A CLCN1 mutation in dominant myotonia congenita impairs the increment of chloride conductance during repetitive depolarization

Myotonia congenita is caused by mutation of the CLCN1 gene, which encodes the human skeletal muscle chloride channel (ClC-1). The ClC-1 protein is a dimer comprised of two identical subunits each incorporating its own separate pore. However, the precise pathophysiological mechanism underlying the abnormal ClC-1 channel gating in some mutants is not fully understood. We characterized a ClC-1 mutation, Pro-480-Thr (P480T) identified in dominant myotonia congenita, by using whole-cell recording. P480T ClC-1 revealed significantly slowed activation kinetics and a slight depolarizing shift in the voltage-dependence of the channel gating. Wild-type/mutant heterodimers exhibited similar kinetic properties and voltage-dependency to mutant homodimers. Simulating myotonic discharge with the voltage clamp protocol of a 50 Hz train pulse, the increment of chloride conductance was impaired in both wild-type/mutant heterodimers and mutant homodimers, clearly indicating a dominant-negative effect. Our data showed that slow activation gating of P480T ClC-1 impaired the increment of chloride conductance during repetitive depolarization, thereby accentuating the chloride conductance reduction caused by a slight depolarizing shift in the voltage-dependence of the channel gating. This pathophysiology may explain the clinical features of myotonia congenita.     

Identification and Functional Characterization of CLCN1 Mutations Found in Nondystrophic Myotonia Patients

Mutations in the gene coding for the skeletal muscle Cl^? channel (CLCN1) lead to dominant or recessive myotonia. Here, we identified and characterized CLCN1 mutations in Costa Rican patients, who had been clinically diagnosed with myotonic dystrophy type 1 but who were negative for DM1 mutations. CLCN1 mutations c.501C>G, p.F167L and c.1235A>C, p.Q412P appeared to have recessive inheritance but patients had atypical clinical phenotypes; c.313C>T, p.R105C was found in combination with c.501C>G, p.F167L in an apparently recessive family and the c.461A>G, p.Q154R variant was associated with a less clear clinical picture. In Xenopus oocytes, none of the mutations exhibited alterations of fast or slow gating parameters or single channel conductance, and mutations p.R105C, p.Q154R, and p.F167L were indistinguishable from wild‐type (WT). p.Q412P displayed a dramatically reduced current density, surface expression and exerted no dominant negative effect in the context of the homodimeric channel. Fluorescently tagged constructs revealed that p.Q412P is expressed inefficiently. Our study confirms p.F167L and p.R105C as myotonia mutations in the Costa Rican population, whereas p.Q154R may be a benign variant. p.Q412P most likely induces a severe folding defect, explaining the lack of dominance in patients and expression systems, but has WT properties once expressed in the plasma membrane.     

Identification of novel MLC1 mutations in Chinese patients with megalencephalic leukoencephalopathy with subcortical cysts (MLC)

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal, recessively inherited disease caused by mutations in the MLC1 gene. Most of the previously published studies have been carried out in ethnic populations other than the Chinese. In this study, the analysis of clinical features and MLC1 mutation screening were performed in 13 Chinese patients for the first time. A total of 10 MLC1 mutations were identified in these patients, including five novel missense mutations (c.65G>A, p.R22Q; c.95C>T, p.A32V; c.218G>A, p.G73E; c.823G>A, p.A275T; c.832T>C, p.Y278H), one novel splicing mutation (c.772-1G>C in IVS9-1), one novel small deletion (c.907_930del, p.V303_L310del), one known nonsense mutation (c.593delCTCA, p.Y198X) and two known missense mutations (c.206C>T, p.S69L; c.353C>T, p.T118M). Mutation c.772-1G>C in IVS9-1, accounting for 27.3% (3/11) of the total number of genetically confirmed patients found in this study, is thus a putative hot-spot mutation in the present study group. The existence of a unique MLC1 mutation spectrum in Chinese MLC patients was shown. A systemic study to assess the mutation spectra in different populations should be undertaken.     

