[This corrects the article DOI: 10.3389/fneur.2024.1358881.].

Charcot-Marie-Tooth disease type 1E (CMT1E) is an inherited autosomal dominant peripheral neuropathy caused by mutations in the peripheral myelin protein 22 (PMP22) gene. The identical leucine-to-proline (L16P) amino acid substitution in PMP22 is carried by the Trembler J (TrJ) mouse and is found in CMT1E patients presenting with early-onset disease. Peripheral nerves of patients diagnosed with CMT1E display a complex and varied histopathology, including Schwann cell hyperproliferation, abnormally thin myelin, axonal degeneration, and subaxonal morphological changes. Here, we have taken an unbiased data-independent analysis (DIA) mass spectrometry (MS) approach to quantify proteins from nerves of 3-week-old, age and genetic strain-matched wild-type (Wt) and heterozygous TrJ mice. Nerve proteins were dissolved in lysis buffer and digested into peptide fragments, and protein groups were quantified by liquid chromatography-mass spectrometry (LC-MS). A linear model determined statistically significant differences between the study groups, and proteins with an adjusted p-value of less than 0.05 were deemed significant. This untargeted proteomics approach identified 3759 quality-controlled protein groups, of which 884 demonstrated differential expression between the two genotypes. Gene ontology (GO) terms related to myelin and myelin maintenance confirm published data while revealing a previously undetected prominent decrease in peripheral myelin protein 2. The dataset corroborates the described pathophysiology of TrJ nerves, including elevated activity in the proteasome-lysosomal pathways, alterations in protein trafficking, and an increase in three macrophage-associated proteins. Previously unrecognized perturbations in RNA processing pathways and GO terms were also discovered. Proteomic abnormalities that overlap with other human neurological disorders besides CMT include Lafora Disease and Amyotrophic Lateral Sclerosis. Overall, this study confirms and extends current knowledge on the cellular pathophysiology in TrJ neuropathic nerves and provides novel insights for future examinations. Recognition of shared pathomechanisms across discrete neurological disorders offers opportunities for innovative disease-modifying therapeutics that could be effective for distinct neuropathies.

Underexpression, overexpression, and point mutations in peripheral myelin protein 22 (PMP22) cause most cases of Charcot-Marie-Tooth disease (CMTD). While its exact functions remain unclear, PMP22 is clearly essential for formation and maintenance of healthy myelin in the peripheral nervous system. This review explores emerging evidence for roles of PMP22 in cholesterol homeostasis. First, we highlight dysregulation of lipid metabolism in PMP22-based forms of CMTD and recently-discovered interactions between PMP22 and cholesterol biosynthesis machinery. We then examine data that demonstrates PMP22 and cholesterol co-traffic in cells and co-localize in lipid rafts, including how disease-causing PMP22 mutations result in aberrations in cholesterol localization. Finally, we examine roles for interactions between PMP22 and ABCA1 in cholesterol efflux. Together, this emerging body of evidence suggests that PMP22 plays a role in facilitating enhanced cholesterol synthesis and trafficking necessary for production and maintenance of healthy myelin.

NMR spectroscopy has played a pivotal role in fragment-based drug discovery by coupling detection of weak ligand-target binding with structural mapping of the binding site. Fragment-based screening by NMR has been successfully applied to many soluble protein targets, but only to a limited number of membrane proteins, despite the fact that many drug targets are membrane proteins. This is partly because of difficulties preparing membrane proteins for NMR-especially human membrane proteins-and because of the inherent complexity associated with solution NMR spectroscopy on membrane protein samples, which require the inclusion of membrane-mimetic agents such as micelles, nanodiscs, or bicelles. Here, we developed a generalizable protocol for fragment-based screening of membrane proteins using NMR. We employed two human membrane protein targets, both in fully protonated detergent micelles: the single-pass C-terminal domain of the amyloid precursor protein, C99, and the tetraspan peripheral myelin protein 22 (PMP22). For both we determined the optimal NMR acquisition parameters, protein concentration, protein-to-micelle ratio, and upper limit to the concentration of D6-DMSO in screening samples. Furthermore, we conducted preliminary screens of a plate-format molecular fragment mixture library using our optimized conditions and were able to identify hit compounds that selectively bound to the respective target proteins. It is hoped that the approaches presented here will be useful in complementing existing methods for discovering lead compounds that target membrane proteins.

