scholarly journals Multiple splicing defects caused by hERG splice site mutation 2592+1G>A associated with long QT syndrome

2011 ◽  
Vol 300 (1) ◽  
pp. H312-H318 ◽  
Author(s):  
Matthew R. Stump ◽  
Qiuming Gong ◽  
Zhengfeng Zhou
Keyword(s):  
Splice Site ◽  
Long Qt Syndrome ◽  
Splice Site Mutation ◽  
Dominant Negative ◽  
Wild Type ◽  
Site Mutation ◽  
Long Qt ◽  
Herg Current ◽  
Qt Syndrome ◽  
Splicing Defects

Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene ( hERG). Cryptic splice site activation in hERG has recently been identified as a novel pathogenic mechanism of LQT2. In this report, we characterize a hERG splice site mutation, 2592+1G>A, which occurs at the 5′ splice site of intron 10. Reverse transcription-PCR analyses using hERG minigenes transfected into human embryonic kidney-293 cells and HL-1 cardiomyocytes revealed that the 2592+1G>A mutation disrupted normal splicing and caused multiple splicing defects: the activation of cryptic splice sites within exon 10 and intron 10 and complete intron 10 retention. We performed functional and biochemical analyses of the major splice product, hERGΔ24, in which 24 amino acids within the cyclic nucleotide binding domain of the hERG channel COOH-terminus is deleted. Patch-clamp experiments revealed that the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that mutant channels did not traffic to the cell surface. Coexpression of wild-type hERG and hERGΔ24 resulted in significant dominant-negative suppression of hERG current via the intracellular retention of the wild-type channels. Our results demonstrate that 2592+1G>A causes multiple splicing defects, consistent with the pathogenic mechanisms of long QT syndrome.

scholarly journals A splice site mutation in hERG leads to cryptic splicing in human long QT syndrome

2008 ◽  
Vol 44 (3) ◽  
pp. 502-509 ◽  
Author(s):  
Qiuming Gong ◽  
Li Zhang ◽  
Arthur J. Moss ◽  
G. Michael Vincent ◽  
Michael J. Ackerman ◽  
...  
Keyword(s):  
Splice Site ◽  
Long Qt Syndrome ◽  
Splice Site Mutation ◽  
Site Mutation ◽  
Long Qt ◽  
Qt Syndrome

Novel Donor Splice Site Mutation in the KVLQT1 Gene is Associated with Long QT Syndrome

1998 ◽  
Vol 9 (6) ◽  
pp. 620-624 ◽  
Author(s):  
JØRGEN K. KANTERS ◽  
LARS ALLAN LARSEN ◽  
MARIANNE ORHOLM ◽  
ERIK AGNER ◽  
PAAL SKYTT ANDERSEN ◽  
...  
Keyword(s):  
Splice Site ◽  
Long Qt Syndrome ◽  
Splice Site Mutation ◽  
Donor Splice Site ◽  
Site Mutation ◽  
Long Qt ◽  
Donor Splice ◽  
Qt Syndrome

Compound heterozygosity for mutations Asp611→Tyr in KCNQ1 and Asp609→Gly in KCNH2 associated with severe long QT syndrome

2005 ◽  
Vol 108 (2) ◽  
pp. 143-150 ◽  
Author(s):  
Masato YAMAGUCHI ◽  
Masami SHIMIZU ◽  
Hidekazu INO ◽  
Hidenobu TERAI ◽  
Kenshi HAYASHI ◽  
...  
Keyword(s):  
Long Qt Syndrome ◽  
Clinical Manifestations ◽  
Torsades De Pointes ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Wild Type ◽  
Long Qt ◽  
Mild Phenotype ◽  
Electrophysiological Properties ◽  
Qt Syndrome

