**6. Genes definitely associated with SQTS**

Pathogenic variants in the *KCNH2*, *KCNQ1* and *KCNJ2* genes are responsible for SQTS type 1, 2 and 3 respectively. These variants are usually of the gain-of-function type and generate prolonged K+ channel activation, accelerated cardiac repolarization with shorter refractory periods, resulting in the short QT phenotype [25].

The *KCNH2* gene (ID: 3757) encodes a voltage-activated potassium channel belonging to the eag family, subfamily H, member 2 (Kv 11.1 α/hERG subunit). It mediates the rapidly activating component of the delayed rectifying potassium current in the heart (IKr) [26–28]. Gain-of-function hERG variants lead to abbreviated ventricular repolarization and SQTS; in contrast, loss-of-function hERG variants are responsible for Long QT Syndrome (LQTS). The pathogenic variants p.Thr618Ile and p.Asn588Lys are the most frequently associated with SQTS [29]. Several functional studies have demonstrated the pathophysiological role of these variants, and their contribution to the SQTS phenotype seems clear [30]. Although other rare variants in the *KCHN2* gene associated with SQTS have been described, many of them require further functional or segregation studies to elucidate their definitive pathological role.

The *KCNQ1* gene (ID: 3784), encodes a voltage-activated potassium channel (Kv7.1 α-subunit) required for repolarization. This protein can form complexes associated with MinK (the *KCNE1* gene) and MiRP2 (the *KCNE3* gene) proteins, both potassium channel. When associated with *KCNE1*, it forms the IKs current, and induces rapid activation of the potassium-selective outward current. It can also associate with the MiRP2 protein and other associated proteins to form the potassium channel [3, 31]. Deleterious variants in this gene are associated with SQT2 and account for less than 5% of SQTS cases. In addition, some *de novo* variants in the *KCNQ1* gene have been associated with a particular phenotype *in utero* with clinical diagnosis of atrial fibrillation (AF), along with concomitant bradycardia and SQTS [32]. The rare variants p.Val141Met and p.Val307Leu have the clearest association with SQT2 to date [22], being potential targets for various therapeutic models. For example, functional and computational simulation studies identified channel-specific blockade of IK1 or IKs as a possible antiarrhythmic strategy in SQT2, depending on the identified deleterious variants (p.Val141Met and p.Val307Leu, respectively) [33, 34].

The *KCNJ2* gene (ID: 37591) encodes the integral membrane protein and an inward rectifier-type potassium channel, subfamily J, Member 2 (Kir2.1 α-subunit). Inward rectifier potassium channels are characterized by a greater tendency to allow potassium to flow into the cell rather than out of it (IK1 current) [4, 35]. Currently, pathogenic variants with the most evidence of causality for SQT3 are the variants p.Asp172Asn and p.Glu299Val [36, 37]. The *KCNJ2* gene has also been associated with other channelopathies, mainly catecholaminergic polymorphic ventricular tachycardia (CPVT) [38].

## **6.1 Gene moderately associated with SQTS**

In 2017, the *SLC4A3* gene (Solute Carrier Family 4 Member 3, ID: 6508) was associated with SQTS, presenting an unusual mechanism for the development of malignant arrhythmia. *SLC4A3* encodes plasma membrane anion exchange protein 3 (AE3) and acts by mediating part of the Cl-/HCO3- exchange in cardiac myocytes. To date, only one rare variant in this gene has been identified in two families (p.Arg370His). This loss-of-function variant would cause an increase in pHi and a decrease in [Cl-]i, shortening the AP duration and reducing the QT interval [39]. This gene is associated with SQT type 8; however, further studies are needed to clarify the definitive role of this gene in SQTS.

#### **6.2 Other genes associated with SQTS**

Loss-of-function alterations in genes encoding different subunits of cardiac Ca2+ channels have been associated with SQTS syndrome with an autosomal dominant inheritance pattern, each accounting for less than 1% of all SQTS cases [40]. However, evidence-based review of this association (ClinGen) leaves the causation of SQTS by mutations in these genes currently in dispute [24].

The *CACNA1C* gene (ID: 775) encodes an alpha-1 subunit of a voltage-dependent calcium channel (calcium channel, voltage-dependent, L-type, alpha 1C subunit, Cav1.2 α subunit). Calcium channels mediate the influx of calcium ions into the cell upon membrane polarization. To date, all variants identified in *CACNA1C* decrease inward currents at early phases of cell repolarization (ICaL) and induce transmural and epicardial dispersion of repolarization, leading to a combined phenotype of SBr and short QTc interval [41]. Currently, more than 10 rare variants in the *CACNA1C* gene have been potentially associated with SQTS, so-called SQT4. However, there is insufficient evidence to establish a definitive association and further studies are needed [22]. Gain-of-function variants in this gene have also been associated with LQTS.

