LQTS-associated mutation A257G in α1-syntrophin interacts with the intragenic variant P74L to modify its biophysical phenotype

The SNTA1-encoded α1-syntrophin (SNTA1) missense mutation, p.A257G, causes long QT syndrome (LQTS) by pathogenic accentuation of Nav1.5’s sodium current (INa). Subsequently, we found p.A257G in combination with the SNTA1 polymorphism, p.P74L in 4 victims of sudden infant death syndrome (SIDS) as well as in 3 adult controls. We hypothesized that p.P74L-SNTA1 could functionally modify the pathogenic phenotype of p.A257G-SNTA1, thus explaining its occurrence in non-LQTS populations. The SNTA1 variants p.P74L, p.A257G, and the combination variant p.P74L/p.A257G were engineered using PCR-based overlap-extension and were co-expressed heterologously with SCN5A in HEK293 cells. INa was recorded using the whole-cell method. Compared to wild-type (WT), the significant increase in peak INa and window current found with p.A257G was reversed by the intragenic variant p.P74L (p.P74L/p.A257G). These results report for the first time the intragenic rescue of an LQT-associated SNTA1 mutation when found in combination with the SNTA1 polymorphism p.P74L, suggesting an ever-increasing picture of complexity in terms of genetic risk stratification for arrhythmia.


Introduction
Long QT syndrome (LQTS) is a heritable cardiac arrhythmia affecting approximately 1 in 2500 individuals. 1LQTS is characterized by a prolongation of the QT interval on the ECG, and is most often manifested clinically as syncope, seizures, or sudden death due to its trademark arrhythmia torsades de pointes. 2 Thus far, 12 genes have been implicated in LQTS, with approximately 75% 3,4 of LQTS caused by mutations in the KCNQ1-encoded potassium channel (LQT1), 5 KCNH2-encoded potassium channel (LQT2) 6 or the SCN5Aencoded sodium channel (LQT3). 7[10][11][12][13][14][15][16] Recently, we discovered that the LQT3-associated cardiac sodium channel, Nav1.5, is connected by the SNTA1-encoded scaffolding protein (α1-syntrophin, SNTA1) to neuronal nitric oxide synthase (nNOS) and the cardiac isoform of the plasma membrane Ca-ATPase (PMCA4b) in a complex in cardiomyocytes.Moreover, we implicated SNTA1 as a novel susceptibility gene for both LQTS 17 and SIDS 16 whereby the SNTA1 mutant disrupted binding with PMCA4b, released inhibition of nNOS, and accentuated both peak and late I Na via Snitrosylation of the cardiac sodium channel.In addition we also have reported a new LQTSassociated SNTA1 missense mutation (c.770C > G, p.A257G) which precipitated a marked increase in peak I Na and altered Nav1.5 channel kinetics in both HEK293 cells and cardiomyocytes through direct interaction of mutant SNTA1 with SCN5A. 18All these findings suggest that SNTA1 is a crucial Nav1.5 channel interacting protein (ChIP) involved in maintaining the normal function of the cardiac sodium channel.
The p.A257G-SNTA1 mutation was identified initially in a separate publication by our group in 3 unrelated LQTS probands out of 39 LQTS patients but absent in 200 ethnicmatched control individuals. 18This study demonstrated that p.A257G-SNTA1 can functionally interact with SCN5A in HEK293 cells without requiring nNOS or PMCA4b.Lastly, in a population-based cohort of 292 SIDS cases, we reported in 4 Caucasian SIDS victims the presence of p.A257G in combination with another single-nucleotide alteration (c.221C > T) that results in the amino acid substitution p.P74L. 16Interestingly, the combined variant of p.P74L/p.A257G was also found in 3 out of 400 additional adult controls not included in the initial study.None of the SIDS cases or the 400 adult controls was positive for either p.P74L-or p.A257G-SNTA1 in isolation. 16ven the following data points: i) that p.A257G-SNTA1 in isolation is an LQTS-associated mutation with abnormal biophysical characteristics, 18 ii) that p.A257G-SNTA1 was identified in an ostensibly healthy population only in the presence of p.P74L-SNTA1, and iii) that in an at-risk population of SIDS victims p.A257G-SNTA1 occurred multiple times, but only in the presence of p.P74L-SNTA1, we hypothesized that p.P74L may rescue the abnormal phenotype caused by p.A257G in isolation, therefore explaining its occurrence both in the general population compared to the LQT-associated single p.A257G missense mutation in isolation, as well as its occurrence at a higher frequency (>1%) in SIDS than would be expected were the combination p.P74L/p.A257G-SNTA1 variant to be a highly pathogenic variant.

