Compound and heterozygous mutations of KCNQ1 in long QT syndrome with familial history of unexplained sudden death: Identified by analysis of whole exome sequencing and predisposing genes

Abstract Introduction Long QT syndrome (LQTS) increases the risk of life‐threatening arrhythmia in young individuals with structurally normal hearts. Sixteen genes such as the KCNQ1, KCNH2, and SCN5A have been reported for association with LQTS. Case presentation We identified the compound heterozygous mutations in the KCNQ1 gene at c. G527A (p.W176X) and c.G1765A (p.G589S) predicted as “damaging.” The in‐silico analysis showed that when compared to the characteristics of mRNA and protein of wild‐type KCNQ1, the mRNA of c.G527A mutation was significantly different in the centroid secondary structure. The subunit coded by W176X would lose the transmembrane domains S3–S6 and helices A‐D. The protein secondary structure of G589S was slightly shortened in helix structure; the protein physics‐chemical parameters of W176X and G589S significantly and slightly changed, respectively. Conclusions The compound heterozygous mutations of W176X and G589S coexisting in KCNQ1 gene of homologous chromosomes, resulting in more severe phenotype, are the likely pathogenic and genetic risks of LQTS and USD in this Chinese family.


Funding information
The study was supported by the Foundation Project of Medical Science and Technology Research in Guangdong Province (A2018043); Science and Technology Project of Zhuhai (20191210E030072); Natural Science Foundation of Guangdong (2016A030313279); the Construction Foundation (ZHLCZD011) of the Key Clinical Specialties in Zhuhai.

| INTRODUC TI ON
Long QT syndrome (LQTS) increases the risk of life-threatening arrhythmia (e.g., torsade de pointes), which leads to syncope, seizures, and unexplained sudden death (USD) in young individuals with structurally normal hearts. LQTS is typically inherited as an autosomal dominant trait while recessive inheritance is observed in rare cases.
Recessive inherited LQTS is characterized by severe cardiac phenotype and multisystem syndrome disorders, such as Ankyrin B syndrome, Andersen-Tawil syndrome, and Timothy syndrome. So far, 16 genes have been reported for association with LQTS, the KCNQ1, KCNH2, and SCN5A are the most common (Nakano & Shimizu, 2016). Here we found a young member of the Chinese Han family characterized as type 1 LQTS and USD. This family was investigated for potential genetic risk by performing the Whole Exome sequencing (WES) and screening candidate genes related to arrhythmia and cardiomyopathies (Lin et al., 2017). • Mutations in KCNQ1 causing loss-of-function and gainof-function can lead to type 1 LQTS, AF, and even fatal arrhythmia.

| C A S E PRE S ENTATI ON
• Some limited reports revealed that compound heterozygous mutations in KCNQ1 genes aggravated the phenotype of LQTS.

Novel Insights
• In our case report, the compound heterozygous mutations of W176X and G589S in KCNQ1 gene were identified in the patient with type 1 LQTs and familial history of unexplained sudden death (USD). The coexisting interaction of both mutations, resulting in more severe phenotype, may be the important pathogenic and genetic risks for LQTS and USD.
F I G U R E 1 Familial pedigree and phenotype old) repeatedly presented palpitation, chest tightness, dizziness, and amaurosis since 7 years old. Subsequently, she further developed more symptoms including syncope, urinary and fecal incontinence, and limb weakness, which could be relieved after persisting for approximately two minutes. The electrocardiogram and 24-hr dynamic electrocardiogram demonstrated the characteristics of type 1 long QT ( Figure 2) with QTc 517 milliseconds and occasional ventricular premature. There were no significant abnormalities in the echocardiogram, test of hearing function, computed tomography of the chest, and thyroid examination. Based on the guidelines and the clinical standard score of familial long QT syndrome (Skinner, 2007), the proband II:4 was evaluated with 5.5 points and diagnosed with long QT syndrome. II:4 was first treated by beta-blocker, but she still repeatedly presented the symptoms while she was suffering from stress. Therefore, she was implanted with an implantable cardioverter defibrillator (ICD) in September 2014 and instructed to avoid as much environmental stress as possible. There was no adverse cardiac event evaluated by ICD monitoring before December 2015. She had no obvious symptom recurrence even without drug therapy during three years of followup. Her sister (II:2) also suffered from similar palpitation, fatigue, amaurosis, and syncope without any medical care and treatment since 10 years old. Consequently, she died of the unexplained sudden death at the age of 23 when she was running. Another sister (II:3) died from the repeated infection at age of 4 years old.

