Comprehensive Open Reading Frame Mutational Analysis of the RYR2-Encoded Ryanodine Receptor/Calcium Channel in Patients Diagnosed Previously with Either Catecholaminergic Polymorphic Ventricular Tachycardia or Genotype Negative, Exercise-Induced Long QT Syndrome

Abstract

Objective

To determine the spectrum and prevalence of mutations in the RYR2-encoded the cardiac ryanodine receptor in cases with exertional syncope and normal QTc.

Background

Mutations in the RYR2 cause type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a cardiac channelopathy with increased propensity for lethal ventricular dysrhythmias. Most RYR2 mutational analyses target 3 canonical domains encoded by < 40% of the translated exons. The extent of CPVT1-associated mutations localizing outside of these domains remains unknown as RYR2 has not been examined comprehensively in most patient cohorts.

Methods

Mutational analysis of all RYR2 exons was performed using PCR, DHPLC, and DNA sequencing on 155 unrelated patients (49% females, 96% white, age at diagnosis 20 ± 15 years, mean QTc 428 ± 29 ms), with either clinical diagnosis of CPVT (n = 110) or an initial diagnosis of exercise-induced long QT syndrome (LQTS) but with QTc < 480 ms and a subsequent negative LQTS genetic test (n = 45).

Results

Sixty-three (34 novel) possible CPVT1-associated mutations, absent in 400 reference alleles, were detected in 73 unrelated patients (47%). Thirteen new mutation-containing exons were identified. Two thirds of the CPVT1-positive patients had mutations that localized to one of 16 exons.

Conclusions

Possible CPVT1 mutations in RYR2 were identified in nearly half of this cohort. 45 of the 105 translated exons are now known to host possible mutations. Considering that ~65% of CPVT1-positive cases would be discovered by selective analysis of 16 exons, a tiered targeting strategy for CPVT genetic testing should be considered.

INTRODUCTION

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal, heritable arrhythmia syndrome often manifesting as exercise-induced ventricular arrhythmias, syncope or sudden death.¹ With mortality rates of 30-50% by age 35 years, CPVT is one of the most malignant cardiac channelopathies expressed predominately in young patients with otherwise structurally normal hearts². While the resting 12-lead electrocardiogram (ECG) is typically normal, the hallmark arrhythmia, bidirectional VT, is often present during exercise stress testing and has been considered pathognomonic for CPVT.^1,3

CPVT stems from an alteration of intracellular calcium handling involving the critical calcium-induced calcium release mechanism of myocardial cells. At the molecular level, gain of function mutations in the cardiac ryanodine receptor encoded by RYR2 account for at least 50% of CPVT cases and is annotated as type 1 CPVT (CPVT1).³ Mutations in CASQ2-encoded calsequestrin are responsible for the very rare, autosomal recessive form known as type 2 CPVT (CPVT2).^2,4

The cardiac ryanodine receptor (RyR2), encoded by the 105-exon-containing RYR2 gene, is one of the largest ion channel proteins comprised of 4967 amino acids; localizes to the sarcoplasmic reticulum, and controls intracellular calcium release and cardiac contraction. Since the sentinel discovery of a CPVT-causing RYR2 mutation⁵, a cluster distribution involving three discrete protein regions has been reported. Based in a potential physiological role for these “hot-spots”, these regions have been termed “domains” I, II and III (Figure 1)^6,7. Similar mutation clustering is observed in the RYR1 gene which encodes the skeletal muscle RyR1 and is linked to malignant hyperthermia and central core disease^8-10. However, since the majority of CPVT cases have not undergone the entire RYR2 scan, the prevalence of mutations residing outside these three canonical domains (i.e. ~61 exons that encode for 2570 amino-acids) remains unknown.

Figure 1. Mutation clustering in the cardiac ryanodine receptor (RyR2).

Mutations are distributed in three “hot-spots” regions, called domains I (N-terminal), II (central) and III (channel region)⁶,⁷. AA: amino-acid number estimated for each domain.

Adapted from George CH, et.al⁷., and Yano M, et. al⁶.

