Congenital Short QT Syndrome

The Short QT Syndrome is a recently described new genetic disorder, characterized by abnormally short QT interval, paroxysmal atrial fibrillation and life threatening ventricular arrhythmias. This autosomal dominant syndrome can afflict infants, children, or young adults; often a remarkable family background of cardiac sudden death is elucidated. At electrophysiological study, short atrial and ventricular refractory periods are found, with atrial fibrillation and polymorphic ventricular tachycardia easily induced by programmed electrical stimulation. Gain of function mutations in three genes encoding K⁺ channels have been identified, explaining the abbreviated repolarization seen in this condition: KCNH2 for I_kr (SQT1), KCNQ1 for I_ks (SQT2) and KCNJ2 for I_k1 (SQT3). The currently suggested therapeutic strategy is an ICD implantation, although many concerns exist for asymptomatic patients, especially in pediatric age. Pharmacological treatment is still under evaluation; quinidine has shown to prolong QT and reduce the inducibility of ventricular arrhythmias, but awaits additional confirmatory clinical data.     

Isoform-Specific Dominant-Negative Effects Associated with hERG1 G628S Mutation in Long QT Syndrome

Background

Mutations in the human ether-a-go-go-related gene 1 (hERG1) cause type 2 long QT syndrome (LQT2). The hERG1 gene encodes a K⁺ channel with properties similar to the rapidly activating delayed rectifying K⁺ current in the heart. Several hERG1 isoforms with unique structural and functional properties have been identified. To date, the pathogenic mechanisms of LQT2 mutations have been predominantly described in the context of the hERG1a isoform. In the present study, we investigated the functional consequences of the LQT2 mutation G628S in the hERG1b and hERG1a_USO isoforms.

Methods

A double-stable, mammalian expression system was developed to characterize isoform-specific dominant-negative effects of G628S-containing channels when co-expressed at equivalent levels with wild-type hERG1a. Western blot and co-immunoprecipitation studies were performed to study the trafficking and co-assembly of wild-type and mutant hERG1 isoforms. Patch-clamp electrophysiology was performed to characterize hERG1 channel function and the isoform-specific dominant-negative effects associated with the G628S mutation.

Conclusions

The non-functional hERG1a-G628S and hERG1b-G628S channels co-assembled with wild-type hERG1a and dominantly suppressed hERG1 current. In contrast, G628S-induced dominant-negative effects were absent in the context of the hERG1a_USO isoform. hERG1a_USO-G628S channels did not appreciably associate with hERG1a and did not significantly suppress hERG1 current when co-expressed at equivalent ratios or at ratios that approximate those found in cardiac tissue. These results suggest that the dominant-negative effects of LQT2 mutations may primarily occur in the context of the hERG1a and hERG1b isoforms.     

Characterization and Functional Restoration of a Potassium Channel Kir6.2 Pore Mutation Identified in Congenital Hyperinsulinism

The inwardly rectifying potassium channel Kir6.2 assembles with sulfonylurea receptor 1 to form the ATP-sensitive potassium (K_ATP) channels that regulate insulin secretion in pancreatic β-cells. Mutations in K_ATP channels underlie insulin secretion disease. Here, we report the characterization of a heterozygous missense Kir6.2 mutation, G156R, identified in congenital hyperinsulinism. Homomeric mutant channels reconstituted in COS cells show similar surface expression as wild-type channels but fail to conduct potassium currents. The mutated glycine is in the pore-lining transmembrane helix of Kir6.2; an equivalent glycine in other potassium channels has been proposed to serve as a hinge to allow helix bending during gating. We found that mutation of an adjacent asparagine, Asn-160, to aspartate, which converts the channel from a weak to a strong inward rectifier, on the G156R background restored ion conduction in the mutant channel. Unlike N160D channels, however, G156R/N160D channels are not blocked by intracellular polyamines at positive membrane potential and exhibit wild-type-like nucleotide sensitivities, suggesting the aspartate introduced at position 160 interacts with arginine at 156 to restore ion conduction and gating. Using tandem Kir6.2 tetramers containing G156R and/or N160D in designated positions, we show that one mutant subunit in the tetramer is insufficient to abolish conductance and that G156R and N160D can interact in the same or adjacent subunits to restore conduction. We conclude that the glycine at 156 is not essential for K_ATP channel gating and that the Kir6.2 gating defect caused by the G156R mutation could be rescued by manipulating chemical interactions between pore residues.     

