A clinical case-control comparison of epidermal innervation density in Rett syndrome

Abstract

Introduction: Rett syndrome (RTT), a rare neurodevelopmental disorder occurring primarily in females (1:10-15,000 female live births), is most often caused by loss-of-function mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2). Clinical observations and preclinical findings indicate apparent abnormal sensory and nociceptive function. There have been no direct investigations of epidermal sensory innervation in patients with RTT.

Methods: We compared 3 mm epidermal punch biopsy specimens from adolescent female RTT patients (N = 4, aged 12-19 years) against an archived approximate age-, sex-, body-site matched comparison sample of healthy adolescent females (N = 8, ages 11-17).

Results: Confocal imaging revealed, on average, statistically significant increased epidermal nerve fiber (ENF) peptidergic (co-stained calcitonin gene-related protein [CGRP]) innervation density compared with healthy female control individuals.

Conclusions: Given the clinical phenotype of disrupted sensory function along with diagnostic criteria specific to cold hands/feet and insensitivity to pain, our preliminary observations of ENF peptidergic fiber density differences warrants further investigation of the peripheral neurobiology in RTT.

Keywords: MECP2; Rett syndrome; epidermal nerve fiber innervation; sensory phenotype.

Figure 1

Confocal images of representative healthy control and Rett syndrome (RTT) comparison skin biopsies. (a) Skin biopsy from a healthy control age/gender/body‐site matched case. Simple epidermal nerve fibers (ENFs) arise from typical subepidermal neural plexus. Nerve fibers appear green or yellow, epidermis appears blue, dermal epidermal junction appears red, and capillaries (CAPs) appear pink or magenta. Scale bar = 100 microns. (b) Skin biopsy from a RTT patient. Epidermal nerve fibers are frequently long and complex. Unusual dermal nerve clusters (DNC) are present in the dermal papilla. The subepidermal neural plexus is dense and robust. A sweat duct (SD) is present in this section. Scale bar = 100 microns. (c) Merkel cell (MC) in a skin biopsy from a healthy control age/gender/body‐site matched case. MC are infrequently observed in biopsies from healthy children and tend to be small. Scale bar = 33.4 microns. (d) MC were frequently observed in all RTT samples, appear large, and robustly stained. Scale bar = 33.4 microns. (e) Arteriole innervation (AI) forms a typical woven pattern surrounding arterioles in skin biopsies from normal healthy age/gender/body‐site matched cases. A dermal nerve bundle (DN) courses in parallel with the vasculature in this image. Arteriole appears red and nerve fibers appear green or yellow Scale bar = 100 microns. (f) Arteriole innervation appears reduced/less complex in RTT specimens. Scale bar = 100 microns. (g) Mast cells (MCs), appearing red, in biopsy specimens from healthy control age/gender/body‐site matched case typically appear quiescent with a more rounded appearance. Scale bar = 50 microns. (h) MCs were elongated in RTT biopsy specimens. Scale bar = 50 microns

Figure 2

Quantification of Rett syndrome epidermal nerve fiber density (RTT ENFd) compared to healthy control comparison group. (a) Group average and individual ENFd values between RTT patients and age‐, gender‐, body‐site matched comparison group. The average ENF density value estimates for the RTT sample was 42.0 ENF/mm (SD = 22.0, range = 12.2–60.3) and for the control sample was 27.3 ENF/mm (SD = 9.7; range = 15.3–41.1) [t = −1.72, p = 0.05, p < 0.10]. (b) Epidermal nerve fiber density values plotted by age for RTT patients and age‐, gender‐, body‐site matched comparison group. (c) Epidermal nerve fiber density values plotted by body surface area for RTT patients and age‐, gender‐, and body‐site matched comparison group