Steffensen AB et al. (2015), High incidence of functional ion-channel abnorm...

XB-ART-50835

Sci Rep 2015 Jan 12;5:10009. doi: 10.1038/srep10009.

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High incidence of functional ion-channel abnormalities in a consecutive Long QT cohort with novel missense genetic variants of unknown significance.

Steffensen AB , Refaat MM , David JP , Mujezinovic A , Calloe K , Wojciak J , Nussbaum RL , Scheinman MM , Schmitt N .

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The Long QT syndrome (LQTS) is a disorder characterized by a prolongation of the QT interval and a propensity to ventricular tachyarrhythmias, which may lead to syncope, cardiac arrest, or sudden death. Our objective was to (1) determine the incidence of variants with unknown significance (VUS) in a cohort of consecutive LQTS patients and (2) to determine the percentage of those with novel missense VUS that have demonstrable functional channel abnormalities from a single referral center. We performed genetic screening of candidate genes in 39 probands with a diagnosis of LQTS to identify mutations and variants. Seven variants of unknown significance were identified, six were missense variants and one was a splice site variant. We investigated the six novel missense VUS in five patients; three missense variants in KCNQ1 (L236R, W379R, Y522S) and three missense variants in KCNH2 (R35W, S620G, V491I). We employed two-electrode voltage-clamp experiments in Xenopus laevis oocytes and confocal imaging to characterize the novel missense mutations functionally. We revealed electrophysiological and trafficking loss-of-function phenotypes. This report emphasizes the frequency of adverse channel function in patients with LQTS and the importance of heterologous studies to define channel function.

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Species referenced: Xenopus laevis
Genes referenced: actl6a arfgap1 kcne1 kcnh2 kcnq1 mmut
GO keywords: voltage-gated potassium channel activity [+]

???displayArticle.disOnts??? long QT syndrome 1 [+]
???displayArticle.omims??? LONG QT SYNDROME 1; LQT1