The *CACNB2* gene (ID: 783) encodes a subunit of a voltage-dependent calcium channel protein, a member of the voltage-gated calcium channel superfamily (Cav1.2 β subunit). The beta subunit of voltage-dependent calcium channels contributes to the calcium channel function by increasing peak calcium current, shifting the voltage dependencies of activation and inactivation, modulating G protein inhibition and controlling the alpha-1 subunit membrane targeting. Only one rare variant in the *CACNB2* gene has been associated with SQTS (p.Ser481Leu) to date, known as SQT5. This variant is also found to be associated with BrS [40].

The *CACNA2D1* gene (ID: 781) encodes a member of the alpha-2/delta subunit family, a protein in the voltage-dependent calcium channel complex (Cav1.2 α2/δ1 subunit). The protein regulates calcium current density and activation/inactivation kinetics of the calcium channel (ICaL) [40]. Only one variant has been identified in this gene (p.Ser755Thr), but its high frequency in the Ashkenazi population and conflicting evidence refutes its pathogenic role in SQTS [24]. It is associated with the so-called SQTS type 6. This gene has also been associated with other channelopathies, mainly LQTS.

The *SCN5*A gene (ID: 6331) encodes the alpha subunit of the type 5 sodium channel (Nav1.5) that mediates voltage-dependent sodium ion permeability in the cardiomyocyte. So far, only a rare variant in the *SCN5*A gene (p.R689H) has been described to be associated with SQTS (called SQT7). Carriers of this variant show a characteristic BrS phenotype with concomitant shortened QT intervals, but without a conclusive clinical diagnosis of SQTS. Therefore, its association is in dispute.

#### **6.3 Gene associated with a SQTS-mimic phenotype**

The *SLC22A5* gene (ID: 6584) encodes a high-affinity sodium ion-dependent carnitine transporter protein (Solute Carrier Family 22 Member 5). So far, only the pathogenic variant p.Phe17Leu has been associated with SQTS, following an autosomal recessive inheritance pattern [42]. However, because the short QT phenotype is reversible with carnitine supplementation, the association of this gene with SQTS remains inconclusive [24].

#### **7. Genetic counselling**

Due to the low number of cases reported worldwide, the real penetrance and incidence of SQTS is difficult to estimate. Although some pathogenic variants exhibit 100% penetrance, approximately 40% of patients may remain asymptomatic [29]. Current guidelines recommend the analysis of four genes: *KCNH2*, *KCNQ1*, *KCNJ2* and *SLC4A3*, despite last gene need more conclusive data concerning definite role [23]. Despite controversial association data between calcium channel genes and SQTS, current guidelines recommend the analysis of *CACNA1C*, *CACNA2D1* and *CACNB2,* frequently associated with BrS. Genetic diagnosis of SQTS has a diagnostic yield of less than 30% [43] with the *KCNH2* gene as the most cost-effective option [10]. Familial genetic analysis is recommended, both to clarify the pathogenic role of newly identified variants and to identify family members at risk for SCD.

#### **8. Risk stratification and management**

Risk stratification is the main current challenge in the clinical setting, especially in asymptomatic patients carrying a pathogenic genetic alteration. In addition, patients with QTc intervals ≤340 ms should be considered at higher risk for SCD, despite the fact that no conclusive results have been published so far. ICD implantation is the treatment of choice for all patients with SQTS, especially for those who have survived aborted cardiac arrest or who have had spontaneous sustained VT [44]. However, there is also a significant risk of device-related complications, mainly due to inappropriate shocks from the over detection of T waves (high and narrow) seen in SQTS. Drugs that prolong the QT interval (quinidine and sotalol) should be considered for all patients at risk for SQTS in both asymptomatic and symptomatic patients who do not have an ICD, especially in young children [43]. Quinidine is currently the agent of choice, since in patients with SQT1, in addition to prolonging the QT interval and ventricular refractory period, it leads to the normalization of ST segments and T waves and the prevention of VF induction. However, the personalized use of drugs aimed at the treatment of patients carrying certain types of variants is becoming increasingly common. A study on human-induced, pluripotent stem cell-derived cardiomyocytes demonstrated that in addition to quinidine, ivabradine, ajmaline and mexiletine may be drug candidates for preventing tachyarrhythmias in patients carrying the p.Asn588Lys variant in the *KCNH2* gene [45]. In addition, modelling studies indicated that high-dose amiodarone may be a potential drug treatment for SQTS2,

especially those patients carrying the p.Val307Leu variant in the *KCNQ1* gene [46]. Recently, a study show that vernakalant (sodium and potassium channel blocker) can prolong action potential and reduce arrhythmias in human-induced pluripotent stem cell-derived cardiomyocytes from a patient diagnosed of SQTS type-1 due to p.Asn588Lys, suggesting an effective candidate drug for treating arrhythmias [47]. Although more studies are needed to confirm these findings, the development of personalized treatments for inherited arrhythmias is currently in expansion.