ClC-3氯通道在二甲双胍抑制鼻咽癌细胞周期进程中的作用

目的:探讨ClC-3氯通道在二甲双胍抑制鼻咽癌细胞周期进程中的作用。方法:采用不同浓度二甲双胍处理低分化鼻咽癌细胞CNE-2Z,CCK-8法检测细胞活力,流式细胞术检测细胞周期分布,Western blot法检测ClC-3氯通道蛋白表达,全细胞膜片钳技术检测细胞氯电流。构建高表达ClC-3氯通道蛋白的质粒pEZ-M03-ClC-3转染CNE-2Z细胞,流式细胞术检测ClC-3氯通道对细胞周期分布的影响。结果:5、10和20 mmol/L浓度的二甲双胍均可有效抑制CNE-2Z细胞的活力。10 mmol/L二甲双胍可阻抑CNE-2Z细胞周期于G0/G1期,并抑制CNE-2Z细胞氯电流及ClC-3氯离子通道蛋白的表达。ClC-3氯通道蛋白高表达可逆转二甲双胍对CNE-2Z细胞周期分布的影响。结论:二甲双胍抑制鼻咽癌CNE-2Z细胞周期进程可能与抑制ClC-3氯通道功能和蛋白表达有关。     

Functional assays testing pathogenicity of 14 cystathionine-beta synthase mutations

In this study, 14 CBS alleles from homocystinuric patients were expressed heterologously in E. coli and their enzyme activities were assayed in vitro. Additionally, mutant CBS proteins were visualized by Western blot from denaturing and non-denaturing polyacrylamide gels. The 14 mutations characterized were: p.R125W (c.373C>T), p.G148R (c.442G>A), p.M173V (c.517A>G), p.T191M (c.572C>T), p.A226T (c.676G>A), p.C275Y (c.824G>A), p.R336C (c.1006C>T), p.R336H (c.1007G>A), p.L338P (c.1013T>C), p.S349N (c.1046G>A), p.R379Q (c.1136G>A), p.L456P (c.1367T>C), p.G522fsX540 (c.1566delG), and p.R548Q (c.1643G>A). Eleven of the mutant alleles exhibited an activity lower than 4% of the wild-type protein. In contrast, mutations p.A226T and p.M173V presented 20% and 40% of the wild-type activity, respectively, whereas the activity of p.R548Q was up to 60% of the wild-type. This suggests that it is a new rare variant rather than a pathogenic mutation. Most of the mutated proteins exhibited a decreased signal in Western blot analyses. The non-denaturing PAGE revealed that the wild-type protein retained the capacity to form a multimeric quaternary structure, whereas in the mutations p.M173V, p.A226T, and p.G548Q, this structure grade was dramatically reduced and was completely absent in the rest of the mutations.     

Regulation of ClC-2 gating by intracellular ATP

ClC-2 is a voltage-dependent chloride channel that activates slowly at voltages negative to the chloride reversal potential. Adenosine triphosphate (ATP) and other nucleotides have been shown to bind to carboxy-terminal cystathionine-ß-synthase (CBS) domains of ClC-2, but the functional consequences of binding are not sufficiently understood. We here studied the effect of nucleotides on channel gating using single-channel and whole-cell patch clamp recordings on transfected mammalian cells. ATP slowed down macroscopic activation and deactivation time courses in a dose-dependent manner. Removal of the complete carboxy-terminus abolishes the effect of ATP, suggesting that CBS domains are necessary for ATP regulation of ClC-2 gating. Single-channel recordings identified long-lasting closed states of ATP-bound channels as basis of this gating deceleration. ClC-2 channel dimers exhibit two largely independent protopores that are opened and closed individually as well as by a common gating process. A seven-state model of common gating with altered voltage dependencies of opening and closing transitions for ATP-bound states correctly describes the effects of ATP on macroscopic and microscopic ClC-2 currents. To test for a potential pathophysiological impact of ClC-2 regulation by ATP, we studied ClC-2 channels carrying naturally occurring sequence variants found in patients with idiopathic generalized epilepsy, G715E, R577Q, and R653T. All naturally occurring sequence variants accelerate common gating in the presence but not in the absence of ATP. We propose that ClC-2 uses ATP as a co-factor to slow down common gating for sufficient electrical stability of neurons under physiological conditions.     

