Myotonia congenita: novel mutations in CLCN1 gene

Myotonia congenita belongs to the group of non-dystrophic myotonia caused by mutations of CLCN1gene, which encodes human skeletal muscle chloride channel 1. It can be inherited either in autosomal dominant (Thomsen disease) or recessive (Becker disease) forms. Here we have sequenced all 23 exons and exon-intron boundaries of the CLCN1 gene, in a panel of 5 unrelated Chinese patients with myotonia congenita (2 with dominant and 3 with recessive form). In addition, detailed clinical analysis was performed in these patients to summarize their clinical characteristics in relation to their genotypes. Mutational analyses revealed 7 different point mutations. Of these, we have found 3 novel mutations including 2 missense (R47W, V229M), one splicing (IVS19+2T>C), and 4 known mutations (Y261C,G523D, M560T, G859D). Our data expand the spectrum of CLCN1 mutations and provide insights for genotype–phenotype correlations of myotonia congenita in the Chinese population.     

Electrophysiological Characteristics of Six Mutations in hClC-1 of Korean Patients with Myotonia Congenita

ClC-1 is a member of a large family of voltage-gated chloride channels, abundantly expressed in human skeletal muscle. Mutations in ClC-1 are associated with myotonia congenita (MC) and result in loss of regulation of membrane excitability in skeletal muscle. We studied the electrophysiological characteristics of six mutants found among Korean MC patients, using patch clamp methods in HEK293 cells. Here, we found that the autosomal dominant mutants S189C and P480S displayed reduced chloride conductances compared to WT. Autosomal recessive mutant M128I did not show a typical rapid deactivation of Cl⁻ currents. While sporadic mutant G523D displayed sustained activation of Cl⁻ currents in the whole cell traces, the other sporadic mutants, M373L and M609K, demonstrated rapid deactivations. V_1/2 of these mutants was shifted to more depolarizing potentials. In order to identify potential effects on gating processes, slow and fast gating was analyzed for each mutant. We show that slow gating of the mutants tends to be shifted toward more positive potentials in comparison to WT. Collectively, these six mutants found among Korean patients demonstrated modifications of channel gating behaviors and reduced chloride conductances that likely contribute to the physiologic changes of MC.     

Involvement of helices at the dimer interface in ClC-1 common gating

ClC-1 is a dimeric, double-pored chloride channel that is present in skeletal muscle. Mutations of this channel can result in the condition myotonia, a muscle disorder involving increased muscle stiffness. It has been shown that the dominant form of myotonia often results from mutations that affect the so-called slow, or common, gating process of the ClC-1 channel. Mutations causing dominant myotonia are seen to cluster at the interface of the ClC-1 channel monomers. This study has investigated the role of the H, I, P, and Q helices, which lie on this interface, as well as the G helix, which is situated immediately behind the H and I helices, on ClC-1 gating. 11 mutant ClC-1 channels (T268M, C277S, C278S, S289A, T310M, S312A, V321S, T539A, S541A, M559T, and S572V) were produced using site-directed mutagenesis, and gating properties of these channels were investigated using electrophysiological techniques. Six of the seven mutations in G, H, and I, and two of the four mutations in P and Q, caused shifts of the ClC-1 open probability. In the majority of cases this was due to alterations in the common gating process, with only three of the mutants displaying any change in fast gating. Many of the mutant channels also showed alterations in the kinetics of the common gating process, particularly at positive potentials. The changes observed in common gating were caused by changes in the opening rate (e.g. T310M), the closing rate (e.g. C277S), or both rates. These results indicate that mutations in the helices forming the dimer interface are able to alter the ClC-1 common gating process by changing the energy of the open and/or closed channel states, and hence altering transition rates between these states.     