LQTS (long QT syndrome) is an inherited cardiac disorder characterized by prolongation of QT interval, torsades de pointes and sudden death. We have identified two heterozygous missense mutations in the KCNQ1 and KCNH2 (also known as HERG) genes [Asp611→Tyr (D611Y) in KCNQ1 and Asp609→Gly (D609G) in KCNH2] in a 2-year-old boy with LQTS. The aim of the present study was to characterize the contributions of the mutations in the KCNQ1 and KCNH2 genes relative to the clinical manifestations and electrophysiological properties of LQTS. Six of 11 carriers of D611Y in KCNQ1 had long QT intervals. D609G in KCNH2 was detected only in the proband. Studies on the electrophysiological alterations due to the two missense mutations revealed that the D611Y mutation in KCNQ1 did not show a significant suppression of the currents compared with wild-type, but the time constants of current activation in the mutants were increased compared with that in the wild-type. In contrast, the D609G mutation in KCNH2 showed a dominant-negative suppression. Our results suggest that the mild phenotype produced by the D611Y mutation in KCNQ1 became more serious by addition of the D609G mutation in KCNH2 in the proband.

scholarly journals Single-Channel Characteristics of Wild-Type IKs Channels and Channels formed with Two MinK Mutants that Cause Long QT Syndrome

1998 ◽  
Vol 112 (6) ◽  
pp. 651-663 ◽  
Author(s):  
Federico Sesti ◽  
Steve A.N. Goldstein
Keyword(s):  
Long Qt Syndrome ◽  
Single Channel ◽  
Xenopus Laevis Oocytes ◽  
Wild Type ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Long Qt ◽  
Conduction Pathway ◽  
Qt Syndrome ◽  
Single Channel Currents

IKs channels are voltage dependent and K+ selective. They influence cardiac action potential duration through their contribution to myocyte repolarization. Assembled from minK and KvLQT1 subunits, IKs channels are notable for a heteromeric ion conduction pathway in which both subunit types contribute to pore formation. This study was undertaken to assess the effects of minK on pore function. We first characterized the properties of wild-type human IKs channels and channels formed only of KvLQT1 subunits. Channels were expressed in Xenopus laevis oocytes or Chinese hamster ovary cells and currents recorded in excised membrane patches or whole-cell mode. Unitary conductance estimates were dependent on bandwidth due to rapid channel “flicker.” At 25 kHz in symmetrical 100-mM KCl, the single-channel conductance of IKs channels was ∼16 pS (corresponding to ∼0.8 pA at 50 mV) as judged by noise-variance analysis; this was fourfold greater than the estimated conductance of homomeric KvLQT1 channels. Mutant IKs channels formed with D76N and S74L minK subunits are associated with long QT syndrome. When compared with wild type, mutant channels showed lower unitary currents and diminished open probabilities with only minor changes in ion permeabilities. Apparently, the mutations altered single-channel currents at a site in the pore distinct from the ion selectivity apparatus. Patients carrying these mutant minK genes are expected to manifest decreased K+ flux through IKs channels due to lowered single-channel conductance and altered gating.

Retention in the Endoplasmic Reticulum as a Mechanism of Dominant-negative Current Suppression in Human Long QT Syndrome

2000 ◽  
Vol 32 (12) ◽  
pp. 2327-2337 ◽  
Author(s):  
Eckhard Ficker ◽  
Adrienne T Dennis ◽  
Carlos A Obejero-Paz ◽  
Pasqualina Castaldo ◽  
Maurizio Taglialatela ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Long Qt Syndrome ◽  
Dominant Negative ◽  
Long Qt ◽  
Negative Current ◽  
Qt Syndrome

scholarly journals Growth Hormone (GH)-Releasing Hormone Increases the Expression of the Dominant-Negative GH Isoform in Cases of Isolated GH Deficiency due to GH Splice-Site Mutations

Endocrinology ◽  
2010 ◽  
Vol 151 (6) ◽  
pp. 2650-2658 ◽  
Author(s):  
Vibor Petkovic ◽  
Michela Godi ◽  
Didier Lochmatter ◽  
Andrée Eblé ◽  
Christa E. Flück ◽  
...  
Keyword(s):  
Splice Site ◽  
Gh Deficiency ◽  
Splice Site Mutation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Pituitary Cells ◽  
Site Mutation ◽  
Dominant Form ◽  
Negative Effect ◽  
Rat Pituitary Cells

