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. 

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.

Nanostring Analysis. 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 PMP22 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.