ELECTROPHYSIOLOGY
An intronic mutation causes long QT syndrome
Li Zhang, MD*,
G. Michael Vincent, MD, FACC*, ,*,
Marco Baralle, PhD ,
Francisco E. Baralle, MD, PhD,
Blake D. Anson, PhD ,
D. Woodrow Benson, MD, PhD||,
Bryant Whiting, BA*,
Katherine W. Timothy, BS,
John Carlquist, PhD*,,
Craig T. January, MD, PhD, FACC,
Mark T. Keating, MD¶ and
Igor Splawski, PhD¶
* LDS Hospital, Salt Lake City, Utah, USA
University of Utah School of Medicine, Salt Lake City, Utah, USA
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
University of Wisconsin, Madison, Wisconsin, USA
|| Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA
¶ Children's Hospital, Harvard Medical School, Howard Hughes Medical Institute, Boston, Massachusetts, USA
Manuscript received April 12, 2004;
revised manuscript received June 3, 2004,
accepted June 7, 2004.
* Reprint requests and correspondence: Dr. G. Michael Vincent, Department of Medicine, LDS Hospital, 324 10th Avenue, Suite 130, Salt Lake City, Utah 84103 (Email: ldgvince{at}ihc.com).
 |
Abstract
|
|---|
OBJECTIVES: The purpose of this research was to determine whether an intronic variant (T1945+6C) in KCNH2 is a disease-causing mutation, and if expanded phenotyping criteria produce improved identification of long QT syndrome (LQTS) patients.
BACKGROUND: Long QT syndrome is usually caused by mutations in conserved coding regions or invariant splice sites, yet no mutation is found in 30% to 50% of families. In one such family, we identified an intronic variant in KCNH2. Long QT syndrome diagnosis is hindered by reduced penetrance, as the long QT phenotype is absent on baseline electrocardiogram (ECG) in about 30%.
METHODS: Fifty-two family members were phenotyped by baseline QTc, QTc maximum on serial ECGs (Ser QTc-max), and on exercise ECGs (Ex QTc-max) and by T-wave patterns. Linkage analysis tested association of the intronic change with phenotype. The consequences of T1945+6C on splicing was studied using a minigene system and on function by heterologous expression.
RESULTS: Expanded phenotype/pedigree criteria identified 23 affected and 29 unaffected. Affected versus unaffected had baseline QTc 484 ± 48 ms versus 422 ± 20 ms, Ser QTc-max 508 ± 48 ms versus 448 ± 10 ms, Ex QTc-max 513 ± 54 ms versus 444 ± 11 ms, and LQT2 T waves in 87% versus 0%. Linkage analysis demonstrated a logarithm of odds score of 10.22. Splicing assay showed T1945+6C caused downstream intron retention. Complementary deoxyribonucleic acid with retained intron 7 failed to produce functional channels.
CONCLUSIONS: T1945+6C is a disease-causing mutation. It alters KCNH2 splicing and cosegregates with the LQT2 phenotype. Expanded ECG criteria plus pedigree analysis provided accurate clinical diagnosis of all carriers including those with reduced penetrance. Intronic mutations may be responsible for LQTS in some families with otherwise negative mutation screening.
|
Abbreviations and Acronyms
| | Ex QTc-max = maximum QTc value during the exercise test, either exercise or recovery | | LQTS = long QT syndrome | | LQT2 = second described variant of LQTS, due to mutations of the KCNH2 (HERG) gene | | RT-PCR = reverse transcription-polymerase chain reaction | | Ser QTc-max = maximum QTc value among serial electrocardiograms during follow-up |
|
Hereditary long QT syndrome (LQTS) is caused by over 250 mutations in five genes, four encoding potassium channel subunits KCNQ1 (KVLQT1, LQT1), KCNH2 (HERG, LQT2), KCNE1 (minK, LQT5), KCNE2 (MiRP1, LQT6), and the cardiac sodium channel gene SCN5A (LQT3) (1). Mutations of ANKB (2) and KCNJ2 (3) have been reported to cause LQT4 and LQT7, respectively. Our recent reports on the ANKB and KCJN2 disorders (4,5) have shown that the mean QTc is within the normal range, the majority of patients have normal to borderline QT intervals, and patients in each disorder have other clinical features that are distinctly different from LQT1 to 3. Thus, classification of ANKB and KCNJ2 mutations as LQTS disorders is uncertain and remains to be fully elucidated. Genetic screens for mutations in coding regions or flanking intronic sequences of the five LQTS genes have been performed frequently during the past decade. Currently, even after improvements in sequence detection technology, a mutation cannot be identified in about 30% of LQTS families. Many of these patients exhibit characteristic T-wave patterns of the known LQTS genotypes (6), prompting us to believe that mutations in noncoding regions of the known genes may be the cause of LQTS in some of these families.