N o n -c o m m e r c i a l u s e o n l y
California, USA).The p.P74L-, p.A257G-, and combination p.P74L/p.A257G-SNTA1 variants were incorporated into WT-SNTA1 using the PCR-based overlap-extension method as previously reported. 16,17All clones were sequenced to confirm integrity and to ensure the presence of the introduced variants and the absence of other substitutions caused by PCR.

Mammalian cell transfection
The WT or mutant SNTA1 in pIRES2EGFP vector was transiently co-transfected with expression vectors containing SCN5A (hNav1.5,Genbank accession no.AB158469) in the dominant splice variant background Q1077del into HEK293 cells with FuGENE6 reagent (Roche Diagnostics, Indianapolis, Indiana, USA) according to manufacturer's instructions.Cells were selected for electrophysiologic studies based on comparable levels of GFP fluorescence, and WT and mutant were transfected on the same day.Persistent or late I Na was measured as the mean between 600 and 700 ms after the initiation of the depolarization from -140 mV to -20 mV for 750 ms after passive leak subtraction as previously described. 13,16,17,19Time course of recovery from inactivation was analyzed by fitting data with a two-exponential (exp) function: normalized

Electrophysiological measurements
where t is time, A f and A s are fractional amplitudes of fast and slow components, respectively, and τ f and τ s are fast and slow time constant, respectively.Decay rates and amplitude component were measured from the trace beginning after peak I Na at 90% of peak I Na to 24 ms and fitted with a sum of   A s exp(-t/τ s )] + offset, where t is time, and A f and A s are fractional amplitudes of fast and slow components, respectively.The other standard voltage clamp protocols are presented with the data and data were measured and analyzed as described previously 13,16,17,19 and with additional details provided in the figure legends.Quantification of window currents was performed by calculating the area under the activation/inactivation curves per cell, and averaging cells for each group to assess for statistical differences.

Statistical analysis
All data points are reported as the mean value and the standard error of the mean (SEM).Determinations of statistical significance were performed using a Student t-test for comparisons of two means or using analysis of variance (ANOVA) for comparisons of multiple groups.Statistical significance was determined by a value of P < 0.05.
To assess the effects of these mutants on late I Na , we measured the level of late I Na after leak subtraction as a percentage of peak I Na elicited by prolonged depolarization.However, p.P74L, p.A257G and p.P74L/p.A257G showed similar percentages of late I Na as WT-SNTA1 (Table 1), suggesting that the direct interaction of these SNTA1 mutants with Nav1.5 does not affect late I Na .
We analyzed the kinetic parameters of activation and inactivation for these 3 SNTA1 variants and compared these data with WT-SNTA1.The p.P74L-SNTA1 variant showed no significant difference in either activation or inactivation compared with WT-SNTA1.As previously reported, the LQTS-associated p.A257G-SNTA1 missense mutation caused a statistically significant hyperpolarizing shift in activation and had comparable inactivation parameters to WT, thus increasing the overlap of the activation and inactivation curves, and resulting in increased window current.However, for p.P74L/p.A257G-SNTA1 both activation and inactivation were significantly shifted toward the hyperpolarizing direction and reversed the increased channel availability caused by p.A257G, significantly decreasing the window current from p.A257G-SNTA1 alone (Figure 1B-1F; Table 1).
The recovery time from fast inactivation was analyzed by fitting the data with a double-exponential equation.p.A257G-SNTA1 showed an increase in the fast component of the time constant τ f (1.99 ± 0.16 ms, n = 20) compared with WT (1.63 ± 0.10 ms, n = 17; P = 0.04) as previously reported, 18 but there was no significant difference in time constant (τ f , τ s ) and fractional amplitudes between WT, p.P74L, and p.P74L/p.A257G (data not shown).Due to the shift of activation parameters, we analyzed the decay of the peak I Na with 2-exponential fits of decay phase of macroscopic I Na .There was no difference in either time constants (τ f , τ s ) or fractional amplitudes of 2 components observed in all mutants.