Currently, among research laboratories and clinical diagnostic laboratories, there is no consensus or clear definition of the “RYR2 targeted scan” resulting in enormous discrepancy in the number of exons studied by each research group or commercial company. This situation has an important impact in “gene-negative” definition, genotype-phenotype correlation and patient quality of care. In the present study, we sought to determine the prevalence of mutations throughout RYR2’s entire open reading frame in a large cohort of unrelated cases referred to 2 different institutions for exertional syncope and, using a combined analysis of the previous reported mutations and the novel mutations found in this cohort, we propose a novel, targeted “genetic approach” for CPVT1 genetic testing.

METHODS

Study Participants

We studied a cohort of 155 unrelated patients referred to either the Windland Smith Rice Sudden Death Genomics Laboratory at Mayo Clinic, Rochester, MN or the Department of Clinical Genetics, Academic Medical Center, University of Amsterdam, Netherlands for genetic testing between August 2001 and June 2008. A clinical diagnosis of CPVT was rendered in 110 patients by either one of the authors (MJA, AAMW) or the referring physician. Of these, 78 were classified as “strong CPVT phenotype” because of exertional syncope plus documentation of bidirectional or polymorphic ventricular tachycardia (BVT/PVT) while 32 were classified as “possible CPVT phenotype” based on the presence of exertional syncope and stress test induced ventricular ectopy but not BVT/PVT. In addition, 45 cases were referred as “possible/atypical long QT syndrome (LQTS)” because of exertional syncope and QTc values < 480 ms. All were genotype negative for the 12 known LQTS-susceptibility genes.

Following receipt of written consent for this Mayo Foundation Institutional Review Board and Amsterdam Academic Medical Center Medical Ethical Committee approved protocol, genomic DNA was extracted from peripheral blood lymphocytes using the Purgene DNA extraction kit (Gentra, Inc, Minneapolis, MN, USA). In cases with suspected mosaicism, additional DNA from saliva was isolated using the ORAgene kit (DNA Genotek, Ottawa, Ontario, Canada) and DNA from skin fibroblasts and hair-roots was isolated using the QIAamp DNA minikit (Qiagen, USA).

Mutational Analysis

Comprehensive open reading frame/splice site mutational analysis of all 105 RYR2 exons was performed using polymerase chain reaction (PCR), denaturing high performance liquid chromatography (DHPLC), and DNA sequencing as described previously.¹¹ The flanking primers used for PCR were published previously or designed with Oligo software (Molecular Biology Insights, Inc., Cascade Colo.) and are available on request. We also searched for large genomic rearrangements affecting exon 3 as reported previously¹².

All putative pathogenic variants must have been absent in 400 reference alleles (100 healthy white and 100 healthy black) obtained from the Human Genetic Cell Repository sponsored by the National Institute of General Medical Sciences and the Coriell Institute for Medical Research (Camden, New Jersey) in order to be considered as potentially disease-related.

Statistical Analysis

We used the JMP Statistical Software (JMP 6.0, 2005; SAS Institute Inc, Cary, NC). All continuous variables are reported as mean ± SD. Differences between continuous variables were evaluated using unpaired Student t tests, and nominal variables were analyzed using chi-square analysis. Statistical significance was considered at p < 0.05.

RESULTS

The demographic characteristics of the 155 unrelated patients are shown in Table 1. 96% were Caucasians, 49% were females, age at symptoms was 20 ± 15 yrs, and average QTc was 428 ± 29 ms. The mean age of onset of symptoms was significantly lower in RYR2 mutation positive subjects compared to those with a negative genetic test (16.7 ± 12.3 vs 23.8 ± 16.6 yrs respectively, p<0.004).

Table 1. Demographics Characteristics of the Cohort.

