Neuropathy due to bi-allelic SH3TC2 variants: genotype-phenotype correlation and natural history.

Recessive SH3TC2 variants cause Charcot-Marie-Tooth disease type 4C (CMT4C). CMT4C is typically a sensorimotor demyelinating polyneuropathy, marked by early onset spinal deformities, but its clinical characteristics and severity are quite variable. Clear relationships between pathogenic variants and the spectrum of disease manifestations are to date lacking. Gene replacement therapy has been shown to ameliorate the phenotype in a mouse model of CMT4C, emphasizing the need for natural history studies to inform clinical trial readiness. Data, including both genetic information and clinical characteristics, were compiled from the longitudinal, prospective dataset of the Inherited Neuropathy Consortium, a member of the Rare Diseases Clinical Research Network (INC-RDCRN). The Charcot Marie Tooth Neuropathy Score (CMTNS), Examination Score (CMTES) and the Rasch-weighted CMTES (CMTES-R) were used to describe symptoms, neurological examinations and neurophysiological characteristics. Standardized response means were calculated at yearly intervals and a mixed model for repeated measures was used to estimate the change in CMTES and CMTES-R over time. Fifty-six individuals (59% female), median age 27 years (range 2-67 years) with homozygous or compound heterozygous variants in SH3TC2 were identified, including 34 unique variants, 14 of which have not previously been published. Twenty-eight participants had longitudinal data available. While there was no significant difference in the CMTES in those with protein truncating versus non-protein truncating variants, there were significant differences in the mean ulnar nerve compound muscle action potential amplitude, the mean radial sensory nerve action potential amplitude, and in the prevalence of scoliosis, suggesting the possibility of a milder phenotype in individuals with one or two non-protein-truncating variants. Overall, the mean value of the CMTES was 13, reflecting moderate clinical severity. There was a high rate of scoliosis (81%), scoliosis surgery (36%), and walking difficulty (94%) among study participants. The CMTES and CMTES-R appeared moderately responsive to change over extended follow-up, demonstrating a standardized response mean of 0.81 standard deviation units or 0.71 standard deviation units, respectively, over 3 years. Our analysis represents the largest cross-sectional and only longitudinal study to date, of the clinical phenotype of both adults and children with CMT4C. With the promise of upcoming genetic treatments, these data will further define the natural history of the disease and inform study design in preparation for clinical trials.

Macrophage depletion blocks congenital SARM1-dependent neuropathy.

Axon loss contributes to many common neurodegenerative disorders. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of programmed axon degeneration. We identified 2 rare NMNAT2 missense variants in 2 brothers afflicted with a progressive neuropathy syndrome. The polymorphisms resulted in amino acid substitutions V98M and R232Q, which reduced NMNAT2 NAD+-synthetase activity. We generated a mouse model to mirror the human syndrome and found that Nmnat2V98M/R232Q compound-heterozygous CRISPR mice survived to adulthood but developed progressive motor dysfunction, peripheral axon loss, and macrophage infiltration. These disease phenotypes were all SARM1-dependent. Remarkably, macrophage depletion therapy blocked and reversed neuropathic phenotypes in Nmnat2V98M/R232Q mice, identifying a SARM1-dependent neuroimmune mechanism as a key driver of disease pathogenesis. These findings demonstrate that SARM1 induced inflammatory neuropathy and highlight the potential of immune therapy as a treatment for this rare syndrome and other neurodegenerative conditions associated with NMNAT2 loss and SARM1 activation.

Team Approach: Management of Brachial Plexus Birth Injury.

Brachial plexus birth injury is an upper-extremity paralysis that occurs from a traction injury to the brachial plexus during birth. Approximately 10% to 30% of children with a brachial plexus birth injury have residual neurologic deficits with associated impact on upper-limb function. Management of brachial plexus birth injuries with a multidisciplinary team allows optimization of functional recovery while avoiding unnecessary intervention. Early occupational therapy should be initiated with a focus on range of motion and motor learning. The need for microsurgical reconstruction of the brachial plexus can be predicted based on early physical examination findings, and reconstruction is generally performed at 3 to 9 months of age. The majority of children with residual neurologic deficits develop associated glenohumeral dysplasia. These children may require secondary procedures, including botulinum toxin injection, subscapularis and pectoralis lengthening, shoulder capsular release, shoulder tendon transfer, and humeral osteotomy.

