A deep intronic variant in MME causes autosomal recessive Charcot–Marie–Tooth neuropathy through aberrant splicing

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.


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.
Splicing variants are increasingly being recognized as a cause of Mendelian disease. 18,19The precise removal of introns (non-coding regions) and inclusion of exons (coding regions) in the final mature mRNA relies on the spliceosome and auxiliary splicing factors recognizing specific sequence motifs, such as the 5 0 donor splice site and 3 0 acceptor splice site.Splice-altering variants can weaken or abolish recognition of the correct splice sites, or alternatively strengthen or create cryptic splice sites that mimic consensus splicing sequences. 20ese variants typically lead to one or more mis-splicing events that result in the skipping of partial or complete exons, and/or the retention of partial or complete introns.  Pathenic splicing variants have been found in several CMT genes including MPZ, 21,30,39,40 MFN2, 22,30,31 LRSAM1, 23 IGHMBP2, 24 INF2, 25 MCM3AP, 26 SH3TC2, 30,32,38 GDAP1, 27,42 SBF1, 28 NDRG1, 37 and FGD4. 29Variants affecting canonical splice donor and acceptor sites have also been described in MME. 4,41on-trapping, also known as a mini-gene assay, is an in vitro technique used to identify exons in a genomic region of interest. 43is is of particular use when relevant patient tissue is unavailable or when relevant transcripts may be unstable or degraded by nonsense mediated decay (NMD). 44The genomic region of interest is cloned into an exon-trapping vector between two known exons.This exontrapping vector is transfected into a cell line where it is transcribed and undergoes a series of post-transcriptional processes that include pre-mRNA splicing to create mature mRNA.This mRNA consists of the 'trapped' exons of the genomic region of interest flanked by the known exons, which can then be Sanger sequenced to characterize the 'trapped' exons.[47][48][49][50][51] Here we report a deep intronic variant in MME

| Variant detection
Genomic DNA for Family 1 was extracted from peripheral blood using the PureGene Kit (Qiagen) following the manufacturer's instructions.
WGS for two individuals in Family 1 (V:1 and V:3) was outsourced to the Garvan Sequencing Platform.Paired-end sequencing reads of 150 base pairs were generated using the Illumina NovaSeq 6000 sequencing machine, with 30-fold average read depth.
In WGS data processing was performed at the Centre for Population Genomics (CPG) following the DRAGEN GATK best practices pipeline.

| Sanger sequencing
For  Sequencing libraries were prepared from 3-5 μg of HMW DNA, using native library prep kit SQK-LSK114, according to manufacturer's instructions.Each library was loaded onto a R10.4.1 flow cell and sequenced on a PromethION device with live target selection/ rejection executed by the ReadFish software package. 57Detailed descriptions of software and hardware configurations used for Read-Fish are provided in a previous publication. 58Samples were run for a maximum duration of 72 h, with nuclease flushes and library reloading performed at approximately 24-and 48-h timepoints for targeted sequencing runs, to maximize sequencing yield.Raw ONT sequencing data was converted to BLOW5 format 59 using slow5tools (v.0.3.0) 60en base-called using Guppy (v6).Resulting FASTQ files were aligned to the hg38 reference genome using minimap2 (v2.14-r883). 61Variants were called using clair3, 62 phased using Whatshap 63 and visualized using the Integrative Genomics Viewer (IGV, v2.17.3). 64

| Cell culture
The human HeLa cervical epithelial cell line (ATCC) was maintained in and 100 μg/mL streptomycin (Gibco) at 37 C in humidified air and 5% CO 2 .

| In vitro exon-trapping
HeLa cells were grown to approximately 70% confluence in a 6-well plate.HeLa cells were separately transfected with either 2 μg of pSpliceExpress-MME WT or 2 μg of pSpliceExpress-MME c.1188+428A>G using Lipofectamine 3000 (ThermoFisher).RNA extraction was performed 48 h following transfection using the RNEasy Mini Kit (Qiagen), and reverse-transcribed template was prepared using the iScript cDNA Synthesis Kit (Bio-Rad).PCR amplification of cDNA was conducted using primers designed to anneal to the flanking Ins2 exons (5 0 -CAGCACCTTTGTGGTTCTCA-3 0 ; 5 0 -CAGTGCCAAGGTCT-GAAGGT-3 0 ).The RT-PCR amplicons were size fractionated using a 1.5% w/v agarose gel.The largest amplicon for each vector was gelpurified using Isolate II PCR and Gel Kit (Bioline) for Sanger Sequencing.