	CPVT Strong Phenotype	CPVT Possible Phenotype	Gene Negative LQTS	Total
No. of Patients	78	32	45	155
Age (yrs) mean ± SD	20 ± 15	20 ± 16	22 ± 14	20 ± 15
QTc (ms) mean ± SD	415 ± 26	434 ± 30	434 ± 27	427 ± 29
%Female	47	44	57	49
RYR2 Positives (%)	n=47 (60.2%)	n=12 (37.5%)	n=14 (31.1%)	n=73 (47.1%)

Overall, 77 (63 unique, 34 novel) putative disease causing mutations were identified in 73 cases (47%, Table 2, Figure 2). 41/73 mutation positive cases (56%) were females. Putative mutations were absent in 400 references alleles and most of the mutated residues exhibit highly conservation across species (Supplemental Table). The yield of the genetic test was significantly higher among the 78 cases classified clinically as “strong CPVT phenotype” compared to the 32 cases diagnosed as “possible CPVT phenotype” (60% vs 37.5%, p < 0.04). Notably, nearly one-third of the 45 “gene negative LQTS” cases had a rare missense mutation in RYR2 (Table 1, Figure 3). Four out of the 73 RYR2 mutation-positive cases hosted multiple mutations (5.5%). As expected, we observed a mutation clustering distribution across RYR2; nevertheless, ten mutations found in 11 cases resided outside the three canonical domains, specifically, between domain I and II; 8 of them exhibited a strong CPVT phenotype. Three large genomic rearrangements comprising exon 3 were detected in three unrelated cases involving a 3.6 kb deletion in one and a 1.1 kb deletion in two cases.

Table 2. Compendium of RYR2 mutations and polymorphisms reported to date.

Putative mutations are indicated in red, n=129 (including 2 large genomic rearrangements involving exon 3, not detectable by regular genetic scan), polymorphisms in blue n=15.