Thoracoscopic thymectomy for juvenile myasthenia gravis.

PURPOSE: A randomized controlled trial of thymectomy in myasthenia gravis demonstrated improved clinical outcomes in adults, but data surrounding juvenile cases, especially those treated with minimally invasive approaches, are limited. Here, we review our experience with thoracoscopic thymectomy for juvenile myasthenia gravis (JMG) in the largest cohort to date. METHODS: All cases of thymectomy for JMG in a single tertiary referral center between 2007 and 2018 were reviewed (N = 50). Patients underwent left thoracoscopic approach with extended dissection and without use of monopolar energy. Demographics, diagnostic criteria, and clinical classification, as well as surgical data were collected. Clinical status and medications were reviewed in follow-up. RESULTS: The mean age at surgery was 10.5 ± 0.8 years. Ocular disease and generalized disease each comprised half of the cohort. No patients suffered complications or increased risk of morbidity or mortality with thymectomy. At any interval of follow-up through 3.5 years, 49.8% of patients were improved compared to their pre-operative presentation, and there was a significant trend towards decreased steroid use. CONCLUSION: Thoracoscopic thymectomy is a safe treatment for juvenile myasthenia gravis in pediatric patients over a wide range of ages, body masses, and symptoms. Our experience adds evidence that pediatric patients likely benefit from thymectomy with improved clinical status and reduced medications.

Variation in SIPA1L2 is correlated with phenotype modification in Charcot- Marie- Tooth disease type 1A.

OBJECTIVE: Genetic modifiers in rare disease have long been suspected to contribute to the considerable variance in disease expression, including Charcot-Marie-Tooth disease type 1A (CMT1A). To address this question, the Inherited Neuropathy Consortium collected a large standardized sample of such rare CMT1A patients over a period of 8 years. CMT1A is caused in most patients by a uniformly sized 1.5 Mb duplication event involving the gene PMP22. METHODS: We genotyped DNA samples from 971 CMT1A patients on Illumina BeadChips. Genome-wide analysis was performed in a subset of 330 of these patients, who expressed the extremes of a hallmark symptom: mild and severe foot dorsiflexion strength impairment. SIPA1L2 (signal-induced proliferation-associated 1 like 2), the top identified candidate modifier gene, was expressed in the peripheral nerve, and our functional studies identified and confirmed interacting proteins using coimmunoprecipitation analysis, mass spectrometry, and immunocytochemistry. Chromatin immunoprecipitation and in vitro siRNA experiments were used to analyze gene regulation. RESULTS: We identified significant association of 4 single nucleotide polymorphisms (rs10910527, rs7536385, rs4649265, rs1547740) in SIPA1L2 with foot dorsiflexion strength (p < 1 × 10-7 ). Coimmunoprecipitation and mass spectroscopy studies identified ß-actin and MYH9 as SIPA1L2 binding partners. Furthermore, we show that SIPA1L2 is part of a myelination-associated coexpressed network regulated by the master transcription factor SOX10. Importantly, in vitro knockdown of SIPA1L2 in Schwannoma cells led to a significant reduction of PMP22 expression, hinting at a potential strategy for drug development. INTERPRETATION: SIPA1L2 is a potential genetic modifier of CMT1A phenotypic expressions and offers a new pathway to therapeutic interventions. ANN NEUROL 2019;85:316-330.

STAC3 variants cause a congenital myopathy with distinctive dysmorphic features and malignant hyperthermia susceptibility.