| In silico splicing analysis of reported MME variants
All reported MME variants in gnomAD v.4.0.0 were detected by searching the gnomAD browser for the genomic region corresponding with the MME gene; '3-155024 124-155183704'(hg38).The MME variants reported in gnomAD were then exported using the 'Export Variants to CSV' function.The consequences of the variants were described by gnomAD using the Variant Effect Predictor (VEP) annotation based on the most deleterious predicted functional effect of each variant. 65These variants were then filtered for a minor allele frequency (MAF) <0.01 and a maximum SpliceAI Δscore >0.8 (high precision splicing change prediction 56 ) as annotated by gnomAD.

| Family 1
Family 1 consists of four affected siblings from a consanguineous family of European (non-Finnish) background (Figure 1A).The phenotype was consistent with a generalized sensorimotor axonal neuropathy and sensory ataxia without cerebellar signs (Table 1), and was confirmed with NCS for individuals V:1 and V:6 (Table S2).S2).

Individual V:3
This 67-year-old woman presented with difficulty running at school, weakness of the ankles, and recurrent ankle sprains.She developed numbness and soreness of the feet and a slowly progressive gait disturbance with recurrent falls.She is now only just able to get around the house using a walker and fixed AFO splints.She also had a distal pattern of weakness and sensory loss.S2).

| Family 2
Family 2 consists of one affected female born to a healthy, nonconsanguineous couple (Figure 1B).There is no history of neuromuscular disorders in the family.The 60-year-old proband presented with slowly progressive bilateral leg weakness and sensory disturbances.
NCS showed evidence of an axonal sensorimotor neuropathy (Table S2).Neurological examination revealed mild distal upper limb weakness and moderate distal lower limb weakness, with muscle atrophy only in the left gastrocnemius muscle (Table 1).Bilateral MRI of the thighs and calves showed multifocal calf muscle T2 hyperintensity, muscle atrophy and T1 hyperintense fatty replacement of the left medial gastrocnemius muscle (Figure S1).The patient had reduced ankle reflexes and showed mild muscle cramps and muscle fatigue.
No homozygous individuals were reported in gnomAD, All of Us, or TOPMED.SpliceAI predicted that MME c.1188+428A>G could create a strong novel splice donor site (Score 0.97; where a SpliceAI score above 0.8 is considered a 'high precision' predicted splice variant 56 ).
This in turn strengthened the SpliceAI prediction for a cryptic splice acceptor site 83 bp upstream of the novel splice donor site (Score 0.99).
Segregation analysis in Family 1 confirmed the biallelic inheritance of the MME c.1188+428A>G variant segregated with the CMT phenotype (Figure 1A).Sanger sequencing of DNA from available individuals showed that all affected individuals were homozygous for the MME c.1188+428A>G variant and unaffected individuals were carriers (Figure S2).
WGS screening using the seqr platform and AIP was conducted in the proband of Family 2 (II:1).The MME c.1188+428A>G variant was detected in a compound heterozygous state with a second MME variant (MME c.467del; p.Pro156Leufs*14), which has previously been reported as pathogenic 41 (Figure 1B).Sanger sequencing validated the presence of each MME variant in the index individual (Figure S3).As parental DNA was unavailable, ONT long-read sequencing was conducted to phase the heterozygous MME variants in the proband (Figure 1C,D).ONT long-read sequencing confirmed that MME c.1188+428A>G (boxed red in Figure 1C) and c.467del (boxed red in Figure 1D) were present on alternative haplotypes (hap-1: red, hap-2: blue) and therefore were in trans in the proband.

| In vitro exon-trapping of MME-pSpliceExpress vectors
Two separate exon-trapping pSpliceExpress vectors were generated to assess the in vitro splicing impact of the MME c.1188+428A>G variant: pSpliceExpress-MME WT (wild-type) and pSpliceExpress-MME c.1188+428A>G (variant).A schematic of the constructs and relevant SpliceAI scores are shown in Figure 2A.RT-PCR products produced following transfection of these vectors were analyzed using gel electrophoresis (Figure 2B), which revealed a visible size difference between the wild-type (381 bp) and MME c.1188+428A>G amplicons (464 bp).
The gel-purified amplicons were Sanger sequenced and the sequencing was aligned to the WT MME mRNA sequence.The sequenced products showed that the pSpliceExpress-MME WT produced a transcript that was correctly spliced between MME exon 12 and 13 (Figure 2C).In contrast, exon-trapping of the pSpliceExpress-MME c.1188+428A>G vector showed that an 83 bp pseudoexon had been spliced between MME exon 12 and 13 (Figure 2D).A BLAT search 70  showed that a PTC was generated in exon 14 (p.Ala397ProfsTer47) at genomic position chr3:155144368 (hg38) (Figure S4).This PTC likely leads to NMD of the MME transcript.

