Novel genetic and nerve imaging characterization of Charcot-Marie-Tooth disease type 4F
Hanna Küpper, Lara G. Stühn, Kathrin Grundmann-Hauser, Alexander Maximilian Grimm, Markus Blankenburg, Tobias B. Haack, Hendrik Rosewich
TL;DR
This paper describes a child with a rare nerve disorder called CMT4F and identifies a new genetic variant that may cause the disease.
Contribution
The study identifies a novel PRX gene variant and a rare variant in the 5’ untranslated region potentially linked to CMT4F.
Findings
A pathogenic PRX variant and a rare variant in the 5’ untranslated region were found in a child with CMT4F-like symptoms.
High-resolution ultrasound showed nerve enlargement and muscle hyperechogenicity consistent with other CMT4 subtypes.
The findings suggest these genetic variants may be causative for CMT4F.
Abstract
Charcot-Marie-Tooth disease type 4F (CMT4F) is a rare hereditary sensorimotor neuropathy, linked to the periaxin (PRX) gene. Early onset, pronounced sensory ataxia and comparatively moderate muscular weakness are characteristic hallmarks. We here report a child with corresponding features carrying a pathogenic PRX variant in trans with a very rare variant of uncertain significance in the 5’ untranslated region predicted to interfere with splicing. High-resolution ultrasound depicted an age-related pattern of nerve enlargement and muscle hyperechogenicity resembling other CMT4 subtypes with similar clinical and histopathological characteristics. Based on this differential analysis, we propose these genetic findings to be possibly causative. The online version contains supplementary material available at 10.1007/s10048-026-00891-6.
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Figure 3- —Universitätsklinikum Tübingen (8868)
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Taxonomy
TopicsHereditary Neurological Disorders · Peripheral Neuropathies and Disorders · Neurofibromatosis and Schwannoma Cases
Introduction
The association of the periaxin gene (PRX) with autosomal-recessive Charcot-Marie-Tooth disease type 4 F (CMT4F) has first been described by Delague et al. in 2000 [1]. An expanding phenotypic and genotypic spectrum is increasingly being recognized in this rare CMT subtype [2–5]. A defining clinical hallmark of CMT4F is a pronounced sensory ataxia from early childhood on [2], typically accompanied by a variable degree of distal weakness with comparably mild muscular atrophy and later-onset foot deformity and scoliosis [2, 4]. Markedly reduced nerve conduction velocities or undetectable nerve action potentials already in early years are characteristic [4] while clinical progression is often slow [6]. To date, little is known about peripheral nerve imaging in CMT4F [7].
We report a 9-year-old girl with a consistent clinical phenotype. Genetic analysis revealed compound heterozygous variants in PRX, one of which represents a previously unreported variant. The diagnosis of CMT4F is further supported by serial nerve ultrasound, demonstrating an age-related pattern of nerve enlargement comparable to other CMT4 patients. This case contributes valuable insights into the natural history of CMT4F and underscores the potential of imaging-based biomarkers for disease characterization.
Materials and methods
A retrospective analysis of clinical, electrophysiological and imaging data was conducted. Clinical evaluation included standardized timed tests [8], the Medical Research Council (MRC) sumscore and CMT Pediatric Scale [9]. Routine nerve conduction studies of upper and lower extremities were conducted at 2 and 9 years of age. High-resolution nerve ultrasound (HRNUS) as well as muscle ultrasound (MUS) were performed with standardized parameters using a Canon aplio a ultrasound system (Canon Medical Systems GmbH, Germany) with a high-frequency linear array probe (18 MHz), according to previous reports [10]. Normative data were used for comparison [10–13]. Quantitative measurement of muscle echogenicity was performed using ImageJ (NIH, Bethesda, USA).
Genetic tests included exome sequencing (ES) and genome sequencing (GS), as well as targeted analysis for spinal muscular atrophy (SMA) and Friedreich’s ataxia, on peripheral blood DNA. For ES, coding genomic regions were enriched using a SureSelectXT HumanAllExon KitV.7 (Agilent Technologies, Santa Clara, USA) for subsequent sequencing as 2 × 100 bp paired-end reads on a NovaSeq6000 system (Illumina, San Diego, USA). Generated sequences were analyzed using the megSAP pipeline (https://github.com/imgag/megSAP). Sequencing library for short-read GS was generated using the TruSeqDNA PCR-Free kit (Illumina) and sequenced on a NovaSeqXPlus system as 150 bp paired-end reads with an average coverage of 46x. Clinical variant prioritisation included different filtering steps, e.g. minor allele frequency (MAF) ≤ 1% in gnomAD (https://gnomad.broadinstitute.org), in-house databases and phenotype-based filtering. DNA variants were interpreted in the context of parents' variants and classified according to the American College of Medical Genetics and Genomics (ACMG) [14]. Sanger sequencing was performed to confirm identified PRX variants and parental carrier status. Primer sequences and PCR conditions are available upon request.