UNASSIGNED: There is growing evidence that severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or COVID-19 infection is associated with the development of immune mediated neuropathies like chronic inflammatory demyelinating polyneuropathy (CIDP), but the impact of SARS-CoV-2 vaccination and COVID-19 infection on genetic disorders such as Charcot-MarieTooth (CMT) remains unclear.
UNASSIGNED: A 42-year-old male with occulted CMT neuropathy type lA (CMT1A) who developed limb numbness and weakness after the second SARS-CoV-2-vaccination was confirmed by identifying characteristic repeats in the p11.2 region of chromosome 17. Due to the progressive deterioration of muscle strength over 8 weeks, limb atrophy, moderately elevated protein counts in the cerebrospinal fluid, and significant improvement with intravenous human immunoglobulin, which were characteristic of acquired inflammatory neuropathies, he was eventually diagnosed with CIDP superimposed on CMT1A. However, after a three-month plateau, the patient contracted COVID-19, which led to repeated and worsening symptoms of limb weakness and atrophy, thus was diagnosed with a recurrence of CIDP and treated with Intravenous immunoglobulin and methylprednisolone 500 mg/d for 5 consecutive days, followed by oral prednisone and mycophenolate mofetil tablets. On 2 month follow-up, he exhibited remarkable clinical improvement and could walk independently with rocking gait. After 1 year of follow-up, the patient\'s condition was stable without further change.
UNASSIGNED: Our case indicates that CMT1A can deteriorate after SARS-CoV-2 vaccination. Thus, SARS-CoV-2 vaccination should be considered a potential predisposing factor for CMT1A worsening. The possible superposition of CMTIA and CIDP in the context of SARS-CoV-2 infection or immunity suggests that any clinical exacerbation in patients with CMT1A should be carefully evaluated to rule out treatable superposition inflammation. In addition, electrophysiological and imaging examination of the proximal nerves, such as the axillary nerve, is helpful for the diagnosis of CIDP.