QT延长综合征的分子生物学机制

遗传性QT延长综合征又称突发性LQTS,是一种以QT间期延长(LQT)、突发性昏厥、惊厥、甚至猝死为特征的先天性心脏病。该将阐述其机制及其研究进展。     

Heteromeric assembly of inward rectifier channel subunit Kir2.1 with Kir3.1 and with Kir3.4

Heteromultimerization of different pore-forming subunits is known to contribute to the diversity of inward rectifier K⁺ channels. We examined if the subunits belonging to different subfamilies Kir2 and Kir3 can co-assemble to form heteromultimers in heterologous expression systems. We observed co-immunoprecipitation of Kir2.1 and Kir3.1 as well as Kir2.1 and Kir3.4 in HEK293T cells. Furthermore, analyses of subcellular localization using confocal microscopy revealed that co-expression of Kir2.1 promoted the cell surface localization of Kir3.1 and Kir3.4 in HEK293T cells. In electrophysiological experiments, co-expression of Kir2.1 with Kir3.1 and/or Kir3.4 in Xenopus oocytes and HEK293T cells did not yield currents with distinguishable features. However, co-expression of a dominant-negative Kir2.1 with the wild-type Kir3.1/3.4 decreased the Kir3.1/3.4 current amplitude in Xenopus oocytes. The results indicate that Kir2.1 is capable of forming heteromultimeric channels with Kir3.1 and with Kir3.4.     

Effects of ACh and adenosine mediated by Kir3.1 and Kir3.4 on ferret ventricular cells

The inotropic effects of ACh and adenosine on ferret ventricular cells were investigated with the action potential-clamp technique. Under current clamp, both agonists resulted in action potential shortening and a decrease in contraction. Under action potential clamp, both agonists failed to decrease contraction substantially. In the absence of agonist, application of the short action potential waveform (recorded previously in the presence of agonist) also resulted in a decrease in contraction. Under action potential clamp, application of ACh resulted in a Ba(2+)-sensitive outward current with the characteristics of muscarinic K+ current (I(K,ACh)); the presence of the muscarinic K+ channel was confirmed by PCR and immunocytochemistry. In the absence of agonist, on application of the short ACh action potential waveform, the decrease in contraction was accompanied by loss of the inward Na(+)/Ca(2+) exchange current (I(NaCa)). ACh also inhibited the background inward K+ current (I(K,1)). It is concluded that ACh activates I(K,ACh), inhibits I(K,1), and indirectly inhibits I(NaCa); this results in action potential shortening, decrease in contraction, and, as a result of the inhibition of I(K,1), minimum decrease in excitability.     

Ablation of Premature Ventricular Complexes Triggering Ventricular Fibrillation in a Patient with Long QT Syndrome

We describe the case of a patient with long QT syndrome and recurrent ventricular fibrillation, triggered by premature ventricular complexes (PVCs) with a left bundle branch block pattern and inferior axis of the QRS. Activation mapping demonstrated the origin of the PVCs to be in the right ventricular outflow tract. Ventricular fibrillation (VF) was successfully treated by catheter ablation of the triggering PVCs and there has been no recurrence of VF during a follow-up period of 14 months.     

