CHRNA1 promotes the pathogenesis of primary focal hyperhidrosis.

The aim of the study was to elucidate the involvement of cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) in the pathogenesis of primary focal hyperhidrosis (PFH). The hyperhidrosis mouse model was constructed using pilocarpine injection. The expression levels of CHRNA1 in sweat gland tissues of PFH patients and hyperhidrosis mice were compared using Western blots and quantitative real-time PCR (qRT-PCR) analyses. Sweat secretion in hyperhidrosis mice treated with small-interfering RNA (siRNA) targeting CHRNA1 (si-CHRNA1) or non-specific siRNA were compared. Sweat secretory granules in the sweat gland cells of hyperhidrosis mice were examined using transmission electron microscopy. The serum level of acetylcholine was measured using enzyme-linked immunosorbent assay, while markers associated with PFH, including Aquaporin 5 (AQP5) and Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C), were assessed using immunohistochemical assay and Western blots. Brain-derived neurotrophic factor (BDNF) and Neuregulin 1 (NRG-1) in sympathetic ganglia axons of hyperhidrosis mice were quantified using Western blots. CHRNA1 up-regulation is a characteristic of the sweat glands of PFH patients and Hyperhidrosis mice. Silencing CHRNA1 decreased sweat secretion and the number of sweat secretory granules of hyperhidrosis mice. Serum acetylcholine, as well as AQP5 and CACNA1C expression in the sweat glands, was reduced by siCHRNA1. BDNF1 and NRG-1 levels in the sympathetic ganglia axons were also attenuated by siCHRNA1 treatment. CHRNA1 up-regulation is a potential biomarker of PFH and downregulating CHRNA1 could alleviate the symptoms of PFH through inactivating the sympathetic system.

Late presentations of congenital myasthenic syndromes: How many do we miss?

INTRODUCTION: Congenital myasthenic syndromes (CMS) usually present neonatally or in early childhood. When they present later, they may be mistaken for seronegative autoimmune myasthenia, and unnecessary immunosuppressive treatment may be administered. METHODS: Patients who met criteria for seronegative generalized myasthenia without congenital or early childhood onset, but with an affected sibling were tested for CMS associated genes using exome and Sanger sequencing. RESULTS: Four sibling pairs from nonconsanguineous families were identified. Three had mutations in the RAPSN gene, and 1 had a mutation in CHRNA1. One sibling of a pair with symptoms of fatigue but no convincing features of neuromuscular dysfunction tested negative on genetic studies. The definite CMS cases comprised 7 of 25 seronegative patients with definite generalized myasthenia in the clinic, and over half had been treated for autoimmune myasthenia. CONCLUSIONS: CMS is probably underdiagnosed in seronegative myasthenic disorders and should be considered in the differential diagnosis. Muscle Nerve 54: 721-727, 2016.

Antisense oligonucleotide-mediated exon skipping of CHRNA1 pre-mRNA as potential therapy for Congenital Myasthenic Syndromes.

CHRNA1 encodes the α subunit of nicotinic acetylcholine receptors (nAChRs) and is expressed at the neuromuscular junction. Moreover, it is one of the causative genes of Congenital Myasthenic Syndromes (CMS). CHRNA1 undergoes alternative splicing to produce two splice variants: P3A(-), without exon P3A, and P3A(+), with the exon P3A. Only P3A(-) forms functional nAChR. Aberrant alternative splicing caused by intronic or exonic point mutations in patients leads to an extraordinary increase in P3A(+) and a concomitant decrease in P3A(-). Consequently this resulted in a shortage of functional receptors. Aiming to restore the imbalance between the two splice products, antisense oligonucleotides (AONs) were employed to induce exon P3A skipping. Three AON sequences were designed to sterically block the putative binding sequences for splicing factors necessary for exon recognition. Herein, we show that AON complementary to the 5' splice site of the exon was the most effective at exon skipping of the minigene with causative mutations, as well as endogenous wild-type CHRNA1. We conclude that single administration of the AON against the 5' splice site is a promising therapeutic approach for patients based on the dose-dependent effect of the AON and the additive effect of combined AONs. This conclusion is favorable to patients with inherited diseases of uncertain etiology that arise from aberrant splicing leading to a subsequent loss of functional translation products because our findings encourage the option of AON treatment as a therapeutic for these prospectively identified diseases.

CHRNA1 and its correlated-myogenesis/cell cycle genes are prognosis-related markers of metastatic melanoma.

