Translating the Effects of CMT2S Variants with Human-on-a-chip Neuromuscular Junction Systems
Caroline Johnson1, Sandra Smieszek1, Bart Przychodzen1, Christina Tyner1, Christos Polymeropoulos1, Gunther Birznieks1, Walker Hagan2, Leticia Lenkiu2, Caitlyn Niccum2, Heather Cannon-Patron2, Rocky Brighton2, Xiufang Guo3, Kenneth Hawkins3, Nesar Akanda3, James Hickman2, Mihael Polymeropoulos1
1Vanda Pharmaceuticals Inc., 2Hesperos, Inc., 3NanoScience Technology Center, University of Central Florida
Objective:
To characterize the neuromuscular phenotype of a specific Charcot-Marie-Tooth disease Type 2S (CMT2S) patient by analyzing the morphology and physiology of diseased motor neurons (MNs) compared to healthy MNs and their subsequent neuromuscular junction (NMJ) differences. 
Background:

CMT2S is a rare Charcot-Marie-Tooth disease subtype caused by immunoglobulin mu‐binding protein 2 (IGHMBP2) variants that result in abnormal RNA processing leading to alpha‐motor neuron degeneration. A patient was reported with variants within IGHMBP2. Whole genome sequencing revealed a paternally inherited cryptic splice site non‐coding variant (c.1235+894 C>A) in intron 8, which leads to nonsense‐mediated decay resulting in IGHMBP2 haploinsufficiency.

Design/Methods:

CMT2S-MNs were differentiated from an iPSC cell line generated from the patient’s fibroblasts. Patch clamp electrophysiology, phase imaging, and immunocytochemistry experiments were performed to characterize CMT2S-MNs and identify morphology differences. To determine NMJ defects, CMT2S-MNs and control iPSCs (WT-MNs) were integrated into a dual-chamber NMJ platform with wild-type iPSC-derived skeletal muscle myofibers.

Results:

Patch clamp electrophysiology revealed hyperexcitability and spontaneous firing of CMT2S-MNs. Similar characteristics can be found in amyotrophic lateral sclerosis-derived MNs (ALS-MNs), though ALS-MNs show hyperexcitability much later than CMT2S-MNs. Analysis of Na+ current normalized by membrane capacitance showed a significant decrease in membrane capacitance and membrane potential in CMT2S-MNs compared to WT-MNs. An NMJ fatigue index (FI) functional readout revealed low FI and quick fatigue in CMT2S. The CMT2S NMJ system presented a dominant phenotype of tetanus, followed by decay.

Conclusions:

CMT2S-MNs revealed a hyperexcitable phenotype with spontaneous firing, comparable to amyotrophic lateral sclerosis. The hyperexcitability observed in CMT2S-MNs may be caused by the observed reduced resting membrane potential, reduced membrane capacitance, and thus reduced Na+ current density. NMJ FI showed quick fatigue which may correlate with a CMT2S clinical phenotype. We are further analyzing this patient-specific model to continue phenotyping CMT2S caused by IGHMBP2 variants.

10.1212/WNL.0000000000205809