SH3 and cysteine-rich domain-containing protein 3 (STAC3) is an essential component of the skeletal muscle excitation-contraction coupling (ECC) machinery, though its role and function are not yet completely understood. Here, we report 18 patients carrying a homozygous p.(Trp284Ser) STAC3 variant in addition to a patient compound heterozygous for the p.(Trp284Ser) and a novel splice site change (c.997-1G > T). Clinical severity ranged from prenatal onset with severe features at birth, to a milder and slowly progressive congenital myopathy phenotype. A malignant hyperthermia (MH)-like reaction had occurred in several patients. The functional analysis demonstrated impaired ECC. In particular, KCl-induced membrane depolarization resulted in significantly reduced sarcoplasmic reticulum Ca2+ release. Co-immunoprecipitation of STAC3 with CaV 1.1 in patients and control muscle samples showed that the protein interaction between STAC3 and CaV 1.1 was not significantly affected by the STAC3 variants. This study demonstrates that STAC3 gene analysis should be included in the diagnostic work up of patients of any ethnicity presenting with congenital myopathy, in particular if a history of MH-like episodes is reported. While the precise pathomechanism remains to be elucidated, our functional characterization of STAC3 variants revealed that defective ECC is not a result of CaV 1.1 sarcolemma mislocalization or impaired STAC3-CaV 1.1 interaction.

A multicenter, retrospective medical record review of X-linked myotubular myopathy: The recensus study.

INTRODUCTION: X-linked myotubular myopathy (XLMTM), characterized by severe hypotonia, weakness, respiratory distress, and early mortality, is rare and natural history studies are few. METHODS: RECENSUS is a multicenter chart review of male XLMTM patients characterizing disease burden and unmet medical needs. Data were collected between September 2014 and June 2016. RESULTS: Analysis included 112 patients at six clinical sites. Most recent patient age recorded was ≤18 months for 40 patients and >18 months for 72 patients. Mean (SD) age at diagnosis was 3.7 (3.7) months and 54.3 (77.1) months, respectively. Mortality was 44% (64% ≤18 months; 32% >18 months). Premature delivery occurred in 34/110 (31%) births. Nearly all patients (90%) required respiratory support at birth. In the first year of life, patients underwent an average of 3.7 surgeries and spent 35% of the year in the hospital. DISCUSSION: XLMTM is associated with high mortality, disease burden, and healthcare utilization. Muscle Nerve 57: 550-560, 2018.

Common and variable clinical, histological, and imaging findings of recessive RYR1-related centronuclear myopathy patients.

Mutations in RYR1 give rise to diverse skeletal muscle phenotypes, ranging from classical central core disease to susceptibility to malignant hyperthermia. Next-generation sequencing has recently shown that RYR1 is implicated in a wide variety of additional myopathies, including centronuclear myopathy. In this work, we established an international cohort of 21 patients from 18 families with autosomal recessive RYR1-related centronuclear myopathy, to better define the clinical, imaging, and histological spectrum of this disorder. Early onset of symptoms with hypotonia, motor developmental delay, proximal muscle weakness, and a stable course were common clinical features in the cohort. Ptosis and/or ophthalmoparesis, facial weakness, thoracic deformities, and spinal involvement were also frequent but variable. A common imaging pattern consisted of selective involvement of the vastus lateralis, adductor magnus, and biceps brachii in comparison to adjacent muscles. In addition to a variable prominence of central nuclei, muscle biopsy from 20 patients showed type 1 fiber predominance and a wide range of intermyofibrillary architecture abnormalities. All families harbored compound heterozygous mutations, most commonly a truncating mutation combined with a missense mutation. This work expands the phenotypic characterization of patients with recessive RYR1-related centronuclear myopathy by highlighting common and variable clinical, histological, and imaging findings in these patients.

A 6-Year-Old With Leg Cramps.

A 6-year-old girl presented with a history of leg pain and cramping that progressively worsened over a 2- to 3-week period of time. Her examination was notable for normal vital signs, limited range of motion of her left hip, and a limp. Inflammatory markers were slightly elevated, but the serum electrolytes, calcium, and magnesium, complete blood cell count and differential, and creatine kinase level were normal. She was hospitalized for further diagnostic evaluation and was noted to have abnormal muscle movements classified as myokymia (continuous involuntary quivering, rippling, or undulating movement of muscles). Electromyography confirmed the myokymia but did not reveal evidence of a myopathy or neuropathy, prompting additional evaluation for a systemic etiology.

Melanocytic tumors express connexin 43 but not 26: immunohistochemical analysis with potential significance in melanocytic oncogenesis.