Results
The patient depicted a primary motor delay with a positive developmental course but a persistent and significant sensory ataxia (Table 1 supplement). Nerve conduction studies at 2 years showed signs of a mixed, predominantly demyelinating neuropathy. At 9 years, upper and lower extremity nerves were inexcitable. HRNUS demonstrated a significant enlargement (Fig. 1), with a diffuse, proximally pronounced pattern and progressive course between 4 and 9 years of age (Table 2 supplement). MUS depicted a mild hyperechogenicity (Table 2 supplement). Brain and spinal MRI showed normal results.Fig. 1. Nerve ultrasound imaging demonstrated a diffuse, but proximally pronounced enlargement pattern of peripheral nerves and cervical roots. Transverse high-resolution imaging are depicted for the brachial plexus in the supraclavicular position (A), the cervical roots C5 and C6 (longitudinal imaging defining the diameter at the proximal measurement site, (B), the cervical roots C5-C7 in the interscalene location (C), the cervical root C5 in the paravertebral localization (D), the vagus nerve (E), the median (F), ulnar (G) and radial (I) nerve in the upper arm, the superficial and deep (PIN) radial nerve (H) at the proximal forearm. Longitudinal imaging of the median (J) and ulnar (K) nerve in the upper arm showed a homogenous enlargement pattern. The size of the cross-sectional area (CSA) or diameter is defined in each image. Muscle ultrasound depicted a mild hyperechogenicity, as quantified by the adjunctive grey scale diagram, of the anterior tibial (L) and peroneus tertius (N) muscles, compared to an age-related patient with a primary bone disease (M, O) without muscle weakness
Trio-ES identified a maternally inherited, previously described, pathogenic stop variant in PRX (ENST00000324001.8: c.2145 T > A, p.Cys715Ter; rs104894707, CADD 17.26) in a heterozygous state. Subsequent GS additionally identified a paternally inherited variant of unknown significance in the 5’untranslated region (UTR) of PRX (c.−243G > A, p.?; Fig. 3), which had remained un-identified in the previous exome analysis due to insufficient ES coverage of the first non-coding exon. This variant is extremely rare (rs2079567584, frequency 0,000013 (gnomAD v4.1.0)) and predicts a loss of the canonical splice donor site of the non-coding exon 1 (SpliceAI Donor Loss delta score 0.40, Pangolin Splice Loss REF score 0.40 to ALT score 0.07). No other causative variants associated with hereditary neuropathies, SMA or Friedrich’s ataxia were detected. All other described intronic PRX variants were frequent and without suggestive splice alteration predictions (Table 4 supplement).
Discussion
The important regulatory role of UTRs in the pathogenicity of human disease has already been outlined [15, 16]. Reporting variants in these non-coding regions is increasingly recommended [17], based on a better accessibility by GS. UTRs are non-coding segments flanking the protein-coding sequence of a gene. Although transcribed into mRNA, they are not translated into protein. Instead, UTRs are pivotal for post-transcriptional gene regulation, and alterations within these regions have been shown to exert profound effects on protein expression [16]. Pathogenic variants within 5’UTR have previously been described for other hereditary neuropathies [18]. The here newly described variant (c.−243G > A, p.?) in the 5’UTR of PRX is predicted to possibly result in altered splicing of the non-coding exon 1. It affects a highly conserved (phyloP score 3.516) nucleotide position and is predicted to impact the splice donor site of the non-coding exon 1 of PRX. Its potential consequences at protein level cannot be determined without functional analyses. However, splicing alterations within 5′UTR have been shown to compromise mRNA stability and impair translational efficiency. In combination with the maternally inherited stop variant (c.2145 T > A, p.Cys715Ter) in exon 7 of the PRX gene and specific clinical features, we hypothesize this change to be associated with autosomal recessive CMT4F.
Loss of function has been described as the underlying mechanism in *PRX-*associated CMT [4, 6], which is also the predominantly described effect of pathogenic variants in 5’UTR [15], further corroborating its possible role here.