One such Caucasian family from the LDS Hospital LQTS program was the stimulus for this study. In 1994, the 27-year-old proband experienced a cardiac arrest triggered by activity and excitement, with subsequent documented torsade de pointes (Fig. 1). He had no prior symptoms, but siblings had experienced recurrent syncope. Electrocardiogram (ECG) screening of the nuclear family showed QTc prolongation and typical LQT2 T-wave patterns (Fig. 2) (6) in several members, suggesting a KCNH2 mutation. Mutation analysis was performed in one affected family member, but failed to find a sequence variation in the coding sequences of any of the five known LQTS genes. The only sequence variant identified was a single nucleotide substitution, T1945+6C, at the 5' splice (donor) site of intron 7 in KCNH2. This substitution was not found in 320 ethnically matched normal control subjects.
View larger version (18K):
[in this window]
[in a new window]
|
Figure 1 Electrocardiogram monitor strip showing torsade de pointes recorded from the proband during resuscitation from cardiac arrest at age of 27 years.
|
|
View larger version (131K):
[in this window]
[in a new window]
|
Figure 2 Examples of typical LQT2 T-wave patterns in the affected family members. (a) Low bifid T waves in the proband, QTc 500 ms. (b and c) Subtle bifid T waves in two other affected members, QTc of 500 ms and 480 ms, respectively. Overall, 87% of T1945+6C mutation carriers presented typical LQT2 T-wave patterns on baseline electrocardiogram.
|
|
An analysis of 400 vertebrate genes (7) has yielded consensus sequences near intron-exon boundaries that are essential for correct splicing. The 10 base pair (bp) (–4 to +6) including the GT at the 5' start of the intron constitute the 5' (donor) splice site. The G and T at positions +1 and +2, respectively, are highly conserved (100%), and, consequently, sequence alterations at these locations are readily recognized as mutations. Several adjacent nucleotides at the splice donor site are less well-conserved, and the nucleotide at position +6 is a T in  50% of the introns.
Intronic KCNH2 mutations in LQTS have been infrequently reported. For example, Splawski et al. (1) reported only three (4%) such mutations in an analysis of 72 KCNH2 mutations. These three mutations altered obligatory (100% conservation) nucleotides within donor or acceptor splice sites and, in the absence of functional analysis, were assumed to alter transcript splicing. By contrast, T1945+6C changes a less highly conserved (50%) intronic nucleotide of a donor splice site and could not be assumed to alter transcript splicing. Therefore, to determine whether this is a disease-causing mutation or a simple polymorphism, we expanded the pedigree, used enhanced ECG and pedigree criteria for phenotyping, tested the association of genotype with phenotype using linkage analysis, and elucidated the splicing effect and molecular mechanism of the T1945+6C through well-established cellular splicing assay and heterologous expression studies.
|
Methods
|
|---|
Phenotyping.
A five-generation pedigree was constructed, and 52 blood-related family members were evaluated by history and ECG (Fig. 3). History evaluation included LQTS-related syncope, cardiac arrest, and sudden death. Electrocardiogram analysis was from supine, resting ECGs, and included baseline QTc interval (Bazett's formula) and T-wave pattern (6) on all subjects. During follow-up, we obtained ECGs at yearly clinic visits and at other times as indicated by clinical needs. Twenty-two members received additional ECG evaluation, either bicycle exercise testing (n = 18) and/or serial ECGs (n = 19) ( 2 ECGs/person). From these, the maximum QTc during exercise or recovery (Ex QTc-max) and on serial ECGs (Ser QTc-max) was evaluated. None of the patients was on beta-blockers at time of initial ECG data acquisition, and the phenotyping was performed without knowledge of genotype.