| In silico splicing analysis of MME variants
All reported MME variants in gnomAD v.4.0.0 were assessed using the integrated SpliceAI scores to determine if splicing variants in MME were a likely underrecognized cause of disease.There were 37 264 total variants reported in MME in gnomAD, of which 35 673 variants had a MAF <0.01.Of these, 88 variants had a maximum SpliceAI Δscore above 0.8 (Table S1).There were no homozygotes reported

| DISCUSSION
Here we report a deep intronic variant, MME c.1188+428A>G causing recessive CMT2T in two unrelated Australian families.This variant results in a pseudoexon that likely leads to NMD of the MME transcript, resulting in a loss-of-function.This is in keeping with previously reported MME variants which have broadly been characterized as 'loss-of-function' variants.To our knowledge, this is the first report of a pathogenic deep intronic variant in MME.Given that a significant portion of patients with axonal CMT remain genetically undiagnosed, [13][14][15][16][17] it's possible that deep intronic variants in MME explain a portion of this diagnostic gap.
Individuals from Family 1 previously underwent both diagnostic WES and research WES, and the proband in Family 2 previously underwent targeted gene panel testing (PathWest neuro v3).These testing methods did not capture deep intronic regions and returned negative results.However, deep intronic regions have previously been shown to have a higher prevalence of variants than coding regions and canonical splice sites. 71Our findings suggest that intronic SNPs should be analyzed to determine if they impact splicing before they are dismissed as benign.Detection and functional validation of deep intronic variants has previously been shown to increase diagnostic rates in other Mendelian diseases including X-linked Alport syndrome, 72 inherited retinal disorders, 73,74 and dystrophinopathy. 75,76Pathogenic deep intronic variants, such as described here, are likely to be underreported amongst CMT-causing genes due to a lack of detection by WES and targeted gene panels, and a lack of functional investigation upon detection.
The MME c.1188+428A>G variant was reported in multiple different genetic ancestry groups in gnomAD v.4.0.0, 67  This is of note as these 'missense' variants are often assumed to result in a single amino acid change, and synonymous variants are often considered functionally neutral, with their effect on splicing typically not assessed. 20Our results suggest that the discovery of any of these 88 variants in a homozygous or compound heterozygous state in an individual with CMT should prompt further functional investigation of their effect on splicing of the MME transcript.
Prior to functional validation, MME c.1188+428A>G was considered a variant of uncertain significance (VUS) according to American College of Medical Genetics and Genomics (ACMG) criteria 77 (BP4, PM2, PM3, PP1), However, the functional evidence generated by the Previously described biallelic MME patients typically have a phenotype consistent with late-onset axonal neuropathy.In contrast, three of the four affected siblings in Family 1 self-described childhood onset and the proband of Family 2 self-described symptom onset in early adulthood.This may reflect an expansion of the phenotype associated with biallelic MME variants in CMT2T.It has also previously been reported that heterozygous MME variants can cause spinocerebellar ataxia type 43 (SCA43) 78 as well as autosomal dominant CMT. 3 However, the individuals in Family 1 who were MME c.1188+428A>G heterozygotes were all clinically assessed as neurologically normal, including an individual who is in their seventh decade.
Homozygous affected individuals in Family 1 were noted to have a sensory ataxia rather than cerebellar ataxia, although MRI brain studies were not conducted.Therefore, our findings here do not support a role for MME c.1188+428A>G to cause SCA43 or autosomal dominant CMT, and further expand the phenotype of recessive CMT2T.
Understanding the specific effects of splice-affecting variants is crucial for developing potential therapeutic strategies.2][93][94][95] As such, individuals with the MME c.1188+428A>G variant may represent a form of CMT that is treatable through personalized ASO therapy and warrants further investigation.

[
chr3:155142758A>G (hg38); MME c.1188+428A>G], found in a recessive state in two Australian families.Exon-trapping revealed that this variant creates a novel splice donor site in MME intron 12 (NM_000902.5)which, along with a preceding existing cryptic splice acceptor site, results in the incorporation of an 83 bp pseudoexon in the MME transcript [chr3:155142675-155 142 757 (hg38); r.1188_1189ins[1188+345_1188+427]].The coding frameshift caused by this pseudoexon leads to a PTC in exon 14 and likely NMD of the MME transcript (p.Ala397ProfsTer47), resulting in a loss of MME function.

2. 5 |
Oxford nanopore technologies (ONT) long read sequencing High molecular weight (HMW) DNA samples of the Proband in Family 2 were transferred to the Garvan Sequencing Platform for targeted long-read sequencing analysis on ONT instruments.Prior to ONT library preparations, DNA was sheared to 20-25 kb fragment size using a MegaRuptor 3 instrument and visualized post-shearing on an Agilent FemtoPulse.

