Background: Loss-of-function variants in MME (membrane metalloendopeptidase) are a known cause of recessive Charcot-Marie-Tooth Neuropathy (CMT). A deep intronic variant, MME c.1188+428A>G (NM_000902.5), was identified through whole genome sequencing (WGS) of two Australian families with recessive inheritance of axonal CMT using the seqr platform. MME c.1188+428A>G was detected in a homozygous state in Family 1, and in a compound heterozygous state with a known pathogenic MME variant (c.467del; p.Pro156Leufs*14) in Family 2. Aims: We aimed to determine the pathogenicity of the MME c.1188+428A>G variant through segregation and splicing analysis. Methods: The splicing impact of the deep intronic MME variant c.1188+428A>G was assessed using an in vitro exon-trapping assay. Results: The exon-trapping assay demonstrated that the MME c.1188+428A>G variant created a novel splice donor site resulting in the inclusion of an 83 bp pseudoexon between MME exons 12 and 13. The incorporation of the pseudoexon into MME transcript is predicted to lead to a coding frameshift and premature termination codon (PTC) in MME exon 14 (p.Ala397ProfsTer47). This PTC is likely to result in nonsense mediated decay (NMD) of MME transcript leading to a pathogenic loss-of-function. Interpretation: To our knowledge, this is the first report of a pathogenic deep intronic MME variant causing CMT. This is of significance as deep intronic variants are missed using whole exome sequencing screening methods. Individuals with CMT should be reassessed for deep intronic variants, with splicing impacts being considered in relation to the potential pathogenicity of variants.

The authors have declared no competing interest.

This work was funded by the Medical Research Future Fund (MRFF) Genomics Health Futures Mission (APP2007681). JMP is supported by the Australian Government Research Training Program. IWD was supported by MRF2025138 and MRF2023126. Genomic analysis was supported by the Centre for Population Genomics (Garvan Institute of Medical Research and Murdoch Childrens Research Institute) and was funded in part by a National Health and Medical Research Council investigator grant (2009982) and the Medical Research Future Fund (MRFF) Genomics Health Futures Mission (2008820).

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Sydney Local Health District Human Ethics Research Committee of Sydney Local Health District gave ethical approval for this work (2019/ETH07839) for Family 1. Human Research Ethics Committee of the Royal Melbourne Hospital (HREC/16/MH/251) gave ethical approval for this work for Family 2.

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