View larger version (27K):
[in this window]
[in a new window]
|
Figure 3 Pedigree structure of the multigenerational family demonstrates the association between the T1945+6C mutation and the LQT2 phenotype. The pedigree was modified to protect patient and family privacy.
|
|
Family members were determined to be phenotypically affected if they met one of the following criteria: 1) a diagnostic QTc interval (470 ms in males or 480 ms in females) (8) with or without the presence of an LQT2 T-wave pattern, on baseline, Ex QTc-max, or Ser QTc-max ECGs; 2) a QTc of 460 ms with an LQT2 T-wave pattern if other family member(s) met the diagnostic criterion 1; and 3) identification as an obligate gene carrier on family pedigree; members could be classified as an obligate gene carrier if offspring plus other blood relative(s) showed a definite LQT2 phenotype (Fig. 3).
Genotyping.
Informed consent (approved by and in accordance with the guidelines of the Institutional Review Board at LDS Hospital of Intermountain Health Care, Inc.) was obtained from all participants before genetic testing.
DNA was collected from buccal swabs and extracted using the QIAamp DNA Mini Kit (Qiagen, Valencia, California). The region containing the T1945+6C intronic variant was amplified at LDS Hospital using previously published oligonucleotides (9). Samples were sequenced by ARUP Laboratories (Associated Regional University Pathologists, Salt Lake City, Utah) using the Applied Biosystems 377 (Foster City, California) deoxyribonucleic acid sequencer with Big Dye Terminator chemistry. The results were read by a single investigator blinded to the phenotype data.
Statistical and linkage analyses.
The QTc on baseline ECG, the Ser QTc-max, and the Ex QTc-max were compared between affected and unaffected members using the Mann-Whitney test. The phenotype-genotype agreement was measured as a Kappa statistic. The statistical analyses were performed using SPSS 10.1 for Windows (SPSS Inc., Chicago, Illinois). Using KCNH2-T1945+6C as an allele, we performed linkage analysis. A two-point logarithm of odds (LOD) score was calculated using LIPED software(Rockefeller University, New York, New York) (10), assuming an allele frequency of 0.1% and disease penetrance of 95%.
Functional splicing assays.
Normal and mutated (T1945+6C change) sequences of the KCNH2 gene were amplified from genomic deoxyribonucleic acid of an individual heterozygous for the mutation using forward primer 5'-ggaattccatatggaatcactgcacctgtcagtgc-3' and reverse primer 5'-ggaattccatatggaattgtacatctgcgctccagc-3'. The amplification generated a fragment that contained exon 7 with 248 bp of 5' and 202 bp of 3' intronic flanking sequences. Both oligonucleotides carry an Nde1 restriction enzyme site in their 5' ends that was used to clone the product into a modified version of the alpha-globin-fibronectin EDB minigene (11). The splicing assay was performed by transfecting 0.5 µg of each minigene plasmid into 3 x 105 He La cells with Qiagen Effectene transfection reagents. Ribonucleic acid extraction and reverse transcription-polymerase chain reaction (RT-PCR) analysis were performed as previously described (11). All of the fragments shown in Figure 4 (panel B) were cloned and sequenced to confirm their identity and exclude polymerase errors.
View larger version (143K):
[in this window]
[in a new window]
|
Figure 4 Diagnostic value of Ex QTc-max in patients with a borderline baseline QTc interval. A 23-year-old male asymptomatic family member shows a baseline QTc of 440 ms during sinus bradycardia. An Ex QTc-max of 480 ms at 9 min of the bicycle exercise test identifies his affected status. Genetic testing revealed he was a T1945+6C mutation carrier.
|
|
The parental U1 snRNA was pG3U1 (WT-U1), a derivative of PHU1 (12). The variant A>G U1 snRNA, complementary to the nucleotide change observed in the patient, was created by replacing the U1 snRNA 5' sequence between the sites Bcl1 and BglII in the pG3U1 plasmid with the mutant oligonucleotides. The effect of T1945+6C mutation on ribonucleic acid processing was studied using a well-documented hybrid minigene system (11,13,14).
Functional expression assays.