应用PCR技术对先天性长QT综合征KCNQ1 基因进行定点突变的研究

利用聚合酶链反应（PCR）技术对长QT综合征（LQTS）KCNQ1基因进行定点突变的研究。首先设计两对引物（包含预定的突变）,通过3轮PCR扩增,扩增出含有所需突变位点的片段,然后将片段克隆入T载体中,通过酶切连接的方法将突变点引入到pIRES2-EGFP-KCNQ1中,随后用Effectene转染试剂介导转染HEK293细胞。结果在真核表达载体pIRES2-EGFP-KCNQ1基础上获得了KCNQ1 cDNA C934T的突变体,测序表明在序列中发生了预期的突变。将含突变点的pIRES2-EGFP-KCNQ1转染HEK293细胞后,在荧光显微镜下观察到被转染的HEK293细胞发出绿色荧光,表明含突变点的pIRES2-EGFP-KCNQ1得到了表达。Abstract: To study PCR site-directed mutagenesis of long QT syndrome KCNQ1 gene in vitro. The site-directed mutagenesis of LQTS gene KCNQ1 was made by PCR. Two sets of primers were designed according to the sequence of KCNQ1 cDNA, and mismatch was introduced into primers. Mutagenesis was performed in a three-step PCR. The amplified fragments from the third PCR which contained the mutation site were subcloned into the T-vecor PCR2.1.Then the fragments containing the mutation site was obtained from PCR2.1 with restriction enzyme digestion and was inserted into the same restriction site of pIRES2-EGFP-KCNQ1. With Effectene Transfection Reagent, pIRES2-EGFP-KCNQ1 was transfected into HEK293 cell. The sequencing analysis showed that the mutation site was correct. Mutation from T to C in 934 site of KCNQ1 cDNA was found. Under the fluorescence microscope, the green fluorescence was spread in the transfected HEK293 cell, meaning the pIRES2-EGFP-KCNQ1 containing the mutation site was expressed correctly.     

Identification of Genetic Alterations,as Causative Genetic Defects in Long QT Syndrome,Using Next Generation Sequencing Technology

Background

Long QT Syndrome is an inherited channelopathy leading to sudden cardiac death due to ventricular arrhythmias. Despite that several genes have been associated with the disease, nearly 20% of cases remain without an identified genetic cause. Other genetic alterations such as copy number variations have been recently related to Long QT Syndrome. Our aim was to take advantage of current genetic technologies in a family affected by Long QT Syndrome in order to identify the cause of the disease.

Methods

Complete clinical evaluation was performed in all family members. In the index case, a Next Generation Sequencing custom-built panel, including 55 sudden cardiac death-related genes, was used both for detection of sequence and copy number variants. Next Generation Sequencing variants were confirmed by Sanger method. Copy number variations variants were confirmed by Multiplex Ligation dependent Probe Amplification method and at the mRNA level. Confirmed variants and copy number variations identified in the index case were also analyzed in relatives.

Results

In the index case, Next Generation Sequencing revealed a novel variant in TTN and a large deletion in KCNQ1, involving exons 7 and 8. Both variants were confirmed by alternative techniques. The mother and the brother of the index case were also affected by Long QT Syndrome, and family cosegregation was observed for the KCNQ1 deletion, but not for the TTN variant.

Conclusions

Next Generation Sequencing technology allows a comprehensive genetic analysis of arrhythmogenic diseases. We report a copy number variation identified using Next Generation Sequencing analysis in Long QT Syndrome. Clinical and familiar correlation is crucial to elucidate the role of genetic variants identified to distinguish the pathogenic ones from genetic noise.     

A COLQ Missense Mutation in Labrador Retrievers Having Congenital Myasthenic Syndrome