Individual V: 4
This 69-year-old man first experienced symptoms as a child with difficulty sitting on crossed legs.He then noticed weakness in the ankles and legs and sensory loss in the feet, which slowly progressed to severe walking difficulty with falls requiring a walker, mobility scooter and AFOs.On examination, he had global muscle weakness, and sensory loss to the knees and elbows, with subtle corticospinal tract signs (increased tone and hyperreflexic knee jerks).Individual V:6This 67-year-old man first developed symptoms in childhood with difficulty running, multiple ankle sprains and difficulty with sport.The symptoms progressed and by his mid-twenties, he developed overt distal weakness and had difficulty with balance, with multiple falls.He currently has severe weakness in the upper and lower extremities with sensory deficits and balance problems and mobilizes with a wheelchair.Neurological examination revealed global weakness in the upper and lower limbs (distal greater than proximal), absent deep tendon reflexes throughout, downgoing plantars, glove and stocking sensory loss, and a positive Romberg's sign.There were no cerebellar or extrapyramidal signs, and the head impulse test was negative.NCS revealed a severe generalized axonal sensorimotor polyneuropathy (Table using the sequence of the trapped pseudoexon revealed alignment to the intronic region directly upstream of the MME c.1188+428A>G variant [chr3:155142675-155 142 757 (hg38)].This suggests that the MME c.1188+428A>G variant creates a novel splice donor site leading to the aberrant inclusion of 83 bp of intronic MME sequence in the final spliced transcript, as predicted by SpliceAI.Prediction of the novel MME coding sequence caused by the introduction of the pseudoexon

1
The MME c.1188+428A>G (NM_000902.5)variant segregates with recessive CMT in two families.(A) Family 1 pedigree showing the associated genotypes for the MME c.1188+428A>G (NM_000902.5)variant.Affected individuals in the fifth generation are homozygous (G/G).Unaffected individuals in the fifth (V) and sixth (VI) generation are heterozygous (A/G).Squares represent males and circles represent females, solid symbol denotes affected individual.The double line in the fourth (IV) generation indicates a consanguineous relationship.The MME c.1188+428 genotype is denoted beneath individuals who underwent Sanger sequencing, with the pathogenic 'G' allele in red text.B) A pedigree showing the associated genotypes for MME c.1188+428A>G and MME c.467del in the index individual in Family 2. C-D) Haplotype phasing showed that the c.1188+428A>G (C) and c.467del (D) variants were present on alternative haplotypes (light red and light blue), thus inherited in trans in the proband of Family 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 & MRF2023126.Genomic analysis was supported by the Centre for Population Genomics (Garvan Institute of Medical Research and Murdoch Children's 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).Open access publishing facilitated by The University of Sydney, as part of the Wiley -The University of Sydney agreement via the Council of Australian University Librarians.
Family 1, PCR amplicons were sent to Garvan Molecular Genetics, Phenotypic characterization of individuals with recessive CMT MME variants reported in this manuscript.any of the 88 MME putative splicing variants.The majority of these predicted splicing variants were predicted to directly change either the canonical splice donor sites (26/88) or canonical splice acceptor sites (30/88) of MME, including an inframe deletion (c.1317_1317+2del) and a frameshift variant (c.957+1del).An additional nine variants were predicted to affect a splicing region, includ- 2. Phased targeted ONT long-read sequencing reads were visualized using the Integrative Genomics Viewer (IGV; v2.17.3).T A B L E 1Abbreviations: AFO, ankle foot orthoses; CMTNS, Charcot-Marie-Tooth Neuropathy Score; GERD, gastroesophageal reflux disease; UL/LL, upper limb/ lower limb.F I G U R E 2 Legend on next page.for

2
An exon-trapping assay was used to analyze splicing changes caused by the MME c.1188+428A>G variant.(A) Schematic of the pSpliceExpress-MME WT (wild-type) and pSpliceExpress-MME c.1188+428A>G (variant) constructs.Both constructs consisted of an RSV LTR promoter region (blue) controlling transcription of a minigene of MME exons 12 and 13 (grey) flanked by Ins2 exon 2 and Ins2 exon 3 (black).The constructs differed in the presence of either an A (wild type: green text) or a G allele (mutant: red text) at MME c.1188+428A>G.i) The wild type sequence was not predicted to contain any strong splice sites when assessed using SpliceAI, with a score of 0.11 for the acceptor site (purple text) and 0.00 for the donor site (green text).ii) SpliceAI predicted that the MME c.1188+428A>G variant (red arrow) would create a strong splice donor site assay now allows for the addition of PVS1 (null variant in a gene where loss-of-function is a known mechanism of disease) and PS3 (well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product) criteria.This allows reclassification of the variant as 'pathogenic'.Therefore, this work demonstrates the importance of functional validation to confirm the effect of candidate variants on splicing to increase diagnostic rates for those with inherited disease.