To study the effects of T1945+6C, human embryonic kidney (HEK-293) cells were transiently transfected with either KCNH2-WT cDNA or KCNH2 complementary deoxyribonucleic acid containing T1945+6C and intron 7 (KCNH2-T>C). Green fluorescent protein (GFP) was cotransfected with each complementary deoxyribonucleic acid as a marker for successful transfection. The KCNH2-WT cDNA expression vector has been described previously (15), and GFP was expressed with the mammalian expression vector pRK-5 (Clonetech, Mansfield, United Kingdom). The KCNH2-T>C expression vector was generated by amplifying T1945+6C genomic deoxyribonucleic acid, restricting at unique Bgl II and Xho I restriction sites in exons 7 and 8, and subsequently ligating into the WT expression vector. All amplified sequences were confirmed to be error-free through Big Dye sequencing. Current levels were measured from GFP-expressing cells 24 to 48 h after transfection using tight-seal whole-cell voltage clamp recording techniques. Peak currents were assessed as peak tail current (Itail) (Fig. 5, arrows) evoked by depolarizing cells from a holding potential of –80 mV to 20 mV for 5 s followed by repolarization to –50 mV for 2.85 s. Details for both the transfection and recording techniques have been described in detail previously (16).
View larger version (30K):
[in this window]
[in a new window]
|
Figure 5 (A) Schematic representation of the KCNH2 exon 7 minigene construct. The human KCNH2 exon 7 was cloned into the Nde I site of the  -globin fibronectin EDB minigene (black shaded and white boxes, respectively, with intervening sequences [IVS] shown as thin lines). The intronic mutation T1945+6C is shown with exonic sequence in upper case and intronic sequence in lower case. The superimposed arrows indicate the primers used in the reverse transcription-polymerase chain reaction (RT-PCR) assay. (B) Agarose gel electrophoresis of the RT-PCR products generated from the splicing assay: T1945+6 (WT) generates a single band of approximately 637 base pairs (bp) (lane 2) while T1945+6C generates three different sized bands (lane 3), whose specific identity was established by cloning and sequencing. On the right of the gel, there is a graphic representation illustrating the DNA content of each band. The 239 bp band contains the fibronectin exons, the 637 bp band contains the fibronectin exons with exon 7 inserted between them, and the 1,302 bp band contains the fibronectin exons, exon 7, as well as the intron 3' to this exon. Note that the mutation causes intron retention and some exon skipping. The modified snRNA (A>G-U1) rescues this splicing defect (lane 4). (C) Upper panel: Base pairing between U1 snRNA (WT-U1) and the 3' end of KCNH2 exon 7. The nucleotide change T1945+6C reduces the base pairing between the RNA and the WT-U1 snRNA. Lower panel: The variant snRNA (A>G-U1) modified to complement the nucleotide change seen in the patient's pre-mRNA, and restored the appropriate base pairing.
|
|
|
Results
|
|---|
Phenotyping.
Phenotype was characterized in the 52 blood-related family members based on medical history, ECG assessment, and pedigree analysis. Twenty-three individuals were considered affected and 29 unaffected (Fig. 3). The QTc on baseline ECG, Ex QTc-max, and the Ser QTc-max were all significantly longer in affected versus nonaffected members (Table 1). A total of 87% of affected members presented typical LQT2 patterns in baseline ECG (Table 1, Fig. 2). No significant gender differences were found in the QTc interval and the T-wave patterns among affected members.
A total of 65% (15 of 23) of affected members were correctly diagnosed by baseline QTc (503 ± 46 ms, range 460 to 610 ms) and the presence of typical LQT2 T-wave patterns in 15 of 15. The other 35% (8 of 23) had a QTc of 449 ± 16 ms (420 to 460 ms) and were classified as "uncertain," as these values overlap with those of normals. The LQTS diagnosis was confirmed in six of the eight by follow-up assessment: Ser QTc-max (3 ± 1 ECGs/person over 1.3 ± 1.9 years of 467 ± 10, range 460 to 480 ms), and Ex QTc-max (482 ± 11 ms, range 470 to 490 ms) (Fig. 4). All six met diagnostic criterion #2, typical T-wave pattern coupled with a QTc of 460 ms or greater, and most met criterion #1. The remaining two members (II-9 and III-21) (Fig. 3), an 86-year-old female and her daughter, both with QTc of 460 ms and absence of an LQT2 T-wave pattern, were identified as obligate mutation carriers by pedigree analysis. Altogether, therefore, 23 affected members were identified, 21 by the expanded ECG phenotype criteria and 2 as obligate carriers by pedigree analysis.