No.	Mutation Number (Figure 1)	Exon	Base Position	Amino-acid Change	Location	Cases hosting the variant (n=108)	AA hosting the variant (n=100)	CC hostingthe variant (n=100)	Reference
1	1	3	1.1kb deletion*	Exon 3 del	NT	2			Bhuiyan12
2	2	3	3.6kb deletion*	Exon 3 del	NT	1			Novel
3	3	3	184 C>T	L62F	NT	1			Novel
4		3	230 C>T	A77V	NT				d’Amati G27
5	4	3	241 A>C	M81L	NT	1			Novel
6	5	8	493 C>T	P164S	NT	1			Choi28
7		8	506 G>A	R169Q	NT				Hsueh29
8	6	8	527 G>A	R176Q	NT	1			Tiso24
9	7	8	556 G>A	V186M	NT	1			Tiso24
10	8	8	567 A>T	E189D	NT	1			Davis30
11		10	6337 G>A	H240R	NT				Tester31
12	9	10	727 G>A	E243K	NT	1			Novel
13	10	12	985 T>C	F329L	NT	1			Novel
14	11	12	994C>T	R332W	NT	1			Novel
15	12	13	1072 G>A	G357S	NT	1			Novel
16	13	13	1129 G>A	V377M	NT		3		Novel
17		14	1240 C>T	R414C	NT				Tester32
18	14	14	1241 G>T	R414L	NT	1			Choi28
19	15	14	1244 C>G	T415R	NT	1			Novel
20		14	1255 A>T	I419F	NT				Choi28
21	16	14	1258 C>T	R420W	NT	3			Bauce33
22	17	14	1259 G>A	R420Q	NT	2			Novel
23		15	1298 T>C	L433P	NT				Tiso24
24	18	15	1396 C>G	P466A	NT	1			Tester14
25	19	16	1519 G>A	V507I	NT	5		4	Novel
26	20	17	1646 C>T	A549V	NT	1			Novel
27		19	Not Reported	S616L	CL				Marjmaa21
28	21	21	2216 G>A	R739H	CL	1			Novel
29	22	26	3038 G>A	R1013Q	CL	1			Novel
30		27	Not Reported	R1051P	CL				Marjmaa21
31	23	28	3320 C>T	T1107M	CL	1			Novel
32	24	28	3407 C>T	A1136V	CL	3		2	Novel
33	25	37	5170 G>A	E1724K	CL	2			Postma34
34	26	37	5509 G>A	E1837K	CL	1			Novel
35	27	37	5654 G>A	G1885E	CL	2	6		Milting26
36	28	37	5656 G>A	G1886S	CL	11	20	9	Milting26
37	29	40	6137 A>G	E2045G	CL	1			Novel
38		41	6337 G>A	V2113M	CL	1			Tester31
39		41	Not Reported	G2145R	CL				Marjmaa35
40	30	42	6467 A>G	Y2156C	CL			1	Novel
41	31	42	6504 C>G	H2168Q	CL	2			Novel
42	32	42	6548 A>T	E2183V	CL		1		Novel
43	33	43		D2216V	CL	1			Novel
44	34	44	6740 C>T	S2246L	CL	1			Priori5
45	35	44	6761 C>T	A2254V	CL	1			Postma34
46		45	6800 G>A	R2267H	CL				Tester36
47	36	45	6886 G>C	E2296Q	FKBP	1			Novel
48		45	6916 G>A	V2306I	FKBP				Laitinen37
49		45	6919 T>C	F2307L	FKBP				Berge38
50		46	6933 G>A	E2311D	FKBP				Priori3
51		46	6992 T>C	V2321M	FKBP				Nishio39
52		46	6982 C>T	P2328S	FKBP				Laitinen37
53		46	6992 T>C	F2331S	FKBP				Creighton40
54		46	7076 G>A	R2359Q	FKBP				Aizawa41
55		47	7157 A>T	N2386I	FKBP				Tiso24
56	37	47	7158 G>A	A2387T	FKBP	3			Tester14
57		47	7158 G>A	A2387P	FKBP				Bagattin42
58	38	47	7165 A>C	M2389L	FKBP		1		Tester43
59		47	7175 A>5	Y2392C	FKBP				Bauce33
60		47	7181 C>G	A2394G	FKBP				Postma34
61	39	47	7202 G>A	R2401H	FKBP	1			Aizawa41
62		47	7207 G>T	R2401L	FKBP				Creighton40
63	40	47	7207 G>A	A2403T	FKBP	1			Choi28
64	41	47	7210 C>A	R2404T	FKBP	1			Beckman44
65	42	48	7258 A>T	R2420W	FKBP	1			Novel
66		49	7422 G>C	P2474S	FKBP				Priori3
67		49	7423 G>T	V2475F	FKBP				Tester32
68		49	7511 C>T	T2504M	FKBP				Tiso24
69		49	Not Reported	L2487I	FKBP				Tester43
70		50	7528 T>C	T2510A	FKBP				Tester31
71		50	7599 C>G	L2534V	FKBP				Hasdemir45
72	43	61	8874 A>G	Q2958R	Cytosol	40	10	36	Tiso24
73		69	Not Reported	N3308S	Cytosol				Marjamaa21
74		75	Not Reported	R3570W	Cytosol				Marjamaa35
