Identification of a novel exon3 deletion of RYR2 in a family with catecholaminergic polymorphic ventricular tachycardia

Abstract Background RYR2, encoding cardiac ryanodine receptor, is the major responsible gene for catecholaminergic polymorphic ventricular tachycardia (CPVT). Meanwhile, KCNJ2, encoding inward‐rectifier potassium channel (IK1), can be the responsible gene for atypical CPVT. We recently encountered a family with CPVT and sought to identify a responsible gene variant. Methods A targeted panel sequencing (TPS) was employed in the proband. Copy number variation (CNV) in RYR2 was identified by focusing on read numbers in the TPS and long‐range PCR. Cascade screening was conducted by a Sanger method and long‐range PCR. KCNJ2 wild‐type (WT) or an identified variant was expressed in COS‐1 cells, and whole‐cell currents (IK1) were recorded using patch‐clamp techniques. Results A 40‐year‐old female experienced cardiopulmonary arrest while cycling. Her ECG showed sinus bradycardia with prominent U‐waves (≥0.2 mV). She had left ventricular hypertrabeculation at apex. Exercise induced frequent polymorphic ventricular arrhythmias. Her sister died suddenly at age 35 while bouldering. Her father and paternal aunt, with prominent U‐waves, received permanent pacemaker due to sinus node dysfunction. The initial TPS and cascade screening identified a KCNJ2 E118D variant in all three symptomatic patients. However, after focusing on read numbers, we identified a novel exon3 deletion of RYR2 (RYR2‐exon3 deletion) in all of them. Functional analysis revealed that KCNJ2 E118D generated IK1 indistinguishable from KCNJ2 WT, even in the presence of catecholaminergic stimulation. Conclusions Focusing on the read numbers in the TPS enabled us to identify a novel CNV, RYR2‐exon3 deletion, which was associated with phenotypic features of this family.


| INTRODUC TI ON
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is characterized by exercise or emotion-induced polymorphic or bidirectional ventricular arrhythmias (VAs) leading to syncope or sudden cardiac death in the absence of structural heart disease and is often accompanied by sinus node dysfunction (SND) or sinus bradycardia. (Leenhardt et al., 1995;Postma et al., 2005) (Miyata, Ohno, Itoh, & Horie, 2018) Several responsible genes for CPVT have been identified so far, and RYR2 which encodes the cardiac ryanodine receptor (RyR2) accounts for approximately 60% of CPVT cases. (Laitinen et al., 2001;Priori et al., 2001).
We recently encountered a family with CPVT. Genetic screening of the proband by a targeted panel sequencing using next-generation sequencer (NGS) identified only a rare KCNJ2 E118D variant.
However, after focusing on the read numbers of the NGS, we could identify a novel exon3 deletion of RYR2 (RYR2-exon3 deletion) without performing multiplex ligation-dependent probe amplification (MLPA) method. To elucidate the causative roles of these variants, we investigated family members and functional role of the KCNJ2 E118D variant.

| Genetic analysis
After obtaining appropriate approval from the institutional review board and written informed consent for the genetic analysis from all the subjects of this study, we performed genetic analysis.
Nucleotides substitution was confirmed by a Sanger method. To detect the copy number variations (CNVs), the depths (read numbers) of the NGS reads obtained by the targeted panel sequencing F I G U R E 1 A pedigree and clinical characteristics of the family. AT, atrial tachycardia; AVB, atrioventricular block; CPA, cardiopulmonary arrest; ICD, implantable cardioverter defibrillator; PPM, permanent pacemaker; Prominent U, Prominent U-waves at rest; SB, sinus bradycardia; SD, sudden death. Carriers of an exon3 deletion of RYR2 (RYR2-exon3 deletion) and a KCNJ2 E118D variant are shown as the graphic symbols in the bottom were compared between control and the proband by using the SureCall software (Agilent Technologies, Santa Clara, CA, USA).
To confirm the CNV in RYR2, we performed long-range PCR using KOD-Plus-Neo (Toyobo, Osaka, Japan). Forward primer (5′-CACAGAACAGGACCAAGTTAGAGGC-3′) and reverse primer (5′-CATTACCTTCCTGACACACTTCATCCTAG-3′) were designed to amplify a region that includes the deleted site of RYR2. The 5' position of the forward primer and the reverse primer was 287,712 and 291,655 in the NCBI Reference Sequence NC_000001.10 (GRCh37), respectively. The PCR products were loaded into 0.8% agarose gels with Tris-Acetate-EDTA buffer and electrophoresed.
Then, the bands were extracted and purified using the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), which were directly sequenced to confirm the precise location of the deletion in RYR2.
Currents were evoked by 100 ms test pulses applied in 10 mV increments to potentials ranging from −140 mV to +50 mV from a holding potential of −60 mV. Current-voltage (I-V) relationships were obtained by the current amplitudes at the end of 100 ms pulses against test potentials.

| Catecholaminergic stimulation
To resemble the catecholaminergic effect (protein kinase A (PKA) activation) on KCNJ2 WT and KCNJ2 E118D, I K1 was also recorded 5 min after the presence of a PKA cocktail containing 100 μmol/L forskolin (Sigma-Aldrich, Missouri, USA) and 10 μmol/L 3-isobutyl-1methylxanthine (Sigma-Aldrich, Missouri, USA) in the Bath solution and 10 min after wash out of it as the same method reported by Vega et al. (Vega, Tester, Ackerman, & Makielski, 2009).

| Statistical analysis
All data are expressed as mean ± SE, and statistical comparisons were tested using the unpaired Student's t test with p < 0.05 considered to be statistically significant.

