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
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Figure 1 Electrocardiogram monitor strip showing torsade de pointes recorded from the proband during resuscitation from cardiac arrest at age of 27 years.
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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.
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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.
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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.
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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.
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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.
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