Nicotinic acetylcholine receptors (CHRNs) expression and their critical role in various types of cancer have been reported. However, it is still unclear which CHRNs and their associated genes play essential roles in metastasis in melanoma patients. Here, we performed bioinformatics analyses on publicly available bulk RNA sequencing (RNA-seq) data of patients with melanoma to identify the CHRNs highly expressed in metastatic melanoma. We found that CHRNA1 was highly expressed in metastatic melanoma samples compared to primary melanoma samples and was strongly associated with CHRNB1 and CHRNG. These muscle-type CHRNs (CHRNA1, CHRNB1, and CHRNG) were correlated with the ZEB1 and Rho/ROCK pathway-related genes in metastatic melanoma samples. Pairwise correlations and enrichment analyses revealed that CHRNA1 was significantly associated with myogenesis/muscle contraction and cell cycle genes. Kaplan-Meier curves illustrated the involvement of CHRNA1, four of its correlated genes (DES, FLNC, CDK1, and CDC20), and the myogenesis gene signature in the prognosis of melanoma patients. Following the bulk RNA-seq analysis, single-cell RNA-seq (scRNA-seq) analysis showed that the CHRNA1-expressing melanoma cells are primarily metastatic and had high expression levels of CHRNB1, CHRNG, and myogenesis/cell cycle-related genes. Our bioinformatics analyses of the bulk RNA-seq and scRNA-seq data of patients with melanoma revealed that CHRNA1 and its correlated myogenesis/cell-related cycle genes are critical prognosis-related markers of metastatic melanoma.

PAI1 inhibits the pathogenesis of primary focal hyperhidrosis by targeting CHRNA1.

BACKGROUND: Primary focal hyperhidrosis (PFH) may be attributed to the up-regulation of the cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) in eccrine glands. Plasminogen activator inhibitor-1 (PAI1, encoded by SERPINE1) is reported to inhibit the expression of CHRNA1, while the role of PAI1 in hyperhidrosis is unknown. METHODS: Serpine1 KO mice, Serpine1-Tg mice, and wild type BALB/c mice were intraperitoneally injected with pilocarpine hydrochloride to induce PFH. Cisatracurium (CIS, antagonist of CHRNA1) or PAI-039 (small-molecule inhibitor of PAI1) was pre-administrated before the induction of hyperhidrosis. On the other hand, Chrna1-expressing AAV was constructed and administered to Serpine1-Tg mice with hydrochloride stimulation. Hydrochloride-related biomarkers, such as acetylcholine (ACH) in the serum, calcium voltage-gated channel subunit alpha1 C (CACNA1C), and aquaporin 5 (AQP5) in sweat glands of mice were assayed with ELISA, RT-PCR, and Western blot. RESULTS: The administration of PAI-039 or Pai1 knock-out increased Chrna1 expression, sweat secretion, and hydrochloride-related biomarkers (ACH, CACNA1C, and AQP5) expression. On the other hand, CIS administration diminished the strengthened hyperhidrosis phenotype induced by Pai1 knock-out with decreased sweat gland secretion. CONCLUSION: PAI1 inhibits CHRNA1-mediated hydrochloride-induced hyperhidrosis, with decreased sweat gland secretion and diminished ACH, AQP5, and CACNA1C expression. These results indicate the potential to utilize PAI1 to alleviate PFH.

A novel phenotype of AChR-deficiency syndrome with predominant facial and distal weakness resulting from the inclusion of an evolutionary alternatively-spliced exon in CHRNA1.

Primary acetylcholine receptor deficiency is the most common subtype of congenital myasthenic syndrome, resulting in reduced amount of acetylcholine receptors expressed at the muscle endplate and impaired neuromuscular transmission. AChR deficiency is caused mainly by pathogenic variants in the ε-subunit of the acetylcholine receptor encoded by CHRNE, although pathogenic variants in other subunits are also seen. We report the clinical and molecular features of 13 patients from nine unrelated kinships with acetylcholine receptor deficiency harbouring the CHRNA1 variant NM_001039523.3:c.257G>A (p.Arg86His) in homozygosity or compound heterozygosity. This variant results in the inclusion of an alternatively-spliced evolutionary exon (P3A) that causes expression of a non-functional acetylcholine receptor α-subunit. We compare the clinical findings of this group to the other cases of acetylcholine receptor deficiency within our cohort. We report differences in phenotype, highlighting a predominant pattern of facial and distal weakness in adulthood, predominantly in the upper limbs, which is unusual for acetylcholine receptor deficiency syndromes, and more in keeping with slow-channel syndrome or distal myopathy. Finally, we stress the importance of including alternative exons in variant analysis to increase the probability of achieving a molecular diagnosis.