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	Figure 1. Clinical and genetic information of Proband-3 carrying mutation KV7.1 Y522S.Circles: female, Squares: male. Arrow indicates Proband-3.
	Figure 2. Characterization of KV11.1âR35W.a: Representative current traces recorded from X. laevis oocytes injected with KV11.1 or KV11.1âR35W cRNA. The current step protocol is shown as inset. b: Currents at the end the steps (points indicated by filled squares and circles in a) were plotted as a function of voltage to depict the current-voltage (IV) relationship for KV11.1 (at 0âmV; 2.9âÂ±â0.3âÎ¼A, nâ=â5) and KV11.1âR35W (at 0âmV; 2.1âÂ±â0.2âÎ¼A, nâ=â17). c: Normalized tail current as measured from peak current indicated by open squares and circles in A resulted in the voltage-dependent activation. For KV11.1 the half-maximal activation voltage (V1/2) was â15.0âÂ±â0.6âmV (nâ=â14) and for KV11.1âR35W: V1/2â=ââ12.2âÂ±â0.3âmV (nâ=â15). d: Activation kinetics were addressed by an envelope of tails protocol (inset). Data were normalized to the maximum amplitude of the tail current and plotted on a log time scale for KV11.1 (49.5âÂ±â2.3 ms, nâ=â15) and KV11.1âR35W (61.3âÂ±â2.3 ms, nâ=â18). e: The kinetics of deactivation (protocol shown as inset) were only altered for the relative contribution of the fast component of KV11.1 (at â70âmV; 0.34âÂ±â0.02, nâ=â13) compared with KV11.1âR35W (at â70âmV; 0.46âÂ±â0.01, nâ=â16). Pâ<â0.05, *Pâ<â0.01.
	Figure 3. Characterization of KV11.1âS620G.a: X. laevis oocytes were injected with KV11.1, KV11.1-S620G, or KV11.1â+âKV11.1âS620G (1:1 molar ratio) cRNA and currents recorded by TEVC. The protocol is shown as inset. b: I/V relationship for KV11.1 (at 0âmV; 2.7âÂ±â0.3âÎ¼A, nâ=â19), hetero (at 0âmV; 1.2âÂ±â0.1âÎ¼A, nâ=â24) and KV11.1-S620G (at 0âmV; 1.2âÂ±â0.1âÎ¼A, nâ=â11). c: Normalized tail current as measured from peak current resulted in the voltage-dependent activation. For KV11.1 the half-maximal activation voltage (V1/2) was â18.9âÂ±â0.7âmV (nâ=â18) and for hetero: V1/2â=ââ20.0âÂ±â0.4âmV (nâ=â24). d: Voltage-dependent recovery from inactivation was determined from the protocol in the inset where a Boltzmann function was fit to the normalized peak current of KV11.1 (V1/2â=ââ69.8âÂ±â2.7âmV, nâ=â17) and of hetero (V1/2â=ââ93.4âÂ±â1.8âmV, nâ=â17) E: Inactivation time was determined from a mono-exponential fit to the tail currents. Pâ<â0.01, *Pâ<â0.001
	Figure 4. Subcellular localization of KV11.1 and mutants.MDCK cells were transiently transfected with KV11.1, KV11.1âR35W, or KV11.1âS620G and grown to confluency. Using a specific KV11.1 antibody the localization of the WT and the MUT channels were visualized with confocal microscopy. Phalloidin (Pha) was used as a membrane marker as it stains the F-actin just underneath the cell membrane. (a-c) All subunits were found capable of trafficking to the cell membrane in some cells. Merged pictures are shown in right panels. (d) Quantification compare the number of channels in the membrane to the number of channels trapped in intracellular compartments (most likely ER) for WT and mutants (pâ=â0.23, nâ=â27-29 cells for each situation).
	Figure 5. Characterization of KV7.1âY522S.KV7.1 or KV7.1âY552S were expressed with KCNE1 in a 1:1 molar ratio in X. laevis oocytes. a: Representative recordings. The voltage protocol is shown as inset. b: IV relationship for KV7.1 measured from points indicated with the filled square in A (at 40âmV; 2.9âÂ±â0.2âÎ¼A, nâ=â36), KV7.1âY522S (at 40âmV; 0.5âÂ±â0.1 Î¼A, nâ=â12), and hetero (at 40âmV; 1.8âÂ±â0.2âÎ¼A, nâ=â27). c: Voltage-dependent activation as measured peak tail current indicated by the open square in A. For KV7.1, the half-maximal activation voltage (V1/2) was 31.1âÂ±â0.7 mV; for KV7.1âY522S: V1/2â=â38.4âÂ±â4.4âmV, and hetero: V1/2â=â28.6âÂ±â1.1âmV. d: Deactivation time constants were obtained by fitting a mono-exponential function to the tail-currents. The protocol is shown as inset. e: Activation time, determined as time-to-half maximal current atâ+â20, 30 or 40âmV. Pâ<â0.05, Pâ<â0.01, **Pâ<â0.001.
	Figure 6. Characterization of KV7.1âL236R.KV7.1 or KV7.1âL236R expressed with KCNE1 in a 1:1 molar ratio in X. laevis oocytes. a: Representative recordings. The voltage protocol is shown as inset. b: IV relationship for KV7.1 (at 40âmV; 2.9âÂ±â0.2âÎ¼A, nâ=â44), KV7.1âL236R (at 40âmV; 0.2âÂ±â0.03âÎ¼A, nâ=â12), and hetero (at 40âmV; 1.0âÂ±â0.1âÎ¼A, nâ=â34). c: Voltage-dependent activation. For KV7.1, the half-maximal activation voltage (V1/2) was 31.1âÂ±â0.7 mV; for KV7.1âL236R: V1/2â=â85.4âÂ±â11.7âmV, and hetero: V1/2â=â31.2âÂ±â1.7âmV. d: Deactivation time constants were obtained by fitting mono-exponential functions to the tail-currents. e: Activation time, determined as the time to half maximal current atâ+â20, 30 or 40 âmV. Pâ<â0.01, *Pâ<â0.001.
	Figure 7. Subcellular localization of KV7.1 and mutants.Horizontal and vertical confocal images of polarized MDCK cells transiently expressing the KV7.1âWT or MUT as indicated and labeled with antibodies against KV7.1 (left panels) and Phalloidin (middle panels). Merged pictures are shown in right panels. Ap; apical; Ba, basolateral. Representative pictures from three independent experiments are shown. Quantification of ratio between channels in the membrane versus channels trapped in intracellular compartments (most likely ER) for WT and mutants (pâ<â0.0001, nâ=â18â22 cells each situation).

References [+] :

Ackerman, Swimming, a gene-specific arrhythmogenic trigger for inherited long QT syndrome. 1999, Pubmed