Discussion
As previously reported, four cases out of a population-based cohort of 292 SIDS victims and three subjects out of 400 control subjects were heterozygous for the combined variants p.P74L-SNTA1 and p.A257G-SNTA1 in α1-syntrophin. 16,18Given the frequency of these two variants, and that neither variant was found in isolation in the SIDS cohort nor in the 400 reported adult controls, it is extremely likely that they are located on the same allele.Conversely, the p.A257G mutation without the p.P74L variant was found in isolation in three patients with LQTS. 1815]20 Because of the anonymized nature of the SIDS study, determination of genetic variants as transmitted or de novo was not possible.
The novel finding in this study is that the intragenic p.P74L-SNTA1 variant distinctively abolished the increased peak I Na caused by the LQTS-associated p.A257G-SNTA1 missense mutation, thus reconciling the pro-arrhythmic nature of the mutation p.A257G-SNTA1 with its discovery in a presumably healthy population as well as in our SIDS cohort at a > 1% frequency.To the best of our knowledge, this is the first report elucidating the functional interaction of a LQTS-associated SNTA1 mutation with an intragenic SNTA1 polymorphism.
Our group recently identified that the LQTSassociated SNTA1 missense mutation p.A257G markedly accentuated peak I Na through the interaction of SNTA1 with Nav1.5 independent of the nNOS-PMCA4b nitrosylation pathway. 18he present investigation showed that the intragenic combination of p.P74L-SNTA1 and p.A257G-SNTA1 significantly reverses the increased peak I Na associated with p.A257G-SNTA1 alone, and exerts an additional channel kinetic modification (negative shift for inactivation) on Nav1.5.The biophysical phenotype of normalized peak I Na in p.P74L/p.A257G suggests that p.P74L plays a protective role, and could account for the persistence of p.P74L/p.A257G in the general population.Meanwhile, it also suggests that like the vast majority of SIDS cases, the pathogenic cause of the 4 SIDS cases with this combined variant remains elusive and the p.P74L/p.A257G combined variant did not contribute to their demise.
Notably, the two variants occurred on either side of the PDZ domain of SNTA1 (Figure 2).Specifically, p.P74L localizes to the first pleckstrin homology 1 (PH1) domain (amino acids 1-86) while p.A257G localizes to the second PH1 domain (amino acids 171-269).At the sarcolemma of the skeletal and cardiac muscles, SNTA1, the most abundant syntrophin isoform in heart muscle, 21,22 was shown to interact through its PDZ domain with many transmem- brane channels such as sodium channels SkM1, SkM2, and Nav1.5, 23,24 potassium channel Kir4.1, 25 and transient receptor potential channels (TRPC). 26More recently, the N-terminal part of SNTA1 containing the PDZ domain was confirmed to be necessary for SNTA1 to normally regulate TRPC1 channel 27 as well as to interact with the α 1D -adrenergic receptor. 28iven its prominent modification of the biophysical phenotype, this interesting combined variation in the PH1 domain surrounding the PDZ domain provides us at least two questions to be further addressed: i) Is the PH1 domain necessary for a functional interaction between SNTA1 and Nav1.5? ii) Do these SNTA1 variants also have special effects on other proteins with PDZ binding sites in the Nav1.5 macromolecular complex in vivo?
In this study, we report for the first time an instance of intragenic complementation modulating the pathogenic phenotype of a mutation in a channelopathy-susceptibility gene.In particular, the functional characterization of the combination variant p.P74L/p.A257G-SNTA1 found in both SIDS and controls demonstrates an intragenic interaction of these two single nucleotide changes in SNTA1 by which the p.A257G mutant phenotype is abolished.To our knowledge, that an arrhythmia-associated mutation, namely p.A257G-SNTA1, in a ChIP is rescued by another intragenic variant has not been previously reported.However, this is certainly not the first report of this biological phenomenon.][31] To what extent this interaction is important in a more native cardiac environment as opposed to a heterologous expression system will need to be explored.However, it should be emphasized that the p.A257G-SNTA1 mutant phenotype appears to be nitrosylation-independent, since pathogenic changes were noted in a heterologous system without the presence of nNOS or PMCA4b, consistent with previous data. 18These changes were abrogated in the presence of p.P74L without recapitulating the nitrosylation complex.Indeed, these findings present a mounting challenge in terms of properly recapitulating the proper sodium channel macromolecular complex for performing such studies.Given the increasing evidence of the interplay between genetic variation among various subunits, caution must be exercised in properly interpreting such data not only from heterologous systems but indeed from any system that attempts to artificially recapitulate a mutant phenotype.An additional limitation to the present study is the lack of high-integrity RNA material to demonstrate the existence of p.P74L and p.A257G on the same SNTA1 allele.
However, neither variant was identified in isolation in either our SIDS cohort or the controls examined.In addition, p.A257G was identified in the previous study in isolation only in LQTSpositive individuals, and p.P74L was not detected in those individuals.Thus it is extremely unlikely in control populations that p.A257G would exist in isolation rather than in linkage disequilibrium with a protective variant.Therefore, the current data support the previously argued pathogenic role of p.A257G-SNTA1 when found in isolation without the accompanying polymorphism p.P74L-SNTA1.
In conclusion, this study highlights the complicated mechanism by which SNTA1 regulates Nav1.5, and emphasizes the potential role of genetic variation in Nav1.5 macromolecular complex-related disorders, as well as the increasingly complicated role that the individual genetic fingerprint plays in modulating the penetrance/expressivity of disease-associated mutations.
Macroscopic voltage-gated I Na was measured 48 hours after transfection with the standard whole-cell patch clamp method at 21°C to 23°C in the HEK293 cells.The extracellular (bath) solution contained the following (in mM): NaCl 140, KCl 4, CaCl 2 1.8, MgCl 2 0.75 and HEPES 5 and was adjusted to pH 7.4 with NaOH.The intracellular (pipette) solution contained the following (in mM): CsF 120, CsCl 2 20, EGTA 2, NaCl 5, and HEPES 5 and was adjusted to pH 7.4 with CsOH.Microelectrodes were manufactured from borosilicate glass using a puller (P-87, Sutter Instrument Co, Novato, California, USA) and were heat polished with a microforge (MF-83, Narishige, Tokyo, Japan).The resistances of microelectrodes ranged from 1.0 to 2.0 MΩ.Voltage clamp data were generated with pClamp software 10.2 and an Axopatch 200B amplifier (Axon Instruments, Foster City, California, USA) with series-resistance compensation.Membrane current data were digitalized at 100 kHz, low-pass filtered at 5 kHz, and then normalized to membrane capacitance.