昆虫钠通道的结构和与击倒抗性有关的基因突变

击倒抗性(kdr)是指昆虫和其他节肢动物由于它们的神经系统对DDT和拟除虫菊酯类杀虫剂的敏感性降低而引起的抗性。电压敏感的钠通道是DDT和拟除虫菊酯类杀虫剂的主要靶标。已知拟除虫菊酯是通过改变位于神经膜上的这类通道而发挥其杀虫效果的,钠通道基因的点突变是产生kdr抗性的主要原因。40年来kdr抗性一直是重要的研究课题,但近10年来在kdr分子生物学方面取得了很大进展。本文主要综述了1996年以来所取得的新进展,着重于钠通道的结构、在14种害虫中与kdr抗性相关的钠通道基因突变及其氨基酸序列的多态性。这些结果有助于对拟除虫菊酯改变钠通道的功能及其机理作进一步探究。     

A novel susceptibility locus in MST1 and gene‐gene interaction network for Crohn's disease in the Chinese population

The incidence of Crohn's disease is increasing in many Asian countries, but considerable differences in genetic susceptibility have been reported between Western and Asian populations. This study aimed to fine‐map 23 previously reported Crohn's disease genes and identify their interactions in the Chinese population by Illumina‐based targeted capture sequencing. Our results showed that the genetic polymorphism A>G at rs144982232 in MST1 showed the most significant association (P = 1.78 × 10^?5; odds ratio = 4.87). JAK2 rs1159782 (T>C) was also strongly associated with Crohn's disease (P = 2.34 × 10^?4; odds ratio = 3.72). Gene‐gene interaction analysis revealed significant interactions between MST1 and other susceptibility genes, including NOD2, MUC19 and ATG16L1 in contributing to Crohn's disease risk. Main genetic associations and gene‐gene interactions were verified using ImmunoChip data set. In conclusion, a novel susceptibility locus in MST1 was identified. Our analysis suggests that MST1 might interact with key susceptibility genes involved in autophagy and bacterial recognition. These findings provide insight into the genetic architecture of Crohn's disease in Chinese and may partially explain the disparity of genetic signals in Crohn's disease susceptibility across different ethnic populations by highlighting the contribution of gene‐gene interactions.     

昆虫击倒抗性基因突变对钠通道功能的影响

该文综述了昆虫钠通道基因的表达与功能特性、击倒抗性突变的功能和这些突变对钠通道门控的影响,以及钠通道基因突变与抗性表现型之间的因果关系;还讨论了这些突变增强击倒抗性的分子机理。     

Sequence CLCN1 and SCN4A in patients with Nondystrophic myotonias in Chinese populations: Genetic and pedigree analysis of 10 families and review of the literature

Myotonia congenita (MC), paramyotonia congenita (PC) and sodium channel myotonias(SCM) were belonged to Non-dystrophic myotonias, in which muscle relaxation is delayed after voluntary or evoked contraction. These diseases can not be simply distinguished only based on symptoms and signs but also on genetics: more than 100 mutations in the CLCN1 gene have been associated with MC, while at least 20 mutations in the SCN4A gene have been associated with PC and SCM. Most of these genetics studies have been conducted outside China, only several MC, PC, and SCM families accepted gene scan were reported in China. Therefore we analyzed genetic mutations in CLCN1 and SCN4A in 10 Chinese families clinically diagnosed with Non-dystrophic myotonias. Our result revealed 12 potential disease-causing mutations(3 mutations were novel) that were present in the probands and affected family members. We also reviewed all available literature on mutations linked to these 3 disease in Chinese populations. Our results may help identify genetic determinants as well as clarify genotype-phenotype relationships.     

Identification of novel mutations of the DAX-1 gene in patients with X-linked adrenal hypoplasia congenita

OBJECTIVE: X-linked adrenal hypoplasia congenita (AHC) is a condition clinically featuring adrenal insufficiency and hypogonadotropic hypogonadism caused by mutations of DAX-1. This study was undertaken to characterize the molecular defects of DAX-1 in 3 unrelated Korean patients with AHC. PATIENTS AND METHODS: Patient 1 is a 6-year-old boy who presented with a salt-losing adrenal crisis in the neonatal period. Patient 2 is a 3-year-old boy who manifested aspiration pneumonia and adrenal insufficiency at the age of 1 month. Patient 3 is a 7-year-old boy who developed an adrenal crisis at the age of 3 days. In each of these patients, DAX-1 was analyzed by direct DNA sequencing after polymerase chain reaction amplification of the entire coding region. RESULTS: Direct sequencing of DAX-1 revealed two novel mutations, 1156_1157delCT in patient 1 and another novel nonsense mutation W105X in patient 2. Patient 3 had complete deletion of DAX-1. In patient 3, serum transaminases and creatine kinase levels were elevated while the glycerol kinase activity of leukocytes was decreased. Markedly elevated glycerol excretion was detected by urine organic acid analysis. Patient 3 was diagnosed as Xp21 contiguous gene syndrome associated with deletions of the entire IL1RAPL, GK genes and the C-terminal region of DMD gene. CONCLUSIONS: Two novel mutations of DAX-1 were detected in 2 unrelated patients with AHC, and complete deletion of DAX-1 in a patient with Xp21 contiguous gene syndrome who also presented with glycerol kinase deficiency, Duchenne muscular dystrophy, and AHC.     