Sudden death of uncertain cause had occurred in 8.7% (2 of 23) before the LQTS diagnosis in the family, an 18-year-old male (II-3) (Fig. 3) and a 59-year-old female (III-16) (Fig. 3). Four (17%) affected members had syncope, and one had resuscitated cardiac arrest (IV-20) (Fig. 1), before treatment with beta-blockers.
Genotyping.
Sequence analyses revealed 22 T1945+6C carriers and 29 noncarriers. An obligate carrier (II-1) (Fig. 3) was phenotyped as affected by baseline ECG (QTc 490 ms with an LQT2 T-wave pattern), but he died at age 85 before genetic testing was available.
Genotype-phenotype correlation: Advantage of the expanded QTc criteria and pedigree analysis.
The T1945+6C variant cosegregated with affected status. Linkage analysis yielded a maximum two-point LOD score of 10.22 at recombination fraction  = 0.0, indicating odds of >1010 that the T1945+6C is linked to the disease phenotype.
Phenotype-genotype correlation showed concordance in 21 of 23 carriers and 29 of 29 noncarriers (Kappa = 0.96). Two obligate carriers were identified by pedigree analysis so that the combination of expanded ECG phenotyping criteria plus identification of obligate carriers revealed all 23 mutation carriers (Fig. 3).
Functional effect of the intron 7 mutation.
The effect of T1945+6C on RNA processing was studied using a well-tested and documented hybrid minigene system (11,13,14) (Fig. 5, panel A). After transfection and expression of the construct in He La cells, the mRNA derived from the minigene was analyzed for the KCNH2 exon 7 splicing pattern by RT-PCR. As shown in Figure 5 (panel B, lanes exon 7 WT and exon 7 T>C), the T1945+6C dramatically affected pre-mRNA processing. In contrast with wild-type, T1945+6C processing resulted in three species of RNA that were unambiguously identified by cloning and complete sequencing. The major band (1,302 bp) shows retention of the intronic sequences downstream of the KCNH2 exon 7 in the mature messenger ribonucleic acid. A lower level of normally spliced mRNA (637 bp) is also present as well as an additional faint band (239 bp) that results from the skipping of the exon.
Previous studies on a similar mutation in the NF-1 gene (14) showed simple exon skipping but not intron retention. However, our hypothesis was that the same basic molecular mechanism was involved, that is, the lack of recognition of the 5' splice site in the mutant gene due to an imperfect base pairing with the U1 snRNP that is required for normal intron-exon splicing. Figure 4, panel C, is a schematic illustrating the decrease in complementarity between the mutant and U1 snRNA when compared with wild-type sequence. To functionally test this hypothesis, we simultaneously introduced into the cells the T1945+6C minigene with a variant U1 snRNA complementary to the mutation (Fig. 5, panel C). This resulted in the almost complete rescue of the correct splicing pattern (Fig. 5, panel B, lane exon 7 T>C + U1 A>G).
The effect of intron retention on KCNH2 current was examined through heterologous expression of KCNH2-WT and KCNH2-T>C cDNA in HEK-293 cells. Representative current tracings from KCNH2-WT or KCNH2-T>C transfected cells are shown in Figure 6 (upper and lower tracings, respectively). All recordings from cells transfected with KCNH2-WT cDNA produced normal KCNH2 currents with a mean peak Itail of 938.3 ± 157.6 pA (n = 15) while none of the cells transfected with KCNH2-T>C complementary deoxyribonucleic acid produced currents above the basal noise level (7.9 ± 2.6 pA; n = 20). Thus, complementary deoxyribonucleic acid with a retained intron 7 failed to produce functional channels.