OBJECTIVE: Hereditary neuropathy with liability to pressure palsy (HNPP) is a rare autosomal dominant peripheral neuropathy, usually caused by heterozygous deletion mutations in the peripheral myelin protein 22 (PMP22) gene. This study aims to investigate the clinical and molecular genetic characteristics of HNPP.
METHODS: HNPP patients in the Department of Neurology at Third Xiangya Hospital of Central South University from 2009 to 2023 were included in this study. The general clinical data, nervous electrophysiological and molecular genetic examination results were collected and analyzed. Molecular genetic examination was to screen for deletion of PMP22 gene using multiplex ligation-dependent probe amplification (MLPA) after extracting genomic DNA from peripheral blood; and if no PMP22 deletion mutation was detected, next-generation sequencing was used to screen for PMP22 point mutations. The related literatures of HNPP were reviewed, and the clinical and molecular genetic characteristics of HNPP patients were analyzed.
RESULTS: A total of 34 HNPP patients from 24 unrelated Chinese Han families were included in this study, including 25 males and 9 females. The average age at illness onset was 22.0 years. Sixty-two point five percent of the families had a positive family history. Among them, 30 patients had symptoms of peripheral nerve paralysis. Patients often presented with paroxysmal single limb weakness with (or) numbness (25/30), and some patients had paroxysmal unilateral recurrent laryngeal nerve (vagus nerve) paralysis (2/30). Physical examination revealed muscle weakness (23/29), hypoesthesia (9/29), weakened or absent ankle reflexes (20/29), distal limb muscle atrophy (8/29) and high arched feet (5/29). Most patients (26/30) could fully recover to normal after an acute attack. Thirty-one patients in our group underwent nervous electrophysiological examination, and showed multiple demyelinating peripheral neuropathies with both motor and sensory nerves involved. Most patients showed significantly prolonged distal motor latency (DML), mild to moderate nerve conduction velocity slowing, decreased amplitude of compound muscle action potential (CMAP) and sensory nerve action potential (SNAP), and sometimes with conduction block. Nerve motor conduction velocity was (48.5±5.5) m/s, and the CMAP amplitude was (8.4±5.1) mV. Nerve sensory conduction velocity was (37.4±10.5) m/s, and the SNAP amplitude was (14.4±15.2) μV. There were 24 families, 23 of whom had the classical PMP22 deletion, the last one had a heterozygous pathogenic variant in the PMP22 gene sequence (c.434delT). By reviewing clinical data and genetic testing results of reported 1 734 HNPP families, we found that heterozygous deletion mutation of PMP22 was the most common pathogenic mutation of HNPP (93.4%). Other patients were caused by PMP22 small mutations (4.0%), PMP22 heterozygous gross deletions (0.6%), and PMP22 complex rearrangements (0.1%). Thirty-eight sorts of HNPP-related PMP22 small mutations was reported, including missense mutations (10/38), nonsense mutations (4/38), base deletion mutations (13/38), base insertion mutations (3/38), and shear site mutations (8/38). HNPP patients most often presented with episodic painless single nerve palsy. Common peroneal nerve, ulnar nerve, and brachial plexus nerve were the most common involved nerves, accounting for about 75%. Only eighteen patients with cranial nerve involved was reported.
CONCLUSIONS: Heterozygous deletion mutation of PMP22 is the most common pathogenic mutation of HNPP. Patients is characterized by episodic and painless peripheral nerve paralysis, mainly involving common peroneal nerve, ulnar nerve, and other peripheral nerves. Nervous electrophysiological examination has high sensitivity and specificity for the diagnosis of HNPP, which is manifested by extensive demyelinating changes. For patients with suspected HNPP, nervous electrophysiological examination and PMP22-MLPA detection are preferred. Sanger sequencing or next generation sequencing can be considered to detect other mutations of PMP22.
目的: 遗传性压力易感性周围神经病(hereditary neuropathy with liability to pressure palsy，HNPP)是一种少见的常染色体显性遗传周围神经病，通常由周围髓鞘蛋白22(peripheral myelin protein 22，PMP22)基因杂合缺失突变引起。本研究旨在探讨HNPP患者的临床和分子遗传学特征。方法: 纳入2009至2023年就诊于中南大学湘雅三医院神经内科的HNPP患者，收集并分析患者的一般临床资料、神经电生理和分子遗传学检查结果。分子遗传学检查为提取外周血基因组DNA后采用多重连接探针扩增技术(multiplex ligation-dependent probe amplification，MLPA)进行PMP22大片段缺失的筛查；若未检测到PMP22缺失突变，则用二代测序法筛查PMP22点突变。进一步对HNPP相关文献进行回顾，分析HNPP患者的临床和分子遗传学特征。结果: 共纳入来自24个无血缘关系的中国汉族家系的34例HNPP患者，包括25名男性和9名女性，平均22.0岁起病，有阳性家族史的家系占62.5%。30例患者出现周围神经麻痹的症状，常表现为发作性单肢无力伴/或麻木(25/30)，亦可表现为发作性单侧喉返神经(迷走神经)麻痹(2/30)。体格检查可发现受累神经相应支配区域的肌肉无力(23/29)和浅感觉减退(9/29)，踝反射减弱或消失(20/29)，肢体远端肌肉萎缩(8/29)及高弓足(5/29)。多数患者(26/30)在急性发作后可完全恢复正常。有31例患者完成神经电生理检查，均表现为多发性周围神经损害，以运动及感觉神经髓鞘损害为主，多数患者可有远端运动潜伏期(distal motor latency，DML)明显延长，轻至中度的神经传导速度减慢，复合肌肉动作电位(compound muscle action potential，CMAP)及感觉神经动作电位(sensory nerve action potential，SNAP)波幅降低，有时可出现传导阻滞。正中神经运动传导速度为(48.5±5.5) m/s，CMAP波幅为(8.4±5.1) mV；正中神经感觉传导速度为(37.4±10.5) m/s，SNAP波幅为(14.4±13.0) μV。在24个HNPP家系中，经MLPA检测证实有23个家系为PMP22杂合缺失突变导致，经二代测序证实剩余1个家系为PMP22 c.434delT突变所致。文献检索到1 734个进行了基因检测的HNPP确诊家系，其基因检测结果再次证实了PMP22杂合缺失突变是最常见的突变类型，占93.4%，其余突变类型包括PMP22微小突变(4.0%)、PMP22杂合不完全缺失突变(0.6%)、PMP22复杂易位突变(0.1%)。目前HNPP相关的PMP22微小突变共报道38种，包括错义突变(10/38)、无义突变(4/38)、碱基缺失突变(13/38)、碱基插入突变(3/38)、剪切位点突变(8/38)。HNPP患者最常表现为发作性无痛性单神经麻痹，腓总神经、尺神经和臂丛神经受累最为常见，约占75%。颅神经受累的患者仅有18例报道。结论: PMP22杂合缺失突变是HNPP最常见的突变类型。HNPP以发作性无痛性单神经麻痹为主要特点，主要累及腓总神经、尺神经等周围神经。神经电生理检查对于诊断本病具有较高的灵敏度和特异度，表现为广泛的脱髓鞘改变。对于怀疑HNPP的患者，首选完善神经电生理检查及PMP22大片段杂合缺失的检测。必要时可以完善Sanger测序或二代测序，对PMP22其他突变类型进行检测以防漏诊。.

Hereditary neuropathy with liability to pressure palsies (HNPP) is a peripheral neuropathy with autosomal dominant inheritance. Diagnosis can be made from the characteristic abnormalities determined by nerve conduction studies (NCS), including subclinical deficits at physiological compression sites. Heterozygous deletion of the chromosome 17p11.2-p12 region including the peripheral myelin protein 22 gene (PMP22) is the cause in the majority of cases. However, the loss of function of PMP22 due to frameshift-causing insertion/deletion, missense, nonsense, or splice-site disrupting variants cause HNPP in some patients. We report a case of a patient diagnosed with HNPP on the basis of clinical features and the results of NCS. No deletions of PMP22 were detected by fluorescence in situ hybridization.
We performed direct nucleotide sequence analysis and identified a heterozygous variant, c.78 + 3G > T, in PMP22. Since this variant is located outside the canonical splice site at the exon 2-intron 2 junction, we investigated whether the variant causes aberrant splicing and leads to the skipping of exon 2 of PMP22 by in vitro minigene splicing assay.
We demonstrated that the c.78 + 3G > T variant causes the skipping of exon 2 and leads to loss of function of the mutant allele.
Searching for sequence variants located outside the canonical splice sites should also be considered even when deletion of PMP22 is not found in a patient with a clinical diagnosis suggesting HNPP.