Identification of two functionally deficient plasma alpha 3-fucosyltransferase (FUT6) alleles

One Indonesian individual without detectable plasma alpha3-fucosyltransferase activity was identified with three point mutations, 730C>G (L244V), 907C>G (R303G), and 370C>T (P124S), in the coding region of one FUT6 allele. Another individual, expressing weak plasma alpha3-fucosyltransferase activity, had the 907C>G together with the 370C>T mutation, but did not have the 730C>G mutation. PCR-RFLP analyses of complete families confirmed the segregation of these alleles and illustrated the existence and inheritance of the [370C>T; 907C>G] mutated allele in three additional families. Altogether, this allele was found heterozygously in nine Indonesian and two Swedish individuals, all with detectable plasma alpha3-fucosyltransferase activities. The FUT6 allele with the three mutations (370C>T; 730C>G; 907C>G) was identified heterozygously in only two Indonesian individuals, both having the inactivating 739G>A mutation in the other allele and both lacking plasma alpha3-fucosyltransferase activity. Enzyme studies made on transiently transfected COS-7 cells demonstrated that the combination of the 370C>T, 730C>G and 907C>G mutations decreased the V(max) by more than 80%, but caused no obvious change of the apparent K(m) values for GDP-fucose and Gal-N-acetyllactosamine. In comparison, chimeric constructs with the isolated 730C>G or 907C>G mutations decreased the V(max) values by about two thirds and one third, respectively.     

A novel alteration of muscle chloride channel gating in myotonia levior

Mutations in the voltage-dependent skeletal muscle chloride channel, ClC-1, result in dominant or recessive myotonia congenita. The Q552R mutation causes a variant of dominant myotonia with a milder phenotype, myotonia levior. To characterise the functional properties of this mutation, homodimeric mutant and heterodimeric wild-type (WT) mutant channels were expressed in tsA201 cells and studied using the whole-cell recording technique. Q552R ClC-1 mutants formed functional channels with normal ion conduction but altered gating properties. Mutant channels were activated by membrane depolarisation, with a voltage dependence of activation that was shifted by more than +90 mV compared to WT channels. Q552R channels were also activated by hyperpolarisation, and this process was dependent upon the intracellular chloride concentration ([  Cl⁻  ]_i). Together, these alterations resulted in a substantial reduction in the open probability at −85 mV at a physiological [  Cl⁻  ]_i. Heterodimeric WT-Q552R channels did not exhibit hyperpolarisation-activated gating transitions. As was the case for WT channels, activation occurred upon depolarisation, but the activation curve was shifted by 28 mV to more positive potentials. The functional properties of heterodimeric channels suggest a weakly dominant effect, a finding that correlates with the inheritance pattern and symptom profile of myotonia levior.     

Biochemical analysis of human POLG2 variants associated with mitochondrial disease

Defects in mitochondrial DNA (mtDNA) maintenance comprise an expanding repertoire of polymorphic diseases caused, in part, by mutations in the genes encoding the p140 mtDNA polymerase (POLG), its p55 accessory subunit (POLG2) or the mtDNA helicase (C10orf2). In an exploration of nuclear genes for mtDNA maintenance linked to mitochondrial disease, eight heterozygous mutations (six novel) in POLG2 were identified in one control and eight patients with POLG-related mitochondrial disease that lacked POLG mutations. Of these eight mutations, we biochemically characterized seven variants [c.307G>A (G103S); c.457C>G (L153V); c.614C>G (P205R); c.1105A>G (R369G); c.1158T>G (D386E); c.1268C>A (S423Y); c.1423_1424delTT (L475DfsX2)] that were previously uncharacterized along with the wild-type protein and the G451E pathogenic variant. These seven mutations encode amino acid substitutions that map throughout the protein, including the p55 dimer interface and the C-terminal domain that interacts with the catalytic subunit. Recombinant proteins harboring these alterations were assessed for stimulation of processive DNA synthesis, binding to the p140 catalytic subunit, binding to dsDNA and self-dimerization. Whereas the G103S, L153V, D386E and S423Y proteins displayed wild-type behavior, the P205R and R369G p55 variants had reduced stimulation of processivity and decreased affinity for the catalytic subunit. Additionally, the L475DfsX2 variant, which possesses a C-terminal truncation, was unable to bind the p140 catalytic subunit, unable to bind dsDNA and formed aberrant oligomeric complexes. Our biochemical analysis helps explain the pathogenesis of POLG2 mutations in mitochondrial disease and emphasizes the need to quantitatively characterize the biochemical consequences of newly discovered mutations before classifying them as pathogenic.     