View larger version (19K):
[in this window]
[in a new window]
|
Figure 6 Representative tight-seal whole-cell voltage clamp recordings from HEK-293 cells transfected with either KCNH2 or KCNH2 T>C cDNA. The upper trace illustrates the voltage clamp protocol while the middle and lower tracings show WT and T>C currents, respectively. Tail currents are denoted by the arrows. Note the complete absence of KCNH2 tail current in the KCNH2-T>C tracing. The inset shows a series of depolarizations from –70 to 40 mV for a cell expressing WT channels. The scale bar applies to the single traces and is 200 pA by 500 ms.
|
|
|
Discussion
|
|---|
These findings demonstrate that the T1945+6C sequence change in intron 7 of KCNH2 is a mutation that causes LQTS with a typical LQT2 phenotype. The functional cellular splicing assay demonstrated that T1945+6C produces a major transcript that retains intron 7. Two minor transcripts include a WT transcript and a transcript resulting from skipping of exon 7. Protein synthesized from the transcript containing intron 7 would be expected to undergo a translational frame-shift at amino acid residue 649 and prematurely truncate 21 amino acid residues further downstream. This would drastically alter the protein's 6th transmembrane domain, completely delete the C-terminus, and most likely render the protein nonfunctional. Whole cell recordings from HEK-293 cells transfected with KCNH2 WT or T>C complementary deoxyribonucleic acid support this conclusion. Although speculative, coassembly of mutant and WT subunits could further inhibit the activity of the residual correctly spliced protein as well as protein synthesized from the other allele. Our results also demonstrated that the impaired splicing of T1945+6C was, at least in part, due to imperfect U1 snRNP recognition of the mutant 5' splice site. This conclusion is supported by the fact that an artificial variant of the U1 snRNA, complementary to the mutant sequence, was able to rescue the correct pre-messenger ribonucleic acid processing.
Mutations in the GT splice donor and AG splice acceptor sites in LQTS genes have been infrequently reported (1,17–20). Because of the high conservation of these consensus sequences, such mutations have been assumed to alter splicing, but, until now, this hypothesis has remained untested. By expanding the genotypic and phenotypic analyses performed in the proband to include a large kindred of 52 individuals, we show that T1945+6C cosegregrates with the LQT2 phenotype. Absence of this nucleotide substitution in 320 ethnically matched control individuals further suggests that it is disease-causing. We have also clearly defined the defective splicing pattern of T1945+6C and provided a mechanism for the molecular dysfunction, making this study the first demonstration that incorrect intron-exon splicing of pre-messenger ribonucleic acid transcripts may result in LQT2. Furthermore, as T1945+6C is a less highly conserved (50%) nucleotide position than either the obligatory GT or AG of the donor and acceptor splice sites, this study clearly indicates that mutations of intronic nucleotides other than invariant splice donor and acceptor sites can cause LQTS. Of the 30% of LQTS families in which no mutation has been found with exon screening, the majority have characteristic T-wave patterns of the known genotypes (6). Consequently, in light of our results, it is reasonable to suggest that some of these families may have intronic mutations in the known LQTS genes rather than defects in unidentified genes.
At present, the function of introns is less well-understood than that of exons. Introns are, on average, much larger than exons and constitute about 25% of the human genome (21). They may harbor promoters or other regulatory modules that modify gene expression and might influence penetrance and expressivity of phenotypes (13,22–28). Thus, it may be that, in some cases, intron variations contribute to the phenotypic heterogeneity of LQTS (8,29,30).
The other important finding of this study is the high accuracy of diagnosis of affected family members using the enhanced QTc and T-wave morphology ECG criteria plus pedigree analysis. The phenotypic heterogeneity and reduced penetrance of the QT phenotype in LQTS (8,29,30) can impede accurate clinical diagnosis, leading to a missed diagnosis in about 30% of LQTS patients by baseline ECG. The enhanced ECG criteria and pedigree analyses used in this study correctly identified all the T1945+6C carriers, including those who had baseline normal-to-borderline QTc intervals of 400 to 460 ms, values that overlap those of normals. Large scale studies regarding the diagnostic value of these enhanced criteria in LQTS patients with nondiagnostic QTc intervals are currently underway. In addition to the usefulness of typical LQT2 T-wave patterns for increased diagnostic accuracy, these patterns are also very accurate for genotype prediction (6). The high accuracy provides a strategy for deoxyribonucleic acid screening by indicating which gene to first examine, thus reducing monetary and time costs.
|
Acknowledgments
|
|---|
The authors are grateful to Jay W. Mason, MD, and Robert L. Lux, PhD, for their support in genotyping some family members.