75		83	11332 C>T	L3778F	Cytosol				Priori3
76	44	83	11399 G>T	C3800F	Cytosol	1			Tester14
77		86	11636 T>C	L3879P	Cytosol				Tester31
78		87	11773 C>G	Q3925E	Cytosol				Tester31
79	45	88	11814 C>A	S3938R	Cytosol	1			Tester14
80		88	11836 G>A	G3946S	Cytosol				Priori3
81		88	Not Reported	G3946A	Cytosol				Davis30
82		88	11876 C>T	S3959L	Cytosol				Tester31
83	46	89	11916 G>T	M3972I	Cytosol	1			Novel
84	47	89	11917 G>C	D3973H	Cytosol	1			Novel
85	48	89	11921 T>A	L3974Q	Cytosol	1			Novel
86	49	90	11989 A>G	K3997E	Cytosol	1			Novel
87	50	90	12028 G>A	V4010M	Cytosol		1		Tester43
88		90	Not Reported	F4020L	Cytosol				Postma34
89	51	90	12226 A>G	E4076K	Cytosol	1			Postma34
90		90	12290 A>G	N4097S	Cytosol				Tester46
91		90	12311 A>T	N4104I	Cytosol				Postma34
92		90	12312 C>G	N4104K	Cytosol				Priori3
93		90	Not Reported	L4105F	Cytosol				Hasdemir47
94	52	90	12322 C>A	H4108N	Cytosol	1			Postma34
95		90	Not Reported	H4108Q	Cytosol				Postma34
96	53	90	12370 A>G	S4124G	Cytosol	1			Novel
97	54	90	12371 G>C	S4124T	Cytosol	1			Tester14
99		90	Not Reported	R4144C	Cytosol				Berge38
100		90	12436 G>A	E4146K	Cytosol				Tester46
101	55	90	12446 A>G	Y4149S†	Cytosol	1			Novel
102	56	90	12470 G>A	R4157Q	Cytosol	1			Novel
103		90	12472 A>C	T4158P	Cytosol				Tester46
104	57	90	12476 A>G	Q4159P	Cytosol	1			Novel
105	58	90	12533 A>G	N4178S	Cytosol	3			Novel
106	59	90	12559 G>C	E4187Q	Cytosol	1			Novel
107	60	90	12586 A>G	T4196A	Cytosol	1			Tester
108		90	12601 C>A	Q4201R	Cytosol				Laitinen37
109	61	90	12845 C>T	A4282V	Cytosol			1	Tester43
110		90	12919 C>T	R4307C	Cytosol				Callis48
111	62	90	12944 G>A	G4315E	Cytosol			1	Novel
112		91	13291 G>A	E4431K	Cytosol				Berge38
113		93	13489 C>T	R4497C	TMD				Priori5
114	63	93	13496 T>G	F4499C	TMD	1			Choi28
115		93	13512 G>A	M4504I	TMD				Bagattin42
116	64	93	13528 G>A	A4510T	TMD	2			Choi28
117		93	Not Reported	F4511L	TMD				Beckmann44
118	65	94	13666 G>A	A4556T	TMD	1			Tester14
119		94	13695 C>A	S4565R	TMD				Tester36
120		95	13819 G>C	A4607P	TMD				Bagattin42
121		95	13831 G>A	E4611K	TMD				Berge38
122		96	Not Reported	W4645R	TMD				Beery49
123	66	96	13948 A>G	K4650E	TMD	1			Novel
124		96	13957 A>G	V4653F	TMD				Laitinen37
125	67	97	13967-13972 Dup	4657-4658 ins EY	TMD	1			Tester14
126		97	Not Reported	G4662S	TMD				Postma34
127	68	97	14011 G>C	G4671R	TMD	1			Choi28
128	69	99	14205-14208 Del	N4736 Del	TMD	1			Novel
129		99	14285 A>C	H4762P	TMD				Postma34
130	70	100	14311 G>A	V4771I	TMD	2			Priori3
131	71	100	14369 G>A	R4790Q	TMD	1			Novel
132	72	100	14414 A>G	K4805R	TMD	1			Novel
133	73	101	14465 G>A	R4822H	TMD	1			Novel
134	74	101	14542 G>A	I4848V	TMD	2			Choi28
135		101	14552 T>G	F4851C	TMD				Aizawa41
136		101	14579 C>G	A4860G	TMD				Priori3
137		102	14601 T>G	I4867M	CT				Priori3
138		102	14639 T>C	V4880A	CT				Bagattin42
139		103	14683 A>G	N4895D	CT				Priori3,5
140		103	14705 C>T	P4902L	CT				Laitinen37
141		103	Not Reported	P4902S	CT				Postma34
142		104	14806 G>A	G4936R	CT				Tester31
143		105	14848 G>A	E4950K	CT				Priori3
144	75	105	14876 G>A	R4959Q	CT	2			Laitinen37