Five novel mutations in the lysosomal sialidase gene (NEU1) in type II sialidosis patients and assessment of their impact on enzyme activity and intracellular targeting using adenovirus-mediated expression

Sialidosis is an autosomal recessive disease resulting from a deficiency of lysosomal sialidase. Type II sialidosis is a rare disease characterized clinically by hydrops fetalis, hepatosplenomegaly, and severe psychomotor retardation. Genomic DNA from four unrelated sialidosis patients was screened for mutations within the sialidase gene NEU1. Five novel mutations were identified. Four are missense and one is nonsense: c.674G>C (p.R225P), c.893C>T (p.A298V), c.3G>A (p.M1?), c.941C>G (p.R341G), and c.69G>A (p.W23X). We have used our findings and diagnostic tools to confirm the presence of a homozygous null allele in a neonate sibling. Recombinant adenoviruses expressing the mutant sialidase alleles in primary cell cultures were utilized to assess the impact of each mutation on enzyme activity and intracellular localization. None of the mutant alleles expressed significant enzymatic activity. The p.R341G mutation exerts its pathological effect by perturbing substrate binding, while the p.A298V and p.R225P mutations appear to impair the folding of the sialidase enzyme. Our findings point to mutation‐sensitive amino acids involved in catalytic function or structural stability and indicate the potential utility of these mutations for molecular diagnosis of this rare disease. Hum Mutat 23:32–39, 2004. © 2003 Wiley‐Liss, Inc.     

Identification and molecular characterization of alpha-L-iduronidase mutations present in mucopolysaccharidosis type I patients undergoing enzyme replacement therapy

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase (IDUA). Mutations in the gene are responsible for the enzyme deficiency, which leads to the intralysosomal storage of the partially degraded glycosaminoglycans dermatan sulfate and heparan sulfate. Molecular characterization of MPS I patients has resulted in the identification of over 70 distinct mutations in the IDUA gene. The high degree of molecular heterogeneity reflects the wide clinical variability observed in MPS I patients. Six novel mutations, c.1087C>T (p.R363C), c.1804T>A (p.F602I), c.793G>C, c.712T>A (p.L238Q), c.1727+2T>A, and c.1269C>G (p.S423R), in a total of 14 different mutations, and 13 different polymorphic changes, including the novel c.246C>G (p.H82Q), were identified in a cohort of 10 MPS I patients enrolled in a clinical trial of enzyme-replacement therapy. Five novel amino acid substitutions and c.236C>T (p.A79V) were engineered into the wild-type IDUA cDNA and expressed. A p.G265R read-through mutation, arising from the c.793G>C splice mutation, was also expressed. Each mutation reduced IDUA protein and activity levels to varying degrees with the processing of many of the mutant forms also affected by IDUA. The varied properties of the expressed mutant forms of IDUA reflect the broad range of biochemical and clinical phenotypes of the 10 patients in this study. IDUA kinetic data derived from each patient's cultured fibroblasts, in combination with genotype data, was used to predict disease severity. Finally, residual IDUA protein concentration in cultured fibroblasts showed a weak correlation to the degree of immune response to enzyme-replacement therapy in each patient.     

