Objective: Our objective was to target a specific cryptic splice site variant affecting immunoglobulin mu‐binding protein 2 (IGHMBP2) protein levels with a novel antisense oligonucleotide (ASO) designed to avoid nonsense‐mediated decay (NMD) and haploinsufficiency in an individual Charcot-Marie-Tooth disease Type 2S (CMT2S) patient.

Background: CMT2S is a rare autosomal recessive type of Charcot-Marie-Tooth disease caused by variants in IGHMBP2 that result in abnormal RNA processing leading to alpha‐motor neuron degeneration. A patient was reported with consequential variants within IGHMBP2; whole genome sequencing (WGS) revealed a paternally inherited cryptic splice site variant (non‐coding variant (c.1235+894 C>A) deep in intron 8). The resulting abnormal transcript undergoes NMD, resulting in haploinsufficiency.

Method: We confirmed the variant with WGS and NMD existence in the patient’s fibroblast cell line. We designed a 19mer ASO targeting deep in intron 8 (c.1235+894 C>A) around sequence CACTTCCAC(A)GGGGGAAGA. ASOs were designed with phosphorothioate methoxyethyl backbones and prioritized based on in silico binding affinity. Fibroblasts underwent ASO treatment (1µM) and 48-hour incubation. Flow cytometry and fluorescein labelled ASO (GFP+99.8%) confirmed cellular entry. Further, electrophysiology studies were done on iPSC cells and motor neurons derived from the patient’s fibroblast cells.

Results: We observed significant IGHMBP2 protein level increases (~30%) in oligo-treated samples compared to control (untreated) samples (WB antibody Sigma SAB2106426). qPCR confirmed increased ratio of restored wild-type transcript to cryptic exon-containing transcript (~1.3-fold).  Preclinical data support this ASO as potential treatment restoring IGHMBP2, with limited off-target effects in silico. Electrophysiology studies revealed hyperexcitability and spontaneous firing of motor neurons, resembling an amyotrophic lateral sclerosis (ALS) phenotype.

Conclusion: Reported cases of CMT2S caused by IGHMBP2 variants are increasing. N-of-1 precision medicine may prove instrumental to designing treatment for this diverse genetic disorder. Patient-specific phenotypic analyses further confirm this genetic heterogeneity, revealing phenotypic resemblance to ALS. This case exemplifies the shifting boundary between rapid WGS based clinical diagnoses and research capabilities allowing for personalized ASO based treatments.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/potential-treatment-for-cmt2s-caused-by-ighmbp2-cryptic-splice-variant-with-aso-based-therapeutic/