Predicted location: NT = Amino-Terminal, CL = Cytoplasmic Loop, FKBP = 12.6 (Calstabin) binding domain, TMD = Transmembrane domain, CT = C Terminal.

^*Large genomic rearrangement comprising intron 2-3 and intron 2-4 resulted in inframe deletion of exon 3. AA: African Americans Controls, CC: Caucasians Controls.

^†Mosaicism.

Figure 2. RyR2 channel topology and localization of mutations and polymorphisms.

Linear topology of the cardiac ryanodine receptor (RyR2); putative pathogenic mutations (yellow circles) and polymorphisms (blue circles) found on this study-cohort are shown in the approximate location. The number within the circle corresponds to the mutation # on Table 1.

Figure 3. Prevalence of RYR2 mutations by subgroups.

The yield from the entire RYR2 scan on this cohort is shown on the left. Bars on the right side show the sensitivity in the 3 different subgroups of this cohort.

One proband had a maternally inherited Y4149S (tyrosine, Y, at position 4149 mutated to serine, S) missense mutation. Although the proband’s mother was asymptomatic and had an unremarkable exercise ECG; germline mosaicism was suspected clinically because more than one offspring was affected. Accordingly, Y4149S mosaicism was detected in her being highest in the hair-roots (~25%), less in leucocytes (~20%) and in fibroblasts and buccal epithelium (~15-18%).

Twelve non-synonymous single nucleotide polymorphisms (6 novel) were also identified, 7 of them were seen only in controls and 5 in cases and controls (Table 2). Four novel polymorphisms localize between domain I and II. The most common polymorphism was Q2958R with an heterozygous prevalence of 34% in Caucasians and 10% in African-Americans; followed by G1886S with a prevalence of 20% (African Americans) and 9% (Caucasians). V377M was found only in African-Americans with a prevalence of 3%. Finally, Y2156C, E2183V, M2389L, V4010M, A4282V and G4315E are rare variants observed only once in different control subjects. Thus, within the exons hosting putative CPVT1-associated mutations, the background prevalence of rare amino acid substitutions among the 200 apparently healthy volunteers was 3% (3/100 Caucasians and 3/100 African Americans, Table 2).

We evaluated the number of mutations in each exon reported to date in the literature (Table 2), excluding exons containing only polymorphisms. As such; 127 unique mutations were analyzed, including those found within this cohort. Sixteen exons hosted > 3 distinct CPVT1-associated mutations; 13 exons had at least 2 mutations reported while an additional 16 exons had, so far, only a single mutation reported (Figure 4). This mutation clustering phenomena might facilitate a tiered strategy that may yield a more cost-effective approach for CPVT genetic testing. If we consider that the average charge for the current RYR2 commercial tests available on the market is approximately $0.40 per coding nucleotide (http://pgxhealth.com, www.preventiongenetics.com), the estimated charge for the entire RYR2 coding region scan would be approximately $6000 per patient, meaning that the commercial charge to analyze this 155 patient cohort in its entirety would have approached $1 million US dollars. In comparison, the total charge to scan only the 45 mutation-hosting exons that have been reported to date exon-containing mutations reported to date would be about 50% less. Further, a reflex tiered strategy would reduce the cost significantly. As modeled here, using a 3-tiered reflex genetic test strategy based on Figure 4, the genetic scan of the first tier of exons in our cohort would cost $190,960.00 (~$1200 per case) and would detect nearly two-thirds of those CPVT cases that are due to mutations in RYR2. The charge to reflex to the second tier genetic scan would add < $1000 per case and combined, nearly 90% of the RYR2-mutation positive cases (CPVT1) would be identified. Reflexing to the third tier would capture the remaining RYR2-positive cases and the charge to do so would be ~$123,225 US dlls ($795.00 US dlls/case, Figure 5).

Figure 4. Possible tiered strategy for reflex genetic testing.

Schematic representation of the 105 coding exons of the RYR2 gene. Boxes in colors: all the exon-containing mutations reported to date. Boxes in white: exons free of reported mutations. The tiered strategy was built based on the number of mutations containing in each exon as shown by three different colors. The 1^st tier included 16 exons, 2^nd tier 13 exons and 3^rd tier 16 exons. Exons containing control variants were not included.

Figure 5. Yield from RYR2 mutational analysis based on a tiered strategy.

Retrospective analysis of the mutations detected in our cohort and in the world-wide compendium of mutations reported to date. The percentage of mutations that would be detected using the tired strategy is shown.

DISCUSSION

Exertional Syncope: LQTS or CPVT?