Characterisation of renal chloride channel, CLCN5, mutations in hypercalciuric nephrolithiasis (kidney stones) disorders

Mutations of the renal-specific chloride channel (CLCN5) gene, which is located on chromosome Xp11.22, are associated with hypercalciuric nephrolithiasis (kidney stones) in the Northern European and Japanese populations. CLCN5 encodes a 746 amino acid channel (CLC-5) that has approximately 12 transmembrane domains, and heterologous expression of wild-type CLC-5 in Xenopus oocytes has yielded outwardly rectifying chloride currents that were markedly reduced or abolished by these mutations. In order to assess further the structural and functional relationships of this recently cloned chloride channel, additional CLCN5 mutations have been identified in five unrelated families with this disorder. Three of these mutations were missense (G57V, G512R and E527D), one was a nonsense (R648Stop) and one was an insertion (30:H insertion). In addition, two of the mutations (30:H insertion and E527D) were demonstrated to be de novo, and the G57V and E527D mutations were identified in families of Afro-American and Indian origin, respectively. The G57V and 30:H insertion mutations represent the first CLCN5 mutations to be identified in the N-terminus region, and the R648Stop mutation, which has been observed previously in an unrelated family, suggests that this codon may be particularly prone to mutations. Heterologous expression of the mutations resulted in a marked reduction or abolition of the chloride currents, thereby establishing their functional importance. These results help to elucidate further the structure-function relationships of this renal chloride channel.      

Genetic characterization of myeloperoxidase deficiency in Italy

Hereditary myeloperoxidase (MPO) deficiency (MPOD) is the most common neutrophil biochemical defect, and is characterized by a lack of peroxidase activity. In order to extend the epidemiological studies on hereditary MPOD in Italy, a population screening was carried out to detect mutations in the MPO gene. Of approximately 40,000 individuals analyzed, seven partial and eight total MPO-deficient subjects were identified. The genetic characterization of the subjects showed the presence of three already-known mutations (c.752T>C, c.1705C>T, and c.1566_1579del14) and six novel mutations: four missense mutations (c.995C>T, c.1112A>G, c.1715T>G, and c.1927T>C), then a deletion of an adenine within exon 3 (c.325delA) and a mutation within the 3' splice site of intron 11 (c.2031-2A>C). The novel missense mutations cause the substitution of the residues p.A332V, p.D371G, p.L572W, and p.W643R, respectively, and the potential structural changes are discussed. The c.325delA deletion causes a shift of the reading frame with the occurrence of a premature stop codon within the propeptide. Then, considering the difficulty in obtaining bone marrow samples from MPO-deficient subjects to study MPO mRNA splicing in vivo, we set up an eukaryotic expression system to investigate how the c.2031-2A>C mutation alters the MPO pre-mRNA splicing. The activation of a cryptic 3' splice site located 109nt upstream of the authentic 3' splice site was observed. The 109nt-insertion causes a shift in the reading frame that should lead to the generation of an abnormal MPO precursor lacking the enzymatic activity.     

Identification of 25 new mutations in 40 unrelated Spanish Niemann-Pick type C patients: genotype-phenotype correlations