Connexins (Cx) are structural proteins that form gap junctions, which are vital to cell-cell communication and help to regulate cell division. The purpose of this study was to evaluate if there are diagnostically important differences in immunostaining for connexins 43 (Cx43) and 26 (Cx26) in melanoma compared with nevi. Formalin-fixed paraffin-embedded sections of 34 histologically well-characterized melanocytic lesions, 17 primary malignant melanomas (MM), and 17 nevi were stained with a polyclonal antibody to Cx43 and a polyclonal antibody to Cx26. Immunoreactivity in tumor cells was evaluated semiquantitatively based on extent (1%-100%) and intensity (0-3) of reactivity. A score of 0-300 was generated by the product of the extent and intensity readings in each case. Significantly higher Cx43 immunoreactivity was detected in MM (mean intensity score = 253.5; 95% confidence interval, 227.9-279.2; P = 0.002) compared with nevi (mean intensity score = 152.4; 95% confidence interval, 104.9-199.8). In contrast, Cx26 immunoreactivity was less than 5% or entirely absent in all melanocytic tumors (n = 34). The significantly higher Cx43 staining in MM when compared with nevi suggests an oncogenic role for this protein in melanocytic tumor progression. Consequently, the evaluation of immunohistochemical staining for Cx43 in conjunction with other ancillary stains and tumor histology may be helpful in distinguishing MM from nevi, although positive Cx26 reactivity suggests that a cutaneous neoplasm is of nonmelanocytic origin.

Aberrant connexin 43 and 26 expression in cervical dysplasia.

OBJECTIVE: To determine whether connexin (Cx) expression is altered in cervical dysplasia. STUDY DESIGN: Cx proteins form gap junctions and are expressed in squamous epithelia including ectocervix. We used multispectral imaging to perform a quantitative immunohistochemical survey of Cx43 Cx26 in 37 archival human cervical specimens. RESULTS: Cx43 expression was very low in normal cervix (100%), but was increased in low-grade squamous intraepithelial lesions (LSILs) (64%), primarily in a parabasal distribution. High-grade squamous intraepithelial lesions (HSILs) showed weak full-thickness Cx43 staining (53%) or lacked Cx43 (47%). An aberrant increase in Cx43 expression was often (62%) present in histologically normal areas of specimens that elsewhere harbored dysplasia. Cx26 was highly expressed in the basal layer of normal ectocervix (100%). In LSIL, 57% showed a decrease in Cx26 and the rest showed no change relative to the normal pattern. In HSIL, Cx26 was expressed in the full thickness of the epithelium, at a high level in 80% of cases and a low level in the rest. CONCLUSION: Cx alteration is moderately consistent in cervical dysplasia, and for Cx43 can precede histologic changes. The resulting changes in Cx signaling may be important in the pathogenesis of cervical intraepithelial neoplasia.

Connexin43 Expression Increases in the Epithelium and Stroma along the Colonic Neoplastic Progression Pathway: Implications for Its Oncogenic Role.

Connexins (Cxs) are critical for normal tissue development, differentiation, and cell proliferation. Normal expression and function of Cxs are considered to play a role in tumor suppression, but abnormal localization and abnormally increased expression of Cxs have been found in a variety of carcinomas. Of the Cx family, Cx43 is a most prevalent member and has been known as a downstream target of ß-catenin, a key component of Wnt signaling pathway. We evaluated the expression of Cx43 in the colonic neoplasia progression sequence with additional attention to the stromal component. Resections of 50 colonic adenocarcinomas were stained immunohistochemically for Cx43 on paraffin embedded sections. Cx43 cytoplasmic expression increased progressively in the colonic adenocarcinoma sequence in both the epithelial [normal (4 ± 1), adenomatous (20 ± 2), cancerous (124 ± 10) (P < 0.01)], and stromal [normal (19 ± 1), cancerous (45 ± 4) (P < 0.01)] components. In the epithelial component, Cx43 was expressed lower in stage I adenocarcinomas (69 ± 12) compared to stage III/IV (158 ± 10, P < 0.01). Additionally, Cx43 was relatively increased in the adenocarcinoma at the invasive tumor front in all stages. Cx43 may play a critical role in the pathogenesis of colon cancer via gap junction or other gap junction independent mechanisms such as the Wnt/ß-catenin pathway.