Congenital myasthenic syndromes (CMSs) are heterogeneous neuromuscular disorders characterized by skeletal muscle weakness caused by disruption of signal transmission across the neuromuscular junction (NMJ). CMSs are rarely encountered in veterinary medicine, and causative mutations have only been identified in Old Danish Pointing Dogs and Brahman cattle to date. Herein, we characterize a novel CMS in 2 Labrador Retriever littermates with an early onset of marked generalized muscle weakness. Because the sire and dam share 2 recent common ancestors, CMS is likely the result of recessive alleles inherited identical by descent (IBD). Genome-wide SNP profiles generated from the Illumina HD array for 9 nuclear family members were used to determine genomic inheritance patterns in chromosomal regions encompassing 18 functional candidate genes. SNP haplotypes spanning 3 genes were consistent with autosomal recessive transmission, and microsatellite data showed that only the segment encompassing COLQ was inherited IBD. COLQ encodes the collagenous tail of acetylcholinesterase, the enzyme responsible for termination of signal transduction in the NMJ. Sequences from COLQ revealed a variant in exon 14 (c.1010T>C) that results in the substitution of a conserved amino acid (I337T) within the C-terminal domain. Both affected puppies were homozygous for this variant, and 16 relatives were heterozygous, while 288 unrelated Labrador Retrievers and 112 dogs of other breeds were wild-type. A recent study in which 2 human CMS patients were found to be homozygous for an identical COLQ mutation (c.1010T>C; I337T) provides further evidence that this mutation is pathogenic. This report describes the first COLQ mutation in canine CMS and demonstrates the utility of SNP profiles from nuclear family members for the identification of private mutations.     

The Application of Root Mean Square Electrocardiography (RMS ECG) for the Detection of Acquired and Congenital Long QT Syndrome

Background

Precise measurement of the QT interval is often hampered by difficulty determining the end of the low amplitude T wave. Root mean square electrocardiography (RMS ECG) provides a novel alternative measure of ventricular repolarization. Experimental data have shown that the interval between the RMS ECG QRS and T wave peaks (RT_PK) closely reflects the mean ventricular action potential duration while the RMS T wave width (TW) tracks the dispersion of repolarization timing. Here, we tested the precision of RMS ECG to assess ventricular repolarization in humans in the setting of drug-induced and congenital Long QT Syndrome (LQTS).

Methods

RMS ECG signals were derived from high-resolution 24 hour Holter monitor recordings from 68 subjects after receiving placebo and moxifloxacin and from standard 12 lead ECGs obtained in 97 subjects with LQTS and 97 age- and sex-matched controls. RT_PK, QT_RMS and RMS TW intervals were automatically measured using custom software and compared to traditional QT measures using lead II.

Results

All measures of repolarization were prolonged during moxifloxacin administration and in LQTS subjects, but the variance of RMS intervals was significantly smaller than traditional lead II measurements. TW was prolonged during moxifloxacin and in subjects with LQT-2, but not LQT-1 or LQT-3.

Conclusion

These data validate the application of RMS ECG for the detection of drug-induced and congenital LQTS. RMS ECG measurements are more precise than the current standard of care lead II measurements.     

Mapping of a Gene for Long QT Syndrome to Chromosome 4q25-27

Long QT syndrome (LQTS) is a heterogeneous inherited disorder causing syncope and sudden death from ventricular arrhythmias. A first locus for this disorder was mapped to chromosome 11p15.5. However, locus heterogeneity has been demonstrated in several families, and two other loci have recently been located on chromosomes 7q35-36 and 3p21-24. We used linkage analysis to map the locus in a 65-member family in which LQTS was associated with more marked sinus bradycardia than usual, leading to sinus node dysfunction. Linkage to chromosome 11p15.5, 7q35-36, or 3p21-24 was excluded. Positive linkage was obtained for markers located on chromosome 4q25-27. A maximal LOD score of 7.05 was found for marker D4S402. The identification of a fourth locus for LQTS confirms its genetic heterogeneity. Locus 4q25-27 is associated with a peculiar phenotype within the LQTS entity.     