CHRNA1 induces sarcopenia through neuromuscular synaptic elimination.

Sarcopenia seriously affects the quality of life of the elderly, but its molecular mechanism is still unclear. Degeneration in muscle innervation is related to age-related movement disorders and muscle atrophy. The expression of CHRNA1 is increased in the skeletal muscle of the elderly, and in aging rodents. Therefore, we investigated whether CHRNA1 induces the occurrence and development of sarcopenia. Compared with the control group, local injection of AAV9-CHRNA1 into the hindlimb muscles decreased the percentage of muscle innervation. At the same time, the skeletal muscle mass decreased, as manifested by a decrease in the gastrocnemius mass index and the cross-sectional area of the muscle fibers. The function of skeletal muscle also decreased, which was manifested by decreases of compound muscle action potential and muscle contractility. Therefore, we concluded that upregulation of CHRNA1 can induce and aggravate sarcopenia.

Case Report: Novel compound heterozygous variants in CHRNA1 gene leading to lethal multiple pterygium syndrome: A case report.

Background: Lethal multiple pterygium syndrome (LMPS) is a rare autosomal recessive inherited disorder typically characterized by intrauterine growth retardation, multiple pterygia, and flexion contractures. Case presentation: We herein report a Chinese case with a history of three adverse pregnancies demonstrating the same ultrasonic phenotypes, including increased nuchal translucency, edema, fetal neck cystoma, reduced movement, joint contractures, and other congenital features. Whole-exome sequencing (WES) revealed novel compound heterozygous variants in the CHRNA1 gene NM_000079.4: c.[1128delG (p.Pro377LeufsTer10)]; [505T>C (p.Trp169Arg)] in the recruited individual, and subsequent familial segregation showed that both parents transmitted their respective mutation. Conclusion: For the first time, we identified an association between the CHRNA1 gene and the recurrent lethal multiple pterygium syndrome (LMPS) in a Chinese family. This finding may also enrich the mutation spectrum of the CHRNA1 gene and promote the applications of WES technology in etiologic diagnosis of ultrasound anomalies in prenatal examination.

Rapid Generation of Pigment Free, Immobile Zebrafish Embryos and Larvae in Any Genetic Background Using CRISPR-Cas9 dgRNPs.

The ability to carry out high-resolution, high-magnification optical imaging of living animals is one of the most attractive features of the zebrafish as a model organism. However, increasing amounts of pigmentation as development proceeds and difficulties in maintaining sustained immobilization of healthy, living animals remain challenges for live imaging. Chemical treatments can be used to suppress pigment formation and movement, but these treatments can lead to developmental defects. Genetic mutants can also be used to eliminate pigment formation and immobilize animals, but maintaining these mutants in lines carrying other combinations of transgenes and mutants is difficult and laborious. In this study, we show that CRISPR duplex guide ribonucleoproteins (dgRNPs) targeting the slc45a2 (albino) and chrna1 (nic1) genes can be used to efficiently suppress pigment formation in and immobilize F0 injected animals. CRISPR dgRNPs can be used to generate pigment-free, immobile zebrafish embryos and larvae in any transgenic and/or mutant-carrying background, greatly facilitating high-resolution imaging and analysis of the many transgenic and mutant lines available in the zebrafish.

Widespread Evolution of Molecular Resistance to Snake Venom α-Neurotoxins in Vertebrates.

Venomous snakes are important subjects of study in evolution, ecology, and biomedicine. Many venomous snakes have alpha-neurotoxins (α-neurotoxins) in their venom. These toxins bind the alpha-1 nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction, causing paralysis and asphyxia. Several venomous snakes and their predators have evolved resistance to α-neurotoxins. The resistance is conferred by steric hindrance from N-glycosylated asparagines at amino acids 187 or 189, by an arginine at position 187 that has been hypothesized to either electrostatically repulse positively charged neurotoxins or sterically interfere with α-neurotoxin binding, or proline replacements at positions 194 or 197 of the nAChR ligand-binding domain to inhibit α-neurotoxin binding through structural changes in the receptor. Here, we analyzed this domain in 148 vertebrate species, and assessed its amino acid sequences for resistance-associated mutations. Of these sequences, 89 were sequenced de novo. We find widespread convergent evolution of the N-glycosylation form of resistance in several taxa including venomous snakes and their lizard prey, but not in the snake-eating birds studied. We also document new lineages with the arginine form of inhibition. Using an in vivo assay in four species, we provide further evidence that N-glycosylation mutations reduce the toxicity of cobra venom. The nAChR is of crucial importance for normal neuromuscular function and is highly conserved throughout the vertebrates as a result. Our research shows that the evolution of α-neurotoxins in snakes may well have prompted arms races and mutations to this ancient receptor across a wide range of sympatric vertebrates. These findings underscore the inter-connectedness of the biosphere and the ripple effects that one adaption can have across global ecosystems.