To better characterize Niemann-Pick type C (NPC) in Spain and improve genetic counselling, molecular analyses were carried out in 40 unrelated Spanish patients. The search identified 70/80 alleles (88%) involving 38 different NPC1 mutations, 26 of which are described for the first time. No patient with NPC2 mutations was identified. The novel NPC1 mutations include 14 amino acid substitutions [R372W (c.1114C>T), P434L (c.1301C>T), C479Y (c.1436G>A), K576R (c.1727G>A), V727F (c.2179G>T), M754K (c.2261T>A), S865L (c.2594C>T), A926T (c.2776G>A), D948H (c.2842G>C), V959E (c.2876T>A), T1036K (c.3107C>A), T1066N (c.3197C>A), N1156I (c.3467A>T) and F1224L (c.3672C>G)], four stop codon [W260X (c.780G>A), S425X (c.1274C>A), C645X (c.1935T>A) and R1059X (c.3175C>T)], two donor splice-site mutations [IVS7+1G>A (g.31432G>A) and IVS21+2insG (g.51871insG)], one in-frame mutation [N961_F966delinsS (c.2882del16bpins1bp)] and five frameshift mutations [V299fsX8 (c.895insT), A558fsX11 (c.1673insG), C778fsX10 (c.2334insT), G993fsX3 (c.2973_78delG) and F1221fsX20 (c.3662delT)]. We also identified three novel changes [V562V (c.1686G>A), A580A (c.1740C>G) and A1187A (c.3561G>T)] in three independent NPC patients and five polymorphisms that have been described previously. The combination of these polymorphisms gave rise to the establishment of different haplotypes. Linkage disequilibrium was detected between mutations C177Y and G993fsX3 and specific haplotypes, suggesting a unique origin for these mutations. In contrast, I1061T mutation showed at least two different origins. The most prevalent mutations in Spanish patients were I1061T, Q775P, C177Y and P1007A (10, 7, 7 and 5% of alleles, respectively). Our data in homozygous patients indicate that the Q775P mutation correlates with a severe infantile neurological form and the C177Y mutation with a late infantile clinical phenotype.     

Characterization of 19 disease-associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator

In order to gain a better insight into the structure and function of the regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, 19 RD missense mutations that had been identified in patients were functionally characterized. Nine of these (I601F, L610S, A613T, D614G, I618T, L619S, H620P, G628R and L633P) resulted in aberrant processing. No or a very small number of functional CFTR proteins will therefore appear at the cell membrane in cells expressing these mutants. These mutations were clustered in the N- terminal part of the RD, suggesting that this subdomain has a folding pattern that is very sensitive to amino acid changes. Mutations that caused no aberrant processing were further characterized at the electrophysiological level. First, they were studied at the whole cell level in Xenopus laevis oocytes. Mutants that induced a whole cell current that was significantly different from wild-type CFTR were subsequently analysed at the single channel level in COS1 cells transiently expressing the different mutant and wild-type proteins. Three mutant chloride channels, G622D, R792G and E822K CFTR, were characterized by significantly lower intrinsic chloride channel activities compared with wild-type CFTR. Two mutations, H620Q and A800G, resulted in increased intrinsic chloride transport activities. Finally, T665S and E826K CFTR had single channel properties not significantly different from wild-type CFTR.      

N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel

Over 1,500 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure-function analyses have only been established for a few of them. This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior. By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Immunofluorescence and whole cell patch-clamp confirmed intracellular retention, thus reflecting a defect of CFTR folding and/or trafficking. In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Nevertheless, whole-cell chloride currents were recorded for all three variants. Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Overall, our results predict a major impact of the CFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology.     

Myofibrillogenesis regulator 1 gene (MR-1) mutation in an Omani family with paroxysmal nonkinesigenic dyskinesia

Two recurrent missense mutations (c.20C>T: A7V; c.26C>T: A9V) in the gene encoding the myofibrillogenesis regulator 1 (MR-1) have been shown to cause autosomal dominant paroxysmal nonkinesigenic dyskinesia (PNKD) in 13 families of primarily European ancestry. Here we report an Omani PNKD family with seven affected family members and autosomal dominant inheritance. Our linkage analysis provided consistent positional evidence that the MR-1 gene could be the responsible disease gene. Sequence analysis identified a MR-1 missense mutation (c.20C>T; A7V) in the affected family members, whereas it was not present in five unaffected family members and 129 population controls. Taking into account that previous haplotype analyses did not reveal evidence for common founders among several PNKD families, our present findings strengthen three implications: (1) autosomal dominant PNKD seems to be a homogenous disorder, for which the MR-1 gene is the major disease gene; (2) mainly two recurrent MR-1 missense mutations (57% V7, 43% V9) account for the genetic variance of familial PNKD; (3) it supports current evidence that some of the recurrent MR-1 mutations may have arisen independently by de novo mutation at functionally convergent key sites of the brain-specific MR-1L isoform.