|
Footnotes
|
|---|
Sources of funding: LDS Hospital/Deseret Foundation grant DF400, Salt Lake City, Utah; Telethon Onlus Foundation grant E1038, Trieste, Italy; NHLBI grant R01 HL60723, Madison, Wisconsin; NIH grants R01 HL46401 and SCOR HL52338, Salt Lake City, Utah.
|
References
|
|---|
- Splawski I, Shen J, Timothy KW, et al. Spectrum of mutations in long-QT syndrome genes KVLQT1, HERG, SCN5A, KCNE1, and KCNE2 Circulation 2000;102:1178-1185.[Abstract/Free Full Text]
- Mohler PJ, Schott JJ, Gramolini AO, et al. Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death Nature 2003;421:634-639.[CrossRef][Medline]
- Tristani-Firouzi M, Jensen JL, Donaldson MR, et al. Functional and clinical characterization of KCNJ2 mutations associated with LQT7 (Andersen syndrome) J Clin Invest 2002;110:381-388.[CrossRef][Medline]
- Zhang L, Probst V, Marec HL, et al. Electrocardiographic abnormalities in patients with ankryin-B gene mutations(abstr) Heart Rhythm 2004;1:S101.
- Zhang L, Tristani-Firouzi M, Ptacek LJ, et al. ECG characteristics in Andersen syndrome patients with KCNJ2 mutations(abstr) J Am Coll Cardiol 2004;43(Suppl A):143A.
- Zhang L, Timothy KW, Vincent GM, et al. The spectrum of ST-T wave patterns and repolarization parameters in congenital long QT syndrome: ECG findings identify genotype Circulation 2000;102:2849-2855.[Abstract/Free Full Text]
- Padgett R, Grabowski P, Konarska M, Seiler S, Sharp P. Splicing of messenger RNA precursors Annu Rev Biochem 1986;55:1119-1150.[CrossRef][Medline]
- Vincent GM, Timothy KW, Leppert M, Keating MT. The spectrum of symptoms and QT intervals in the carriers of the gene for the long-QT syndrome N Engl J Med 1992;327:846-852.[Abstract]
- Splawski I, Shen J, Timothy KW, Vincent GM, Lehmann MH, Keating MT. Genomic structure of three long QT syndrome genes: KVLQT1, HERG, and KCNE1 Genomics 1998;51:86-97.[CrossRef][Medline]
- Cox NJ. Genetic Analysis Workshop II: results of segregation analyses using POINTER and linkage analyses using LIPED Genet Epidemiol 1984;1:167-170.[Medline]
- Pagani F, Buratti E, Stuani C, et al. Splicing factors induce cystic fibrosis transmembrane regulator exon 9 skipping through a nonevolutionary conserved intronic element J Biol Chem 2000;275:21041-21047.[Abstract/Free Full Text]
- Lund E, Dahlberg JE. True genes for human U1 small nuclear RNACopy number, polymorphism, and methylation. J Biol Chem 1984;259:2013-2021.[Abstract/Free Full Text]
- Pagani F, Buratti E, Stuani C, Bendix R, Dork T, Baralle FE. A new type of mutation causes a splicing defect in ATM Nat Genet 2002;30:426-429.[CrossRef][Medline]
- Baralle M, Baralle D, del Conte L, et al. Identification of a mutation that perturbs NF-1 gene splicing using genomic DNA samples and a minigene assay J Med Genet 2003;40:220-222.[Free Full Text]
- Zhou Z, Gong Q, Ye B, et al. Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature Biophys J 1998;74:230-241.[Abstract/Free Full Text]
- Anson BD, Ackerman MJ, Tester DJ, et al. Molecular and functional characterization of common polymorphisms in HERG (KCNH2) potassium channels. Am J Physiol Heart Circ Physiol 2004;286:H2434–41..
- Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating MT. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome Cell 1995;80:795-803.[CrossRef][Medline]
- Berthet M, Denjoy I, Donger C, et al. C-terminal HERG mutations: the role of hypokalemia and a KCNQ1-associated mutation in cardiac event occurrence Circulation 1999;99:1464-1470.[Abstract/Free Full Text]
- Murray A, Donger C, Fenske C, et al. Splicing mutations in KCNQ1: a mutation hot spot at codon 344 that produces in frame transcripts Circulation 1999;100:1077-1084.[Abstract/Free Full Text]
- Ackerman MJ, Tester DJ, Porter CJ. Swimming, a gene-specific arrhythmogenic trigger for inherited long QT syndrome Mayo Clin Proc 1999;74:1088-1094.[Medline]
- Castresana J. Estimation of genetic distances from human and mouse introns Genome Biol 2002;3:0028.1-0028.7.