Gene Polymorphisms for Both Auto-antigen and Immune-Modulating Proteins Are Associated with the Susceptibility of Autoimmune Myasthenia Gravis.

Myasthenia gravis (MG) is an antibody-mediated autoimmune disease against antigens at the neuromuscular junction. Both genetic and environmental factors contribute to the susceptibility of MG. We undertook a case-control study to explore the contribution of genes of the auto-antigen and immune-modulating proteins in the pathogenesis of MG. We enrolled 389 adult MG patients and 487 healthy controls. Eighteen SNPs were selected from genes of cholinergic receptor nicotinic alpha 1 (CHRNA1), autoimmune regulator (AIRE), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), protein tyrosine phosphatase nonreceptor type 22 (PTPN22), and interleukin-10 (IL-10). Rs16862847 and rs2229957 in CHRNA1, rs3761389 in AIRE, and rs733618 in CTLA-4 were significantly associated with MG, with the highest association in SNPs of CHRNA1. Carrier of rs16862847 G allele was found to be an independent risk factor in predicting high-level acetylcholine receptor (AChR) antibodies (P = 0.003, OR = 10.296). Genetic interaction analysis revealed a synergistic effect of CHRNA1 (rs16862847), AIRE (rs3761389), and CTLA-4 (rs733618) in the susceptibility of MG (P < 0.0001, OR = 1.95). These findings highlight the role of auto-antigen gene (CHRNA1) in the autoimmune reactions against AChR and reveal synergistic contribution of genes of both auto-antigen and immune-regulating proteins (AIRE and CTLA-4) in the pathogenesis of MG.

Juvenile-onset myasthenia gravis: autoantibody status, clinical characteristics and genetic polymorphisms.

Myasthenia gravis (MG) is an autoimmune disorder mediated by antibodies against proteins at the neuromuscular junction. Juvenile-onset MG (JMG) has been reported to have special characteristics. It is still unclear whether there are any pathogenic and genetic differences between juvenile and adult MG. In this study, we evaluated the clinical characteristics, autoantibody status (antibodies against AChR, MuSK, LRP4, titin and RyR) and genetic susceptibility (CHRNA1, CTLA4 and AIRE) in 114 Chinese JMG patients, and compared with 207 young adult MG patients (onset age 18-40 years). JMG patients were classified into two subgroups: the very early onset group (<8 years) and puberty onset group (8-18 years). The very early onset MG patients had a higher proportion of ocular MG and thymus hyperplasia, compared with puberty onset MG and young adult MG (P < 0.05). AChR antibodies were found in majority of JMG patients and were associated with more severe disease (P < 0.05), while other antibodies were rare in JMG. Moreover, the very early onset MG had a more prominent genetic predisposition than puberty and adult MG, affecting the susceptible genes CHRNA1 and CTLA4. JMG has the same pathogenic background as adult MG, but has typical clinical features and a prominent genetic predisposition in very early onset patients (<8 years). Specific therapeutic considerations are needed.

Role of SLCO1B1, ABCB1, and CHRNA1 gene polymorphisms on the efficacy of rocuronium in Chinese patients.

This study explored the role of SLCO1B1, ABCB1, and CHRNA1 gene polymorphisms on the efficacy and duration of action of rocuronium in Chinese patients. Two hundred seven unrelated Chinese patients scheduled for elective surgery were recruited, and 200 completed the study. Their ABCB1, SLCO1B1, and CHRNA1 genotypes were determined. Demographic and clinical non-genetic data also were collected. The SLCO1B1, ABCB1, and CHRNA1 variants did not affect the onset time of rocuronium. Clinical duration and recovery time of rocuronium were prolonged in patients with the ABCB1 rs1128503TT and SLCO1B1 rs2306283 AG and GG genotypes. We demonstrate that the SLCO1B1 and ABCB1 gene variants could affect the pharmacodynamics of rocuronium. The ABCB1 rs1128503 C>T genotype was the most important factor on the efficacy of rocuronium.