The clinical phenotype, characterized by predominant sensory ataxia, with electrophysiological evidence of a severe sensorimotor neuropathy, is consistent with PRX-associated CMT4F [4, 6, 19, 20]. This distinct clinical profile is further reflected in a moderate-to-severe impairment level of the CMT Pediatric Scale, which incorporates measures of sensory ataxia. In contrast, muscle strength was only mildly affected, as demonstrated by near-normal results in the MRC sum score and 6-min walk test. Notably, the sensorimotor trajectory in our patient closely parallels a 5-year-old patient harboring a homozygous deletion in exon 7 of PRX [7].
To date, only a single nerve ultrasound examination in a child with PRX-associated CMT4F has been published, reporting nerve sizes within normal range [7]. However, comparing normative HRNUS in childhood [11], these measurements reported by Cartwright et al. [21] fall within the upper reference range. Indeed, the nerve dimensions of this 5-year-old patient were similar to those of our patient at 4 years 2 months (Table 2 supplement), who exhibited the most pronounced enlargement from 6 years onward (Fig. 2). This age-dependent pattern parallels pediatric patients with CMT4D (publication in submission), an equally early-onset sensorimotor neuropathy with comparable histopathological features (Fig. 2). Further evidence of nerve enlargement in CMT4F can be found in an autopsy case of an adult patient [19] and histological analyses [4, 22].Fig. 2. Longitudinal follow-up of the nerve size pattern, as depicted by high-resolution nerve ultrasound for the cross sectional area (CSA) of the median nerve at the forearm (A), elbow (B) and distal third of the upper arm (C), as well as the vagus nerve (D) and diameter of the cervical roots C5 (E) and C6 (F), in comparison to other CMT4 subtypes, differentiated for age. Consecutive measurements in a single patient are connected by a line. A significant, age-related enlargement pattern is visualized for the CMT4F patient, similar to in-house data of pediatric patients with CMT4D. Age-related normative values are used for comparison^5−8^. Longitudinal follow-up of muscle echogenicity, as depicted for the anterior tibial (G) and peroneus tertius muscle (H), derived as a mean grey scale value of three measurements with standardized imaging settings. Consecutive measurements in a single patient are connected by a line. For comparison, in-house data from pediatric patients with other CMT4 subtypes are demonstrated
In summary, this report underscores the potential pathogenicity of untranslated regions in PRX-associated CMT4F and broadens the knowledge on clinical and imaging characterization of this rare disease. As major limitations of this study, the genetic findings need to be validated by functional RNA analysis and the imaging results by ultrasound studies in further CMT4F patients (Fig. 3).Fig. 3. Panel A shows the pedigree of the family with the index patient carrying compound heterozygous variants in the PRX gene. Sanger sequencing of the non-coding exon 1 containing the identified variant in the 5′UTR of PRX. The variant position is highlighted in grey. The figure shows exemplary sequencing chromatograms for the index, father, and mother. The exonic region is highlighted in blue, and the intronic region is highlighted in yellow. Both the index and the father carry the variant c.−243G > A in a heterozygous state, whereas the mother is homozygous for the wild-type allele at this position. Panel** B** shows a schematic representation of the PRX gene and protein structure. White boxes represent non-coding exons, and black boxes indicate coding exons. Latin numerals indicate the exons, whereas Roman numerals indicate the introns. The positions of the two variants identified in the index are indicated. Below, a schematic overview of all previously reported variants in the coding region of the PRX gene and their respective positions within L(ong)-Periaxin are indicated. Most variants are located in the exon 7 of the gene which is the largest exon encoding 90% of L-periaxin which contains 4 major domains: PDZ which is important for the interaction with proteins, peptides, lipidosomes, NLS (nuclear localization signal) which mediates the export of L-periaxin from the nucleus to cytoplasm as well as the repeat and acidic domain which enable L-periaxin to bind to the cytoskeleton of Schwann cells and imply a stabilizing role in the myelin formation31. Loss of the acidic domain has been characterized as the main aetiological factor causing CMT4F. A detailed list of the previously reported pathogenic variants, indicating the nucleotide and protein position, is enclosed in Table 3 of the appendix
Supplementary Information
Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 15 KB)Supplementary file2 (XLSX 13 KB)Supplementary file3 (XLSX 19 KB)Supplementary file4 (XLSX 15 KB)Supplementary file5 (MP4 10709 KB)Supplementary file6 (MP4 7735 KB)Supplementary file7 (MP4 6688 KB)