Expression of GIRK (Kir3.1/Kir3.4) channels in mouse fibroblast cells with and without beta1 integrins

G protein-activated K+ channel (GIRK) subunits possess a conserved extracellular integrin-binding motif (RGD) and bind directly to beta1 integrins. We expressed GIRK1/GIRK4 channels labeled with green fluorescent protein in fibroblast cell lines expressing or lacking beta1 integrins. Neither plasma membrane localization nor agonist-evoked GIRK currents were affected by the absence of beta1 integrins or by incubation with externally applied RGD-containing peptide. Mutation of the aspartate (D) of RGD impaired currents, GIRK glycosylation, and membrane localization, but the interaction with beta1 integrins remained intact. Thus, beta1 integrins are not essential for functional GIRK expression; and the GIRK-integrin interactions involve structural elements other than the RGD motif.     

Presence of Macrovolt T Wave Alternans and Short Coupled PVC Simultaneously in a Patient with Long QT Syndrome

This report presents a patient with macrovolt T wave alternans, PVC with R on T or a long-short sequence followed by torsades de pointes.     

The selectivity filter may act as the agonist-activated gate in the G protein-activated Kir3.1/Kir3.4 K+ channel

The Kir3.1/Kir3.4 channel is activated by Gbetagamma subunits released on binding of acetylcholine to the M2 muscarinic receptor. A mechanism of channel opening, similar to that for the KcsA and Shaker K+ channels, has been suggested that involves translocation of pore lining transmembrane helices and the opening of an intracellular gate at the "bundle crossing" region. However, in the present study, we show that an extracellular gate at the selectivity filter is critical for agonist activation of the Kir3.1/Kir3.4 channel. Increasing the flexibility of the selectivity filter, by disrupting a salt bridge that lies directly behind the filter, abolished both selectivity for K+ and agonist activation of the channel. Other mutations within the filter that altered selectivity also altered agonist activation. In contrast, mutations within the filter that did not affect selectivity had little if any effect on agonist activation. Interestingly, mutation of bulky side chain phenylalanine residues at the bundle crossing also altered both agonist activation and selectivity. These results demonstrate a significant correlation between agonist activation and selectivity, which is determined by the selectivity filter, and suggests, therefore, that the selectivity filter may act as the agonist-activated gate in the Kir3.1/Kir3.4 channel.     

Novel Mechanisms of Trafficking Defect Caused by KCNQ1 Mutations Found in Long QT Syndrome

Long QT syndrome (LQTS) is a hereditary arrhythmia caused by mutations in genes for cardiac ion channels, including a potassium channel, KvLQT1. Inheritance of LQTS is usually autosomal-dominant, but autosomal-recessive inheritance can be observed in patients with LQTS accompanied by hearing loss. In this study, we investigated the functional alterations caused by KCNQ1 mutations, a deletion (delV595) and a frameshift (P631fs/19), which were identified in compound heterozygous state in two patients with autosomal-recessive LQTS not accompanied by hearing loss. Functional analyses showed that both mutations impaired cell surface expression due to trafficking defects. The mutations severely affected outward potassium currents without apparent dominant negative effects. It was found that delV595 impaired subunit binding, whereas P631fs/19 was retained in endoplasmic reticulum due to the newly added 19-amino acid sequence containing two retention motifs (R⁶³³GR and R⁶⁴⁶LR). This is the first report of novel mechanisms for trafficking abnormality of cardiac ion channels, providing us new insights into the molecular mechanisms of LQTS.     

Identification and Functional Analysis of a Novel MIP Gene Mutation Associated with Congenital Cataract in a Chinese Family