Dominant Cx26 mutants associated with hearing loss have dominant-negative effects on wild type Cx26.

Mutations in GJB2, the gene encoding the human gap junction protein connexin26 (Cx26), cause either non-syndromic hearing loss or syndromes affecting both hearing and skin. We have investigated whether dominant Cx26 mutants can interact physically with wild type Cx26. HeLa cells stably expressing wild type Cx26 were transiently transfected to co-express nine individual dominant Cx26 mutants; six associated with non-syndromic hearing loss (W44C, W44S, R143Q, D179N, R184Q, and C202F) and three associated with hearing loss and palmoplantar keratoderma (G59A, R75Q, and R75W). All mutants co-localized and co-immunoprecipitated with wild type Cx26, indicating that they interact physically, likely by forming admixed heteromeric/heterotypic channels. Furthermore, all nine mutants inhibited the transfer of calcein in cells stably expressing Cx26, demonstrating that they each have dominant effects on wild type Cx26. Taken together, these results show that dominant-negative effects of these Cx26 mutants likely contribute to the pathogenesis of hearing loss.

A novel recessive Nefl mutation causes a severe, early-onset axonal neuropathy.

OBJECTIVE: To report the first cases of a homozygous recessive mutation in NEFL, the gene that encodes the light subunit of neurofilaments. METHODS: Clinical and electrophysiologic data were evaluated, and a sural nerve biopsy from one affected child was examined by immunohistochemistry and electron microscopy. The ability of the mutant protein to form filaments was characterized in an established cell culture system. RESULTS: Four of five siblings developed of a severe, progressive neuropathy beginning in early childhood. Serial nerve conduction studies showed progressively reduced amplitudes with age and pronounced slowing at all ages. Visual-evoked responses were slowed in three children, indicating that central nervous system axons were subclinically involved. All four affected children were homozygous for a nonsense mutation at glutamate 210 (E210X) in the NEFL gene; both parents were heterozygous carriers. A sural nerve biopsy from an affected patient showed markedly reduced numbers of myelinated axons; the remaining myelinated axons were small and lacked intermediate filaments. The E210X mutant protein did not form an intermediate filament network and did not interfere with the filament formation by wild-type human light subunit of neurofilaments in SW-13 vim(-) cells. INTERPRETATION: This is the first demonstration of a recessive NEFL mutation, which appears to cause a simple loss of function, resulting in a severe, early-onset axonal neuropathy with unique features. These results confirm that neurofilaments are the main determinant of axonal caliber and conduction velocity, and demonstrate for the first time that neurofilaments are required for the maintenance of myelinated peripheral nervous system axons.

Cellular mechanisms of connexin32 mutations associated with CNS manifestations.

Both oligodendrocytes and myelinating Schwann cells express the gap junction protein connexin32 (Cx32). Mutations in the gene encoding Cx32 (GJB1) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX). Although most CMTX patients do not have clinical central nervous system (CNS) manifestations, subclinical evidence of CNS dysfunction is common. We investigated the cellular effects of a subgroup of GJB1/Cx32 mutations that have been reported to cause clinical CNS dysfunction. We hypothesized that these mutants have dominant-negative effects on other connexins expressed by oligodendrocytes, specifically Cx45. We expressed these and other Cx32 mutants in communication-incompetent as well as Cx45-expressing HeLa cells, and analyzed the transfected cells by immunocytochemistry and immunoblotting. In communication-incompetent cells, the mutants associated with CNS phenotypes failed to reach the cell membrane and were instead retained in the endoplasmic reticulum (A39V, T55I) or Golgi apparatus (M93V, R164Q, R183H), although rare gap junction plaques were found in cells expressing M93V or R183H. In HeLa cells stably expressing Cx45, these Cx32 mutants showed a similar expression pattern, and did not alter the pattern of Cx45 expression. These results indicate that Cx32 mutants that are associated with a CNS phenotype do not interact with Cx45, but may instead have other toxic effects in oligodendrocytes.