| WES and predisposing gene analysis
We conducted the WES or Sanger sequencing of DNA extracted from peripheral blood of the family members except II:2, II:3, and II:5 who had died or refused the genetic test. SNPs and InDels were annotated using a pipeline, in which all insertion and deletion variants occurring at coding regions were considered damaging. Nonsynonymous SNPs were predicted by Sorting Intolerant From Tolerant (SIFT, http://sift.jcvi. org/www/); (Kumar, Henikoff, & Ng, 2009) and PolyPhen-2 algorithms (Polymorphism Phenotyping v2, http://genet ics.bwh.harva rd.edu/ pph2/); (Adzhubei et al., 2010). Variants in predisposing genes that are associated with hereditary arrhythmias and cardiomyopathies were screened. The filtering criteria are as follows: (a) same variants in the WES data; (b) missense, nonsense, insertion, and deletion variants; (c) SNPs with minor allele frequency not more than 0.01 according to the SNP database of National Center for Biotechnology Information (NCBI) (Smigielski, Sirotkin, Ward, & Sherry, 2000;Via, Gignoux, & Burchard, 2010). We obtained 897 mutations in exon and splicing regions, including 8 mutations of genes predisposing to arrhythmic and cardiomyopathies (Table 1). In this list of the predisposing genes, the KCNQ1 gene encodes a voltage-gated potassium channel required for repolarization phase of the cardiac action potential, and of which more than 200 mutants ( Figure 3) associates with type 1 LQTS, familial atrial fibrillation (AF), Jervell and Lange-Nielsen syndrome (JLNS), short QT syndrome (SQTs), and Beckwith-Wiedemann syndrome (BWS) ( Table 2). We acquired one stop-codon mutation of W176X and nonsynonymous mutation of G589S in KCNQ1 gene of homologous chromosomes, carried by II:4 as heterozygous patterns, respectively, which were not existing in the population according to the 1,000 genome database. The other family members only carried one mutation of W176X or G589S ( Figure 1). The G589S mutation was predicted as "damaging" by the Polyphen2 algorithm.

| HOLTER RECORDING S
The Holter recordings There was no cardiac event during the recordings of I:2, III:1, III:2, and III:3. The 12-lead ECGs of all three single G589S mutation carriers were shown in Figure S1.

| IN -S ILICO ANALYS IS
In-silico analysis was used to evaluate the changes of the RNA-protein secondary structure and the protein physics-chemical parameters (Qureshi et al., 2013).

| RNA secondary structure prediction
The RNA secondary structure was predicted by RNAfold WebSever.
The difference of the minimum free energy (MFE) in the centroid secondary structure was evaluated between the mutant mRNA and wild mRNA. The MFE of c.G527A mutation (−585.36 kcal/mol) was much lower than that of the wild type (−359.16 kcal/mol), which thus lead to an improvement of the structural stability. Whereas the MFE of c.G1765A mutation (−333.70 kcal/mol) was approximately similar to that of the wild type, which therefore probably induced no obvious change in the centroid secondary structure (Table 3).

| Protein secondary structure prediction
As evaluated by the Phyre2 software, the protein secondary structure of KCNQ1 p.W176X was completely lost since the 175th position and the secondary structure before the 175th position also showed significant changes (Figure5a,b). In addition, the protein F I G U R E 3 The common pathogenic mutants of KCNQ1 gene in Clinvar database secondary structure of G589S was only slightly shortened in its previous helix structure compared with the wild type (Figure5c,d).

| Protein physics and chemical parameters prediction
Predicted by the ProtParam tool, it can be seen that compared with the wild type, the Theoretical pI, Instability index, Aliphatic index, and Grand average of hydropathicity (GRAVY) of W176X showed an increasing trend. The Instability index of G589S also showed a slight increase (Table 4).

