profiling of sorted cell types in mouse embryonic skin.30 Total RNA was extracted from skin biopsies by homogenization/purification with the TRIzol reagent and then further purified using the Qiagen RNeasy MinElute Cleanup Kit (catalog no.: 74204; Qiagen, Hilden, Germany). Samples were assessed for concentration, purity, and quality by NanoDrop and Bioanalyzer (Agilent RNA6000 PicoChip; Agilent Technologies, Santa Clara, CA). The nCounter gene expression assay was performed by Nanostring (Seattle, WA). Each RNA sample (100ng) was added to the CodeSet in hybridization buffer and incubated at 65°C for 16 hours. The custom CodeSet consists of Reporter and Capture probes that hybridize to the target sequences of interest, forming a tripartite complex. After hybridization/washing, transcript counts were obtained using the MAX System Digital Analyzer. For most samples, the two punches were combined before purification of RNA, but for selected samples, the two punches were purified separately.
description: # Skin transcript biomarkers for Charcot-Marie-Tooth Disease
 [https://doi.org/10.5061/dryad.hqbzkh1st](https://doi.org/10.5061/dryad.hqbzkh1st) ## Description of the data and file structure Nanostring panel data for skin biopsies from control and CMT1A The spreadsheet (2019_nanostring_paper_samples_(jpsvaren_wisc.edu).xlsx) contains raw data from the custom Nanostring panel assays of control and CMT1A skin biopsies: Schwann cell transcript biomarkers for hereditary neuropathy skin biopsies Annals Neurology. 2019 Jun;85(6):887-898. doi: 10.1002/ana.25480. Epub 2019 Apr 22. PMID: 30945774 PMCID: PMC7050273 (also available as free PMC article [https://pmc.ncbi.nlm.nih.gov/articles/PMC7050273/](https://pmc.ncbi.nlm.nih.gov/articles/PMC7050273/)) The first tab has design data for the targeted human transcripts, including the target sequence and predicted melting temperature. 2nd tab has the pilot data from Figure 2 (21 CMT1A, 4 control), which includes raw count data generated from the Nanostring analysis. The 3rd tab contains raw nanostring data from 11 control skin biopsies and 32 CMT1A skin biopsies, about figures 3-6 of the publication. However, the data were from different runs of the same panel, and one sample (37) was assayed 3 times. In addition, we prepped up two independent biopsies from the same patient in some cases, and the highlighted samples have A and B skin biopsies. For the analysis of expression relative to control, we used A samples. However, duplicate samples were analyzed in Figure 4 using alternate normalization methods. The each column contains the sample ID as the bolded number: e.g. 20180522_UW2_**37**_10.RCC is sample 10 And the other numbers indicate the date of the run, the specific 12-sample panel (UW2), and the indicated slot (10) of the panel for the indicated sample. #### Code/Software Nanostring nCounter Analysis Software #### Access information Other publicly accessible locations of the data: * NA Data was derived from the following sources: * NA