Novel Mutations in the CLCN1 Gene of Myotonia Congenita: 2 Case Reports

Introduction: Myotonia Congenita is an inherited myotonia that is due to a mutation in the skeletal muscle chloride channel CLCN1. These mutations lead to reduced sarcolemmal chloride conductance, causing delayed muscle relaxation that is evident as clinical and electrical myotonia.Methods: We report the clinical presentations of two individuals with Myotonia Congenita (MC).Results: Patient 1 has been diagnosed with the recessive form of MC, known as the Becker variant, and Patient 2 has been diagnosed with the dominant form of MC, known as the Thomsen variant. In both patients, the diagnosis was made based on the clinical presentation, EMG and CLCN1 gene sequencing. Patient 1 also had a muscle biopsy.Conclusions: Genetic testing in both patients reveals previously unidentified mutations in the CLCN1 gene specific to Myotonia Congenita. We report the salient clinical features of each patient and discuss the effects and common types of CLCN1 mutations and review the literature.     

Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations

Missense mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene cause autosomal-recessive Parkinson's disease. To date, little is known about the intrinsic catalytic properties of PINK1 since the human enzyme displays such low kinase activity in vitro. We have discovered that, in contrast to mammalian PINK1, insect orthologues of PINK1 we have investigated-namely Drosophila melanogaster (dPINK1), Tribolium castaneum (TcPINK1) and Pediculus humanus corporis (PhcPINK1)-are active as judged by their ability to phosphorylate the generic substrate myelin basic protein. We have exploited the most active orthologue, TcPINK1, to assess its substrate specificity and elaborated a peptide substrate (PINKtide, KKWIpYRRSPRRR) that can be employed to quantify PINK1 kinase activity. Analysis of PINKtide variants reveal that PINK1 phosphorylates serine or threonine, but not tyrosine, and we show that PINK1 exhibits a preference for a proline at the +1 position relative to the phosphorylation site. We have also, for the first time, been able to investigate the effect of Parkinson's disease-associated PINK1 missense mutations, and found that nearly all those located within the kinase domain, as well as the C-terminal non-catalytic region, markedly suppress kinase activity. This emphasizes the crucial importance of PINK1 kinase activity in preventing the development of Parkinson's disease. Our findings will aid future studies aimed at understanding how the activity of PINK1 is regulated and the identification of physiological substrates.     

Adeno-associated virus-mediated gene delivery approaches for the treatment of CNS disorders

Over the last few years, a large number of preclinical and clinical studies have demonstrated the potential of gene therapy applications using adeno-associated viral (AAV) vectors. Gene transfer via AAV vectors has been particularly successful for the treatment or adjunct therapy of several CNS disorders. The present review summarizes the progress on AAV gene delivery models for three different CNS disorders. In particular, we discuss advances in AAV-mediated gene transfer strategies in animal models of Parkinson's disease, Alzheimer's disease and spinal cord trauma and summarize the results from the first clinical studies using AAV systems.     

Differential regulation of amyloid precursor protein sorting with pathological mutations results in a distinct effect on amyloid‐β production

The deposition of amyloid‐β (Aβ) peptide, which is generated from amyloid precursor protein (APP), is the pathological hallmark of Alzheimer's disease (AD). Three APP familial AD mutations (D678H, D678N, and H677R) located at the sixth and seventh amino acid of Aβ have distinct effect on Aβ aggregation, but their influence on the physiological and pathological roles of APP remain unclear. We found that the D678H mutation strongly enhances amyloidogenic cleavage of APP, thus increasing the production of Aβ. This enhancement of amyloidogenic cleavage is likely because of the acceleration of APP_D678H sorting into the endosomal‐lysosomal pathway. In contrast, the APP_D678N and APP_H677R mutants do not cause the same effects. Therefore, this study indicates a regulatory role of D678H in APP sorting and processing, and provides genetic evidence for the importance of APP sorting in AD pathogenesis.