An autosomal dominant form of isolated GH deficiency (IGHD II) can result from heterozygous splice site mutations that weaken recognition of exon 3 leading to aberrant splicing of GH-1 transcripts and production of a dominant-negative 17.5-kDa GH isoform. Previous studies suggested that the extent of missplicing varies with different mutations and the level of GH expression and/or secretion. To study this, wt-hGH and/or different hGH-splice site mutants (GH-IVS+2, GH-IVS+6, GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GC-GHRHR). Upon GHRH stimulation, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed reduced hGH secretion and intracellular GH content when compared with cells expressing only wt-hGH, confirming the dominant-negative effect of 17.5-kDa isoform on the secretion of 22-kDa GH. Furthermore, increased amount of 17.5-kDa isoform produced after GHRH stimulation in cells expressing GH-splice site mutants reduced production of endogenous rat GH, which was not observed after GHRH-induced increase in wt-hGH. In conclusion, our results support the hypothesis that after GHRH stimulation, the severity of IGHD II depends on the position of splice site mutation leading to the production of increasing amounts of 17.5-kDa protein, which reduces the storage and secretion of wt-GH in the most severely affected cases. Due to the absence of GH and IGF-I-negative feedback in IGHD II, a chronic up-regulation of GHRH would lead to an increased stimulatory drive to somatotrophs to produce more 17.5-kDa GH from the severest mutant alleles, thereby accelerating autodestruction of somatotrophs in a vicious cycle.

scholarly journals Early somatic mosaicism is a rare cause of long-QT syndrome

2016 ◽  
Vol 113 (41) ◽  
pp. 11555-11560 ◽  
Author(s):  
James Rush Priest ◽  
Charles Gawad ◽  
Kristopher M. Kahlig ◽  
Joseph K. Yu ◽  
Thomas O’Hara ◽  
...  
Keyword(s):  
Long Qt Syndrome ◽  
Sodium Current ◽  
Somatic Mosaicism ◽  
Mendelian Inheritance ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Causal Mechanism ◽  
Long Qt ◽  
Gene Panel Testing ◽  
Qt Syndrome

Somatic mosaicism, the occurrence and propagation of genetic variation in cell lineages after fertilization, is increasingly recognized to play a causal role in a variety of human diseases. We investigated the case of life-threatening arrhythmia in a 10-day-old infant with long QT syndrome (LQTS). Rapid genome sequencing suggested a variant in the sodium channel NaV1.5 encoded by SCN5A, NM_000335:c.5284G > T predicting p.(V1762L), but read depth was insufficient to be diagnostic. Exome sequencing of the trio confirmed read ratios inconsistent with Mendelian inheritance only in the proband. Genotyping of single circulating leukocytes demonstrated the mutation in the genomes of 8% of patient cells, and RNA sequencing of cardiac tissue from the infant confirmed the expression of the mutant allele at mosaic ratios. Heterologous expression of the mutant channel revealed significantly delayed sodium current with a dominant negative effect. To investigate the mechanism by which mosaicism might cause arrhythmia, we built a finite element simulation model incorporating Purkinje fiber activation. This model confirmed the pathogenic consequences of cardiac cellular mosaicism and, under the presenting conditions of this case, recapitulated 2:1 AV block and arrhythmia. To investigate the extent to which mosaicism might explain undiagnosed arrhythmia, we studied 7,500 affected probands undergoing commercial gene-panel testing. Four individuals with pathogenic variants arising from early somatic mutation events were found. Here we establish cardiac mosaicism as a causal mechanism for LQTS and present methods by which the general phenomenon, likely to be relevant for all genetic diseases, can be detected through single-cell analysis and next-generation sequencing.

Long QT syndrome and associated gene mutation carriers in Japanese children: results from ECG screening examinations

2009 ◽  
Vol 117 (12) ◽  
pp. 415-424 ◽  
Author(s):  
Kenshi Hayashi ◽  
Noboru Fujino ◽  
Katsuharu Uchiyama ◽  
Hidekazu Ino ◽  
Kenji Sakata ◽  
...  
Keyword(s):  
Ion Channel ◽  
Long Qt Syndrome ◽  
High Probability ◽  
Dominant Negative ◽  
Carrier Status ◽  
Japanese Children ◽  
Long Qt ◽  
Mutation Carriers ◽  
Qt Intervals ◽  
Qt Syndrome