	Figure 1. Clinical and genetic information of Proband-3 carrying mutation KV7.1 Y522S.Circles: female, Squares: male. Arrow indicates Proband-3.
	Figure 2. Characterization of KV11.1âR35W.a: Representative current traces recorded from X. laevis oocytes injected with KV11.1 or KV11.1âR35W cRNA. The current step protocol is shown as inset. b: Currents at the end the steps (points indicated by filled squares and circles in a) were plotted as a function of voltage to depict the current-voltage (IV) relationship for KV11.1 (at 0âmV; 2.9âÂ±â0.3âÎ¼A, nâ=â5) and KV11.1âR35W (at 0âmV; 2.1âÂ±â0.2âÎ¼A, nâ=â17). c: Normalized tail current as measured from peak current indicated by open squares and circles in A resulted in the voltage-dependent activation. For KV11.1 the half-maximal activation voltage (V1/2) was â15.0âÂ±â0.6âmV (nâ=â14) and for KV11.1âR35W: V1/2â=ââ12.2âÂ±â0.3âmV (nâ=â15). d: Activation kinetics were addressed by an envelope of tails protocol (inset). Data were normalized to the maximum amplitude of the tail current and plotted on a log time scale for KV11.1 (49.5âÂ±â2.3 ms, nâ=â15) and KV11.1âR35W (61.3âÂ±â2.3 ms, nâ=â18). e: The kinetics of deactivation (protocol shown as inset) were only altered for the relative contribution of the fast component of KV11.1 (at â70âmV; 0.34âÂ±â0.02, nâ=â13) compared with KV11.1âR35W (at â70âmV; 0.46âÂ±â0.01, nâ=â16). Pâ<â0.05, *Pâ<â0.01.
	Figure 3. Characterization of KV11.1âS620G.a: X. laevis oocytes were injected with KV11.1, KV11.1-S620G, or KV11.1â+âKV11.1âS620G (1:1 molar ratio) cRNA and currents recorded by TEVC. The protocol is shown as inset. b: I/V relationship for KV11.1 (at 0âmV; 2.7âÂ±â0.3âÎ¼A, nâ=â19), hetero (at 0âmV; 1.2âÂ±â0.1âÎ¼A, nâ=â24) and KV11.1-S620G (at 0âmV; 1.2âÂ±â0.1âÎ¼A, nâ=â11). c: Normalized tail current as measured from peak current resulted in the voltage-dependent activation. For KV11.1 the half-maximal activation voltage (V1/2) was â18.9âÂ±â0.7âmV (nâ=â18) and for hetero: V1/2â=ââ20.0âÂ±â0.4âmV (nâ=â24). d: Voltage-dependent recovery from inactivation was determined from the protocol in the inset where a Boltzmann function was fit to the normalized peak current of KV11.1 (V1/2â=ââ69.8âÂ±â2.7âmV, nâ=â17) and of hetero (V1/2â=ââ93.4âÂ±â1.8âmV, nâ=â17) E: Inactivation time was determined from a mono-exponential fit to the tail currents. Pâ<â0.01, *Pâ<â0.001
	Figure 4. Subcellular localization of KV11.1 and mutants.MDCK cells were transiently transfected with KV11.1, KV11.1âR35W, or KV11.1âS620G and grown to confluency. Using a specific KV11.1 antibody the localization of the WT and the MUT channels were visualized with confocal microscopy. Phalloidin (Pha) was used as a membrane marker as it stains the F-actin just underneath the cell membrane. (a-c) All subunits were found capable of trafficking to the cell membrane in some cells. Merged pictures are shown in right panels. (d) Quantification compare the number of channels in the membrane to the number of channels trapped in intracellular compartments (most likely ER) for WT and mutants (pâ=â0.23, nâ=â27-29 cells for each situation).
	Figure 5. Characterization of KV7.1âY522S.KV7.1 or KV7.1âY552S were expressed with KCNE1 in a 1:1 molar ratio in X. laevis oocytes. a: Representative recordings. The voltage protocol is shown as inset. b: IV relationship for KV7.1 measured from points indicated with the filled square in A (at 40âmV; 2.9âÂ±â0.2âÎ¼A, nâ=â36), KV7.1âY522S (at 40âmV; 0.5âÂ±â0.1 Î¼A, nâ=â12), and hetero (at 40âmV; 1.8âÂ±â0.2âÎ¼A, nâ=â27). c: Voltage-dependent activation as measured peak tail current indicated by the open square in A. For KV7.1, the half-maximal activation voltage (V1/2) was 31.1âÂ±â0.7 mV; for KV7.1âY522S: V1/2â=â38.4âÂ±â4.4âmV, and hetero: V1/2â=â28.6âÂ±â1.1âmV. d: Deactivation time constants were obtained by fitting a mono-exponential function to the tail-currents. The protocol is shown as inset. e: Activation time, determined as time-to-half maximal current atâ+â20, 30 or 40âmV. Pâ<â0.05, Pâ<â0.01, **Pâ<â0.001.
	Figure 6. Characterization of KV7.1âL236R.KV7.1 or KV7.1âL236R expressed with KCNE1 in a 1:1 molar ratio in X. laevis oocytes. a: Representative recordings. The voltage protocol is shown as inset. b: IV relationship for KV7.1 (at 40âmV; 2.9âÂ±â0.2âÎ¼A, nâ=â44), KV7.1âL236R (at 40âmV; 0.2âÂ±â0.03âÎ¼A, nâ=â12), and hetero (at 40âmV; 1.0âÂ±â0.1âÎ¼A, nâ=â34). c: Voltage-dependent activation. For KV7.1, the half-maximal activation voltage (V1/2) was 31.1âÂ±â0.7 mV; for KV7.1âL236R: V1/2â=â85.4âÂ±â11.7âmV, and hetero: V1/2â=â31.2âÂ±â1.7âmV. d: Deactivation time constants were obtained by fitting mono-exponential functions to the tail-currents. e: Activation time, determined as the time to half maximal current atâ+â20, 30 or 40 âmV. Pâ<â0.01, *Pâ<â0.001.
	Figure 7. Subcellular localization of KV7.1 and mutants.Horizontal and vertical confocal images of polarized MDCK cells transiently expressing the KV7.1âWT or MUT as indicated and labeled with antibodies against KV7.1 (left panels) and Phalloidin (middle panels). Merged pictures are shown in right panels. Ap; apical; Ba, basolateral. Representative pictures from three independent experiments are shown. Quantification of ratio between channels in the membrane versus channels trapped in intracellular compartments (most likely ER) for WT and mutants (pâ<â0.0001, nâ=â18â22 cells each situation).