Charcot-Marie-Tooth disease subtype 1A (CMT1A) is one of the most prevalent demyelinating peripheral neuropathies worldwide, caused by duplication of the peripheral myelin protein 22 (PMP22) gene, which is expressed primarily in Schwann cells (SCs). PMP22 overexpression in SCs leads to intracellular aggregation of the protein, which eventually results in demyelination. Unfortunately, previous biochemical approaches have not resulted in an approved treatment for CMT1A disease, compelling the pursuit for a biophysical approach such as electrical stimulation (ES). However, the effects of ES on CMT1A SCs have remained unexplored. In this study, we established PMP22-overexpressed Schwannoma cells as a CMT1A in vitro model, and investigated the biomolecular changes upon applying ES via a custom-made high-throughput ES platform, screening for the condition that delivers optimal therapeutic effects. While PMP22-overexpressed Schwannoma exhibited intracellular PMP22 aggregation, ES at 20 Hz for 1 h improved this phenomenon, bringing PMP22 distribution closer to healthy condition. ES at this condition also enhanced the expression of the genes encoding myelin basic protein (MBP) and myelin-associated glycoprotein (MAG), which are essential for assembling myelin sheath. Furthermore, ES altered the gene expression for myelination-regulating transcription factors Krox-20, Oct-6, c-Jun and Sox10, inducing pro-myelinating effects in PMP22-overexpressed Schwannoma. While electroceuticals has previously been applied in the peripheral nervous system towards acquired peripheral neuropathies such as pain and nerve injury, this study demonstrates its effectiveness towards ameliorating biomolecular abnormalities in an in vitro model of CMT1A, an inherited peripheral neuropathy. These findings will facilitate the clinical translation of an electroceutical treatment for CMT1A.

Data from gnomAD indicate that a missense mutation encoding the T118M variation in human peripheral myelin protein 22 (PMP22) is found in roughly one of every 75 genomes of western European lineage (1:120 in the overall human population). It is unusual among PMP22 variants that cause Charcot-Marie-Tooth (CMT) disease in that it is not 100% penetrant. Here, we conducted cellular and biophysical studies to determine why T118M PMP22 predisposes humans to CMT, but with only incomplete penetrance. We found that T118M PMP22 is prone to mistraffic but differs even from the WT protein in that increased expression levels do not result in a reduction in trafficking efficiency. Moreover, the T118M mutant exhibits a reduced tendency to form large intracellular aggregates relative to other disease mutants and even WT PMP22. NMR spectroscopy revealed that the structure and dynamics of T118M PMP22 resembled those of WT. These results show that the main consequence of T118M PMP22 in WT/T118M heterozygous individuals is a reduction in surface-trafficked PMP22, unaccompanied by formation of toxic intracellular aggregates. This explains the incomplete disease penetrance and the mild neuropathy observed for WT/T118M CMT cases. We also analyzed BioVU, a biobank linked to deidentified electronic medical records, and found a statistically robust association of the T118M mutation with the occurrence of long and/or repeated episodes of carpal tunnel syndrome. Collectively, our results illuminate the cellular effects of the T118M PMP22 variation leading to CMT disease and indicate a second disorder for which it is a risk factor.

Peripheral myelin protein 22 (PMP22) and epithelial membrane proteins (EMP-1, -2, and -3) belong to a small hydrophobic membrane protein subfamily, with four transmembrane structures. PMP22 and EMPs are widely expressed in various tissues and play important roles in cell growth, differentiation, programmed cell death, and metastasis. PMP22 presents its highest expression in the peripheral nerve and participates in normal physiological and pathological processes of the peripheral nervous system. The progress of molecular genetics has shown that the genetic changes of the PMP22 gene, including duplication, deletion, and point mutation, are behind various hereditary peripheral neuropathies. EMPs have different expression patterns in diverse tissues and are closely related to the risk of malignant tumor progression. In this review, we focus on the four members in this protein family which are related to disease pathogenesis and discuss gene mutations and post-translational modification of them. Further research into the interactions between structural alterations and function of PMP22 and EMPs will help understand their normal physiological function and role in diseases and might contribute to developing novel therapeutic tools.