LQTS (long QT syndrome) is caused by mutations in cardiac ion channel genes; however, the prevalence of LQTS in the general population is not well known. In the present study, we prospectively estimated the prevalence of LQTS and analysed the associated mutation carriers in Japanese children. ECGs were recorded from 7961 Japanese school children (4044 males; mean age, 9.9±3.0 years). ECGs were examined again for children who had prolonged QTc (corrected QT) intervals in the initial ECGs, and their QT intervals were measured manually. An LQTS score was determined according to Schwartz's criteria, and ion channel genes were analysed. In vitro characterization of the identified mutants was performed by heterologous expression experiments. Three subjects were assigned to a high probability of LQTS (3.5≤ LQTS score), and eight subjects to an intermediate probability (1.0< LQTS score ≤3.0). Genetic analysis of these II subjects identified three KCNH2 mutations (M124T, 547–553 del GGCGGCG and 2311–2332 del/ins TC). In contrast, no mutations were identified in the 15 subjects with a low probability of LQTS. Electrophysiological studies showed that both the M124T and the 547–553 del GGCGGCG KCNH2 did not suppress the wild-type KCNH2 channel in a dominant-negative manner. These results demonstrate that, in a random sample of healthy Japanese children, the prevalence of a high probability of LQTS is 0.038% (three in 7961), and that LQTS mutation carriers can be identified in at least 0.038% (one in 2653). Furthermore, large-scale genetic studies will be needed to clarify the real prevalence of LQTS by gene-carrier status, as it may have been underestimated in the present study.

scholarly journals Occipital Horn Syndrome as a Result of Splice Site Mutations in ATP7A. No Activity of ATP7A Splice Variants Missing Exon 10 or Exon 15

2021 ◽  
Vol 14 ◽  
Author(s):  
Lisbeth Birk Møller ◽  
Mie Mogensen ◽  
David D. Weaver ◽  
Per Amstrup Pedersen
Keyword(s):  
Splice Site ◽  
Splice Variants ◽  
Splice Site Mutation ◽  
Early Death ◽  
Wild Type ◽  
Site Mutation ◽  
Functional Protein ◽  
Protein Variants ◽  
Occipital Horn ◽  
Exon 10

Disease-causing variants in ATP7A lead to two different phenotypes associated with copper deficiency; a lethal form called Menkes disease (MD), leading to early death, and a much milder form called occipital horn syndrome (OHS). Some investigators have proposed that an ATP7A transcript missing exon 10 leads to a partly active protein product resulting in the OHS phenotype. Here, we describe an individual with OHS, a biology professor, who survived until age 62 despite a splice site mutation, leading to skipping of exon 15. ATP7A transcripts missing exon 10, or exon 15 preserve the reading frame, but it is unknown if either of these alternative transcripts encode functional protein variants. We have investigated the molecular consequence of splice site mutations leading to skipping of exon 10 or exon 15 which have been identified in individuals with OHS, or MD. By comparing ATP7A expression in fibroblasts from three individuals with OHS (OHS-fibroblasts) to ATP7A expression in fibroblasts from two individuals with MD (MD-fibroblasts), we demonstrate that transcripts missing either exon 10 or exon 15 were present in similar amounts in OHS-fibroblasts and MD-fibroblasts. No ATP7A protein encoded from these transcripts could be detected in the OHS and MD fibroblast. These results, combined with the observation that constructs encoding ATP7A cDNA sequences missing either exon 10, or exon 15 were unable to complement the high iron requirement of the ccc2Δ yeast strain, provide evidence that neither a transcript missing exon 10 nor a transcript missing exon 15 results in functional ATP7A protein. In contrast, higher amounts of wild-type ATP7A transcript were present in the OHS-fibroblasts compared with the MD-fibroblasts. We found that the MD-fibroblasts contained between 0 and 0.5% of wild-type ATP7A transcript, whereas the OHS-fibroblasts contained between 3 and 5% wild-type transcripts compared with the control fibroblasts. In summary these results indicate that protein variants encoded by ATP7A transcripts missing either exon 10 or exon 15 are not functional and not responsible for the OHS phenotype. In contrast, expression of only 3-5% of wild-type transcript compared with the controls permits the OHS phenotype.

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