It has been reported that nearly 30% of CPVT cases have been misdiagnosed as “LQTS with normal QT intervals” or “concealed LQTS”.¹³ Recently, we demonstrated that nearly 6% of 269 LQTS genotype negative patients hosted a putative CPVT1-causing RyR2 mutation¹⁴. Here, we included only referral cases of “atypical/possible LQTS” with a phenotype of exertional syncope and QTc < 480 ms. Herein, the yield of RYR2 mutations for these 45 cases was 31%; indicating the critical importance of properly distinguishing between CPVT and LQTS. CPVT-related arrhythmias can be easily reproduced during an exercise stress test, isoproterenol infusion or by other forms of adrenergic stimulation^15,16. The induction of polymorphic ventricular tachycardia or bidirectional VT, characterized by 180° alternating QRS axis on a beat-to-beat basis, sets CPVT apart from “concealed” or “borderline” LQTS.

RYR2 genetic approach: Targeted scan and tiered strategy

Our results confirm that mutation clustering exists. The functional significance of mutation clustering remains unclear. It has been suggested, however, that a domain-domain interaction is crucial for channel function^17-19 and a defective inter-molecular interaction may be crucial in disease phenotypes. Interestingly, in this study 11/64 (17%) of the putative mutations localize outside the considered canonical domains.

Based upon our results and after analyzing a large publicly available compendium of the 127 RYR2 putative mutations known to date (Table 2), we propose an expanded genetic approach for research/investigational laboratories. A reasonable RYR2 scan will include the analysis of at least 45 exons in total known to host all published mutations reported to date. Since some exons (19) imbibed in the hot-spot region remain free of mutations so far, a more ambitious and “comprehensive” RYR2 genetic test would include these exons as well resulting in a 64-exon scan (exons 3-28, 37-50, 75 and 83-105).

The mutation clustering phenomena might facilitate a tiered strategy that may yield a more cost-effective approach for CPVT genetic testing. Figure 4 summarizes this proposed tiered strategy. The approach was developed considering the number of mutations in each exon reported to date in the literature. The first tier comprises those exons (N=16) now known to host > 3 unique CPVT-associated mutations. The second tier includes 13 exons with at least 2 mutations reported while the third tier consists of the final 16 exons where, so far, only a single mutation within that exon has been reported. Considering that ~65% of the RYR2 mutation-positive cases might have a mutation in the first tier of 16 RYR2 exons, the charge of the genetic analysis in this group could be reduced by approximately half (predicted $1232.00 US dlls/case for the first tier of 16 exons vs $3019.00 US dlls/case for the entire sequencing of exons-containing reported mutation).

In case of negative results, we suggest that the pseudo-comprehensive (64 exon) RYR2 scan mentioned previously (exons 3-28, 37-50, 75 and 83-105) be performed. Additional “rare” although documented causes of CPVT should also be considered, like large RYR2 genomic rearrangements involving exon 3 and mutations in calsequestrin 2 (CASQ2) and Kir2.1 (KCNJ2)²⁰. The area surrounding exon 3 is highly susceptible to large Alu-repeat-mediated genomic rearrangements; we documented 3 unrelated cases hosting large heterozygous deletions involving exon 3 that could not be detected by regular genetic screening using DHPLC or direct DNA sequencing. Validating this observation, exon 3 deletion was also reported recently in a different cohort where 2 unrelated cases (out of 33), hosted a 1.1kb deletion, including exon 3²¹.

Polymorphisms in RYR2, not that rare and with potential functional effect

It has been considered that RYR2 is not a polymorphic gene. However, 15/142 (10.5%) missense variants reported to date were found in controls. We did not scan the entire RYR2 gene in control subjects. Instead, since we focused on the exon-containing mutations, the rate of non-synonymous genetic variation throughout all of RYR2 may be higher. Importantly however, among the exons now known to host possible CPVT1-associated missense mutations, similarly rare amino acid substitutions were found in only 6 of the 200 control subjects examined in this study. Although not a true case-control genetic epidemiologic study, if validated, this would suggest that among cases where CPVT is strongly suspected, there would be a 95% estimated probability that the identification of a rare missense mutation would likely represent the pathogenic basis for the patient’s CPVT rather than merely being only a rare amino acid substitution.