Figure 1 .
Figure 1.Co-transfection of SNTA1 with SCN5A.A) Representative whole-cell current traces of HEK293 cells co-expressing WT or mutant SNTA1 and SCN5A, showing increased peak I Na for p.A257G-SNTA1.The currents were recorded at membrane potentials between -120 to +60 mV in 10-mV increments from a holding potential of -140 mV as depicted in the protocol inset.B) Activation was measured using this protocol: currents were elicited by step depolarization for 24 ms to different potentials between -120 mV to 60 mV from a holding potential of -140 mV.The curves were fit with a Boltzmann function where G Na = [1+exp (V 1/2 -V) /K] -1 , where V 1/2 and k are the midpoint and slope factor, respectively.G/G Na = I Na (norm)/ (V -Vrev) where Vrev is the reversal potential and V is the membrane potential.p.A257G-and p.P74L/p.A257G-SNTA1 mutations caused a statistically significant hyperpolarizing shift in steady-state activation by 2.1-2.7 mV.C) Steady-state availability from inactivation was measured using the protocol shown in the inset and was determined by fitting the data to the Boltzmann function: I Na = I Na-Max [1+exp (Vc -V 1/2 ) / K] -1 , where V 1/2 and k are the midpoint and the slope factor, respectively, and Vc is the membrane potential.p.P74L/p.A257G-SNTA1 mutation had a statistically significant hyperpolarizing shift in steady-state inactivation by 2.6 mV.D) The peak current activation data are replotted as a conductance (G) curve with steady-state inactivation relationships to show p.A257G increases the overlap of these relationships (window current).E) The peak current activation data are replotted as a conductance (G) curve with steady-state inactivation relationships to show p.P74L/p.A257G reverses the increased overlap of these relationships.Circles indicate activation, whereas squares indicate inactivation.Lines represent fits to Boltzmann equations with parameters of the fit and n numbers in Table 1.F) shows an increase in quantified window current area for p.A257G-SNTA1 compared with both WT-SNTA1 as well as p.P74L-p.A257G-SNTA1.*P<0.05 compared with WT and p.P74L/p.A257G.The experiment numbers are shown within each bar.

*
I Na = Sodium current.°P < 0.05 versus WT-SNTA1.# P < 0.05 versus p.A257G-SNTA1.V1/2, pA/pF, and % Late I Na are shown as the mean value and the standard error of the mean while K parameters are given as mean value.N o n -c o m m e r c i a l u s e o n l y exponentials(exp): I Na (t) = 1 -[A f exp(-t/τ f ) +

Figure 2 .
Figure 2. Localization of SNTA1 variants.Illustrated is the linear topology for SNTA1 with variant localization.