- Himes SR, Tagoh H, Goonetilleke N, et al. A highly conserved c-fms gene intronic element controls macrophase-specific and regulated expression J Leukoc Biol 2001;70:812-820.[Abstract/Free Full Text]
- D'Souza I, Schellenberg GD. Tau exon 10 expression involves a biapartite intron 10 regulatory sequence and weak 5' and 3' splice sites J Biol Chem 2002;277:26587-26599.[Abstract/Free Full Text]
- Kim SY, Lee JH, Shin HS, Kang HJ, Kim YS. The human elongation factor 1 alpha (EF-1 alpha) first intron highly enhances expression of foreign gene from the murine cytomegalovirus promoter J Biotechnol 2002;93:183-187.[CrossRef][Medline]
- Fontemaggi G, Gurtner A, Strano S, et al. The transcriptional repressor ZEB regulates p73 expression at the crossroad between proliferation and differentiation Mol Cell Biol 2001;21:8461-8470.[Abstract/Free Full Text]
- Rowntree RK, Vassuax G, McDowell TL, et al. An element in intron 1 of the GFTR gene augments intestinal expression in vivo Hum Mol Genet 2001;10:1455-1464.[Abstract/Free Full Text]
- Mun JH, Lee SY, Yu HJ, et al. Petunia actin-depolymerizing factor is mainly accumulated in vascular tissue and its gene expression is enhanced by the first intron Gene 2002;292:233-243.[CrossRef][Medline]
- Zieler H, Huynh CQ. Intron-dependent stimulation of marker gene expression in cultured insect cells Insect Mol Biol 2002;11:87-95.[CrossRef][Medline]
- Vincent GM. Heterogeneity in the inherited long QT syndrome J Cardiovasc Electrophysiol 1995;6:137-146.[Medline]
- Priori SG, Napolitano C, Schwartz PJ. Low penetrance in the long QT syndrome: clinical impact Circulation 1999;99:529-533.[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
W. Lieb, B. Mayer, I. R. Konig, I. Borwitzky, A. Gotz, S. Kain, C. Hengstenberg, P. Linsel-Nitschke, M. Fischer, A. Doring, et al.
Lack of Association Between the MEF2A Gene and Myocardial Infarction
Circulation,
January 15, 2008;
117(2):
185 - 191.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. M. Roden
Long-QT Syndrome
N. Engl. J. Med.,
January 10, 2008;
358(2):
169 - 176.
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. M. Albert, E. G. Nam, E. B. Rimm, H. W. Jin, R. J. Hajjar, D. J. Hunter, C. A. MacRae, and P. T. Ellinor
Cardiac Sodium Channel Gene Variants and Sudden Cardiac Death in Women
Circulation,
January 1, 2008;
117(1):
16 - 23.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Buratti, M. Baralle, and F. E. Baralle
Defective splicing, disease and therapy: searching for master checkpoints in exon definition
Nucleic Acids Res.,
July 19, 2006;
34(12):
3494 - 3510.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. Nerbonne and R. S. Kass
Molecular Physiology of Cardiac Repolarization
Physiol Rev,
October 1, 2005;
85(4):
1205 - 1253.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D Baralle and M Baralle
Splicing in action: assessing disease causing sequence changes
J. Med. Genet.,
October 1, 2005;
42(10):
737 - 748.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. P. Delisle, J. K. Slind, J. A. Kilby, C. L. Anderson, B. D. Anson, R. C. Balijepalli, D. J. Tester, M. J. Ackerman, T. J. Kamp, and C. T. January
Intragenic Suppression of Trafficking-Defective KCNH2 Channels Associated with Long QT Syndrome
Mol. Pharmacol.,
July 1, 2005;
68(1):
233 - 240.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. M. Scheinman and E. Keung
The year in clinical electrophysiology
J. Am. Coll. Cardiol.,
March 1, 2005;
45(5):
790 - 795.
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Methods