We have learned that common polymorphisms in other ion channels have the potential to modify the clinical phenotype^22,23; polymorphisms in RYR2 may have the same potential. RyR2-Q2958R is the most common RYR2 polymorphism; was described for the first time 9 years ago²⁴ and is particularly common in Caucasians (34%). The second most common polymorphism in RYR2 is G1886S (20% African Americans, 9% Caucasians) followed by G1885E (6% Caucasians). Interestingly, in vitro studies in heterologous systems have demonstrated that both G1885E and G1886S polymorphisms caused a significant increase in the cellular Ca(2+) oscillation activity compared with RyR2 wild-type channels. Further, when both polymorphisms were introduced in the same RyR2 subunit, the store-overload-induced calcium release activity was nearly completely abolished²⁵. The clinical consequences of this “RyR2 loss of function” in vitro phenotype is not clear, however, compound heterozygosity involving these two polymorphisms has been reported in right ventricular dysplasia²⁶. The potential functional effects of the 6 novel polymorphisms identified in this study are unknown.

It is important to remark that none of the novel mutations detected on this study have been functionally characterized to further bolster the contention of pathogenicity. However, less than 15% of the mutations reported to date in RYR2 have been studied in vitro, pathogenicity has been suspected based on co-segregation with the disease and absence in control subjects. Here, co-segregation with the disease data was not available for all cases. Instead, the prevalence of putative mutations amongst strong cases (~60%) was markedly higher than in controls (~3%) and all putative mutations were absent in 400 reference alleles. Thus, although the precise contribution of each discrete mutation to the phenotype remains to be determined, statistically, the estimated probability for pathogenicity for RYR2 mutations found in strong cases is quite high (~95%).

Mosaicism in RYR2

This is the first report involving RYR2 mosaicism which was transmitted to descendants, presumably causing sudden death in two children and full blown CPVT in one child from the age of 9 years. RYR2 mutations, in many circumstances (~20% in our cohort) are de novo in origin, but it could also be present in a mosaic form in the asymptomatic parents, which requires attention during genetic counseling as well as during genetic screening.

Clinical Significance

This study represents the first analysis of RYR2 mutation distribution in a large cohort of cases. Our results contribute to a better delineation of the “hot spot” region with important consequences in “gene negative” definition. The identification of novel common variants in control subjects will allow a better interpretation of the CPVT genetic test and the detection of RYR2 mosaicism and confirmation of exon 3 deletion in different patients-cohort, provide novel genetic possibilities in the pathogenesis of CPVT. Moreover, the possibility of a tiered strategy for RYR2 genetic scan may enable a more cost-effective genetic approach to analyzing one of the largest genes in the human genome. Finally, we emphasize the critical importance of properly distinguishing between CPVT and LQTS (including Andersen-Tawil syndrome), two different diseases with a similar clinical presentation but different clinical outcomes and different responsiveness to pharmacotherapy.

CONCLUSION

Although intimidating as one of the largest genes in the human genome, results from this comprehensive open reading frame analysis involving one of the largest cohorts of unrelated patients examined, combined with a detailed analysis of all published CPVT1-associated mutations indicate that to date, only 45 of RYR2’s 105 translated exons host a putative CPVT1-associated mutation thus far. Moreover, an initial targeting of only 16 exons would allow the identification of putative mutations in ~65% of the RYR2-mutation positive cases, though compound heterozygosity may be missed. Finally, given the present estimate of 3% frequency for rare missense mutations among controls, one must be cognizant of the possibility of a “false positive” especially as the pre-test probability of a CPVT diagnosis decreases. The ~33% yield that was observed among the “possible” cases of CPVT indicates that perhaps 90% of the mutations, identified among cases labeled as “possible CPVT” or so-called “atypical LQTS” with exercise-induced syncope and QTc < 480 ms, are pathogenic whereas 10% of those mutations may represent “false positives”.