| D ISCUSS I ON
In this Chinese family, the coexisting compound heterozygous mutations of W176X and G589S in KCNQ1 gene may be the potentially pathogenic and genetic risks for the proband of type 1 LQTS with a familial history of USD.
KCNQ1 protein is in the form of tetramer as an intact functional channel. KCNQ1 channel plays an important role in numerous tissues, including the heart, inner ear, stomach, and colon. It mediates the slowly activating potassium current (I ks ) characterized by outward rectification, which contributes to repolarization in cells of atria and ventricles (Kinoshita et al., 2014;Schroeder et al., 2000).
Moreover, the regulation of the voltage-gated K + channel can be mainly divided into two important parts: On one hand, as an ion channel, the functional structure of KCNQ1 lies in the transmembrane domains of S1-S6. The voltage sensor domain (VSD) works for voltage-dependent movements comprising of transmembrane helices S1-S4, and the pore-gate domain (PGD) works for activation F I G U R E 5 The Changes of protein secondary structure predicted by Phyre2 software. Note: A: secondary structure nears the W176 residue in protein of wild-type KCNQ1. B: secondary structure nears the W176 residue in protein of W176X mutation of KCNQ1. C: secondary structure nears the G589 residue in protein of wild-type KCNQ1. D: secondary structure nears the G589 residue in protein of G589S mutation of KCNQ1 gate movements comprising S5-S6. The interaction between VSD and PGD makes the movements feasible (Cui, 2016). In addition, KCNE1 also interacts with VSD, especially the domain S4 and alters the VSD movement drastically (Nakajo & Kubo, 2015). On the other hand, helices A-B in C-terminal mediate the binding of KCNQ1 and CaM in charge of gating, folding, and membrane trafficking of the channel (Kinoshita et al., 2014). More importantly, helix D provides a leucine zipper (LZ) motif for the interaction with Yotiao (also called A-kinase anchoring protein 9, AKAP9), which plays a key role in the sympathetic nervous system (SNS) regulation of cardiac action potential duration (APD). This means exercise or emotional fluctuation can trigger cardiac events if mutations in this domain lead to severe damage (Jespersen, Grunnet, & Olesen, 2005). Mutations in KCNQ1 causing loss-of-function and gain-of-function can lead to type 1 LQTS, AF, and even fatal arrhythmia when calcium channels are reactivated causing early afterdepolarizations (Chen, Xu, & Bendahhou, 2003;Keating & Sanguinetti, 2001).

| Compound heterozygous mutations associated with long QT syndrome
Some limited reports revealed that compound heterozygous muta- protein secondary structure, and protein physics-chemical parameters prediction distinctly altered the normal role in the gene. In addition, I:2 who carried W176X had a longer QTc than III:1, III:2, and III:3 members who carried G589S, which probably means W176X can cause more damage to the I ks channel. Interestingly, the Q530X had caused complete loss of I ks channel function according to previous research (Westenskow et al., 2004). Therefore, W176X as a truncated mutation also causing complete loss of I ks function is one of the important pathogenic factors in this patient.
The mutation G589S, located in the helix D domain of the channel subunit, was first identified in our study. Two mutations G589D and A590T, which were nearby and have similar significance to G589S, were previously reported (Lupoglazoff, Denjoy, & Villain, 2004;Marx, Kurokawa, & Reiken, 2002). As mentioned above, β-adrenergic receptor activation of SNS mediates an increase in cAMP leading PKA stimulation and thereby PKA interacts with KCNQ1 through Yotiao causing the phosphorylation of residue S27. It is believed that the blocking between KCNQ1 and Yotiao by mutation G589D (G589 is the first "e" position in the LZ motif) may lead to the turbulence of SNS modulation which shortens APD and increases the risk of cardiac events such as USD (Marx et al., 2002). Thus, G589S which is the same position as G589D may cause the alike consequence. On the other hand, functional analyses showed that A590T causes a reduction in I ks density and voltage-sensitivity, which can prolong the QT interval. Mutation G589D has also been reported to cause a similar effect on voltage-sensitivity (Marx et al., 2002). Besides, immunocytochemical and immunoblot analyses demonstrated A590T reduced cell surface expression.
These findings suggest that A590 residue, even the near residues, has an important effect on the maintenance of channel surface expression and function (Kinoshita et al., 2014). This mechanism may be another potential pathogenic reason for the G589S.  Määttänen et al., 2013), and thus stress management should be a key link in the treatment of LQTS.

| CON CLUS IONS
The compound heterozygous mutations of W176X and G589S in KCNQ1 gene from homologous chromosomes were identified in the patient with type 1 LQTS and familial history of USD. Compared with the wild-type KCNQ1, both mutations significantly changed the RNA and protein secondary structure, protein physics-chemical parameters predicted by bioinformation algorithms. The coexisting interaction of both mutations, resulting in more severe phenotype, may be the important pathogenic and genetic risks for LQTS and USD.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no competing interests.

AUTH O R S ' CO NTR I B UTI O N S
XL and YL conceived and designed the study. YL and TZ investigated relevant family information, analyzed, and interpreted the patient data regarding the cardiac disease, and wrote the article. SH, JH, and QL organized the data and produced the figures and tables. NY performed the experiment and provided the mutants. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The dataset supporting the conclusions of this article are included within the article [and its Additional file(s)].

This study was approved by the Guangdong Medical Institutional
Review Board and Medical Ethics Committees [No.GDREC2016001H (R1)]. All participants gave informed consent.

CO N S E NT FO R PU B LI C ATI O N
The written informed consent or parental consent for publication was obtained from all participants.