| Functional analysis of the KCNJ2 E118D variant
Since KCNJ2 variants can be the cause of an atypical type of CPVT, (Tester et al., 2006) there remained a possibility that the KCNJ2 E118D variant might cause the CPVT phenotype of this and after wash out of it, as reported by Vega et al.(Vega et al., 2009) As shown in Figure 6, PKA activation affected neither KCNJ2 WT nor KCNJ2 E118D. Contrary to the report by Vega et al., (Vega et al., 2009) PKA activation did not reduce even I K1 produced by KCNJ2 WT.

| Identification of a rare KCNJ2 E118D variant and a novel exon3 deletion of RYR2
Our genetic screening by the initial targeted panel sequencing on the proband with CVPT identified only a rare KCNJ2 E118D variant. However, focusing on the read numbers of the targeted panel sequencing led us think of the presence of the CNV, then we could finally identify a novel massive deletion of RYR2 including exon3.
An MLPA method has been reported to be useful for identifying CNVs for many diseases including arrhythmogenic disorders. However, it is noteworthy that we identified CNV in RYR2 by ( Barc et al., 2011;Cox et al., 2011;Sonoda et al., 2017Sonoda et al., , 2018 Therefore, our study underlies the diagnostic importance of examining the read numbers in targeted panel sequencing to rule out the presence of pathological CNVs, even when a conventional method identifies candidate variants that are not actually disease-causing. Although there have been several reports regarding massive deletion of RYR2 including exon3, direct sequencing of the PCR products ( Figure 4e) from the proband revealed that the deletion site of our RYR2-exon3 deletion was different from those of previous reports. (Bhuiyan et al., 2007;Marjamaa et al., 2009;Medeiros-Domingo et al., 2009;Ohno et al., 2014;Szentpali et al., 2013).

| Pathophysiological roles of U-waves in patients with CPVT
In patients with RYR2-related CPVT, intracellular Ca 2+ overload or oscillations in the heart are thought to be induced by catecholaminergic stimulation through an increased diastolic Ca 2+ leakage from SR, which may lead to DAD or DAD-induced triggered activity. (Jiang et al., 2004;Katra & Laurita, 2005;Katra et al., 2007;Paavola et al., 2007) Clinical examinations of CPVT patients reported that DADs recorded in monophasic action potential of ventricular myocardium could be augmented by catecholaminergic stimulation, and concomitant U-wave changes on ECG were associated with the DADs. (Nakajima et al., 1997;Paavola et al., 2007) In the present case, catecholaminergic stimulation by isoproterenol infusion augmented Uwaves, followed by PVC. These findings appear to be consistent with these concepts. Intriguingly, Aizawa et al. reported that U-waves in CPVT patients could also be affected by some specific conditions such as after ventricular pacing, after the exercise test and after sinus arrest. (Aizawa et al., 2006) Therefore, there may be a high probability of the relationship between U-waves and arrhythmogenicity in CPVT patients; however, it needs to be further investigated.
In this family, the proband and her elder sister experienced cardiac events during exercise. The proband had sinus bradycardia, prominent U-waves at rest, and hypertrabeculation localized at LVA. Her father and paternal aunt received PPM due to SND, and had prominent U-waves at rest. Her father also had AT with AVB.
These family members, except for her deceased sister, carried both the RYR2-exon3 deletion and the KCNJ2 E118D, which obscured pathophysiological roles of each variant. On the other hand, ECG of her daughter (age 11), who carried only the RYR2-exon3 deletion, showed no prominent U-waves but inverted T-waves. We speculate that normal juvenile pattern of T-waves might obscure U-waves. (Surawicz, 1998) She had been asymptomatic, and exercise stress test induced only one premature ventricular contraction.

| Possible functional role of the RYR2-exon3 deletion and the KCNJ2 E118D variant
The RYR2-exon3 deletion may produce mutant RyR2 proteins without frameshift, thus may cause their functional abnormalities.
Actually, functional analysis of exon3 deletion of RyR2 using heterologous expression in HEK293 cells or HL-1 cells revealed that it reduced the endoplasmic reticulum or SR luminal Ca 2+ threshold at which Ca 2+ release terminates and increased the fractional Ca 2+ release, and also enhanced the amplitude of store overload-induced Ca 2+ transients. (Tang, Tian, Wang, Fill, & Chen, 2012) Considering it together with clinical reports of patients with exon3 deletion of RYR2, various phenotypic manifestations in this family could be attributable to the RYR2-exon3 deletion. (Bhuiyan et al., 2007;Marjamaa et al., 2009;Medeiros-Domingo et al., 2009;Ohno et al., 2014;Szentpali et al., 2013).
Regarding the KCNJ2 E118D variant, it caused no functional abnormalities of I K1 based on the patch-clamp study, even in the presence of PKA activation. These findings suggested that prominent U-waves at rest in this family might be associated with the RYR2-exon3 deletion.

| CON CLUS IONS
A novel RYR2-exon3 deletion and a rare KCNJ2 E118D variant were identified in a family with CPVT. The RYR2-exon3 deletion could be identified by focusing on the read numbers of the targeted panel sequencing without performing the MLPA method, which emphasizes the importance of focusing on the read numbers when performing genetic analysis using NGS. The RYR2-exon3 deletion may produce mutant RyR2 proteins without frameshift, thus cause RyR2 dysfunction, but the KCNJ2 E118D variant did not cause I K1 dysfunction.
Therefore, the novel RYR2-exon3 deletion may be associated with phenotypic features of this family.

ACK N OWLED G M ENTS
We would like to thank to Ms. Miki Matsui and Ms. Takako Kobayashi for their technical expertise.