Congenital cataracts are major cause of visual impairment and blindness in children and previous studies have shown about 1/3 of non-syndromic congenital cataracts are inherited. Major intrinsic protein of the lens (MIP), also known as AQP0, plays a critical role in transparency and development of the lens. To date, more than 10 mutations in MIP have been linked to hereditary cataracts in humans. In this study, we investigated the genetic and functional defects underlying a four-generation Chinese family affected with congenital progressive cortical punctate cataract. Mutation screening of the candidate genes revealed a missense mutation at position 448 (c.448G>C) of MIP, which resulted in the substitution of a conserved aspartic acid with histidine at codon 150 (p.D150H). By linkage and haplotype analysis, we obtained positive multipoint logarithm of odds (LOD) scores at microsatellite markers D12S1632 (Z_max = 1.804 at α = 1.000) and D12S1691 (Z_max = 1.806 at α = 1.000), which flanked the candidate locus. The prediction results of PolyPhen-2 and SIFT indicated that the p.D150H mutation was likely to damage to the structure and function of AQP0. The wild type and p.D150H mutant AQP0 were expressed in HeLa cells separately and the immunofluorescence results showed that the WT-AQP0 distributed at the plasma membrane and in cytoplasm, while AQP0-D150H failed to reach the plasma membrane and was mainly retained in the Golgi apparatus. Moreover, protein levels of AQP0-D150H were significantly lower than those of wide type AQP0 in membrane-enriched lysates when the HEK-293T cells were transfected with the same amount of wild type and mutant plasmids individually. Taken together, our data suggest the p.D150H mutation is a novel disease-causing mutation in MIP, which leads to congenital progressive cortical punctate cataract by impairing the trafficking mechanism of AQP0.     

Base of pore loop is important for rectification, activation, permeation, and block of Kir3.1/Kir3.4

The Kir3.1/Kir3.4 channel is an inward rectifier, agonist-activated K(+) channel. The location of the binding site within the channel pore that coordinates polyamines (and is thus responsible for inward rectification) and the location of the gate that opens the channel in response to agonist activation is unclear. In this study, we show, not surprisingly, that mutation of residues at the base of the selectivity filter in the pore loop and second transmembrane domain weakens Cs(+) block and decreases selectivity (as measured by Rb(+) and spermine permeation). However, unexpectedly, the mutations also weaken inward rectification and abolish agonist activation of the channel. In the wild-type channel and 34 mutant channels, there are significant (p < 0.05) correlations among the K(D) for Cs(+) block, Rb(+) and spermine permeation, inward rectification, and agonist activation. The significance of these findings is discussed. One possible conclusion is that the selectivity filter is responsible for inward rectification and agonist activation as well as permeation and block.     

A COLQ Missense Mutation in Sphynx and Devon Rex Cats with Congenital Myasthenic Syndrome

An autosomal recessive neuromuscular disorder characterized by skeletal muscle weakness, fatigability and variable electromyographic or muscular histopathological features has been described in the two related Sphynx and Devon Rex cat breeds (Felis catus). Collection of data from two affected Sphynx cats and their relatives pointed out a single disease candidate region on feline chromosome C2, identified following a genome-wide SNP-based homozygosity mapping strategy. In that region, we further identified COLQ (collagen-like tail subunit of asymmetric acetylcholinesterase) as a good candidate gene, since COLQ mutations were identified in affected humans and dogs with endplate acetylcholinesterase deficiency leading to a synaptic form of congenital myasthenic syndrome (CMS). A homozygous c.1190G>A missense variant located in exon 15 of COLQ, leading to a C397Y substitution, was identified in the two affected cats. C397 is a highly-conserved residue from the C-terminal domain of the protein; its mutation was previously shown to produce CMS in humans, and here we confirmed in an affected Sphynx cat that it induces a loss of acetylcholinesterase clustering at the neuromuscular junction. Segregation of the c.1190G>A variant was 100% consistent with the autosomal recessive mode of inheritance of the disorder in our cat pedigree; in addition, an affected, unrelated Devon Rex cat recruited thereafter was also homozygous for the variant. Genotyping of a panel of 333 cats from 14 breeds failed to identify a single carrier in non-Sphynx and non-Devon Rex cats. Finally, the percentage of healthy carriers in a European subpanel of 81 genotyped Sphynx cats was estimated to be low (3.7%) and 14 control Devon Rex cats were genotyped as wild-type individuals. Altogether, these results strongly support that the neuromuscular disorder reported in Sphynx and Devon Rex breeds is a CMS caused by a unique c.1190G>A missense mutation, presumably transmitted through a founder effect, which strictly and slightly disseminated in these two breeds. The presently available DNA test will help owners avoid matings at risk.