The demographic characteristics of the 155 unrelated patients are shown in . 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).
Demographics Characteristics of the Cohort
Overall, 77 (63 unique, 34 novel) putative disease causing mutations were identified in 73 cases (47%, , ). 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
(, ). 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.
Compendium of RYR2 mutations and polymorphisms reported to date
RyR2 channel topology and localization of mutations and polymorphisms
Prevalence of RYR2 mutations by subgroups
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 (). 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, ).
We evaluated the number of mutations in each exon reported to date in the literature (), 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 (). 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
), 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 , 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, ).
Possible tiered strategy for reflex genetic testing
Yield from RYR2 mutational analysis based on a tiered strategy