     

Computational screening of disease associated mutations on NPC1 gene and its structural consequence in Niemann-Pick type-C1

Niemann-Pick disease type C1 （NPC1）, caused by mutations of NPC1 gene, is an inherited lysosomal lipid storage disorder. Loss of functional NPC1 causes the accumulation of free cholesterol （FC） in endocytic organelles that comprised the characteristics of late endosomes and/or lysosomes. In this study we analyzed the pathogenic effect of 103 nsSNPs reported in NPC1 using computational methods. Rl186C, S940L, R958Q and I1061T mutations were predicted as most deleterious and disease associated with NPC1 using SIFT, Polyphen 2.0, PANTHER, PhD-SNP, Pmut and MUTPred tools which were also endorsed with previous in vivo experimental studies. To understand the atomic arrangement in 3D space, the native and disease associated mutant （Rl186C, S940L, R958Q and I1061T） structures were modeled. Quantitative structural and flexibility analysis was conceded to observe the structural consequence of prioritized disease associated mutations （R1186C, S940L, R958Q and I1061T）. Accessible surface area （ASA）, free folding energy （FFE） and hydrogen bond （NH bond） showed more flexibility in 3D space in mutant structures. Based on the quantitative assessment and flexibility analysis of NPC1 variants, I1061T showed the most deleterious effect. Our analysis provides a clear clue to wet laboratory scientists to understand the structural and functional effect of NPCI gene upon mutation.     

Identification of three novel mutations in the COL2A1 gene in four unrelated Chinese families with spondyloepiphyseal dysplasia congenita

Introduction

Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant skeletal dysplasia characterized by short stature, abnormal epiphyses, and flattened vertebral bodies. The condition occurs through a mutation in the COL2A1 gene that encodes the type II procollagen alpha1 chain (proalpha1 (II)).

Method and Results

We investigated nine affected individuals from four unrelated Chinese families with SEDC. We screened for COL2A1 gene mutations, and identified found four missense mutations (G447A, G456A, R789C and G1152D). The G447A, G456A and G1152D mutations are novel and the R789C mutation has been reported previously in several other studies with a strikingly similar phenotype.

Conclusions

Our study extends the mutation spectrum of SEDC and is helpful in early molecular diagnoses of SEDC.     

Oculocutaneous albinism type 4: six novel mutations in the membrane‐associated transporter protein gene and their phenotypes

Oculocutaneous albinism type 4 (OCA4) is an autosomal recessive hypopigmentary disorder caused by mutations in the Membrane‐Associated Transporter Protein gene (SLC45A2). The SLC45A2 protein is a 530‐amino‐acid polypeptide that contains 12 putative transmembrane domains, and appears to be a transporter that mediates melanin synthesis. Eighteen pathological mutations have been reported so far. In this study, six novel mutations, p.Y49C (c.146A > G), p.G89R (c.265G > A), p.C229Y (c.686G > A), p.T437A (c.1309A > G), p.T440A (c.1318A > G) and p.G473D (c.1418G > A) were found in eight Japanese patients with various clinical phenotypes. The phenotypes of OCA4 were as various as the other types of OCA and probably depended on the mutation sites in the SLC45A2 gene.     

Functional studies of the effect of NO donor on human CLCN1 polymorphism/mutants expressed in Xenopus laevis oocytes

In this study, we investigated the effect of NO donor, diethylamine/nitric oxide (DEA/NO), on the electrophysiological behavior of human skeletal muscle chloride channel (CLCN1). The wild-type and variants of CLCN1, including one polymorphism (P727L) and four mutants (T631I, D644G, G482R, and S471F), were expressed in Xenopus oocytes and the ionic current was measured by two-electrode voltage-clamp method. Our results revealed that there is no significant difference in the current-voltage relationships and half-voltage values of open probability between wild-type and variants of CLCN1 except for G482R. Application of the DEA-NO (0.1 mM) significantly increases the channel conductance of wild-type, T631I, D644G, and S471F, but not P727L. This indicates that P727L polymorphism causes loss of sensitivity of CLCN1 to the DEA/NO treatment, which could be due to a conformational change caused by proline substitution. The data suggest that the polymorphic changes may affect the function of CLCN1 in response to the treatment of chemical compounds.