Genetic testing for long-QT syndrome: distinguishing pathogenic mutations from benign variants

Abstract

Background: Genetic testing for long-QT syndrome (LQTS) has diagnostic, prognostic, and therapeutic implications. Hundreds of causative mutations in 12 known LQTS-susceptibility genes have been identified. Genetic testing that includes the 3 most commonly mutated genes is available clinically. Distinguishing pathogenic mutations from innocuous rare variants is critical to the interpretation of test results. We sought to quantify the value of mutation type and gene/protein region in determining the probability of pathogenicity for mutations.

Methods and results: Type, frequency, and location of mutations across KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3) were compared between 388 unrelated "definite" (clinical diagnostic score >or=4 and/or QTc >or=480 ms) cases of LQTS and >1300 healthy controls for each gene. From these data, estimated predictive values (percent of mutations found in definite cases that would cause LQTS) were determined according to mutation type and location. Mutations were 10 times more common in cases than controls (0.58 per case versus 0.06 per control). Missense mutations were the most common, accounting for 78%, 67%, and 89% of mutations in KCNQ1, KCNH2, and SCN5A in cases and >95% in controls. Nonmissense mutations have an estimated predictive value >99% regardless of location. In contrast, location appears to be critical for characterizing missense mutations. Relative frequency of missense mutations between cases and controls ranged from approximately 1:1 in the SCN5A interdomain linker to infinity in the pore, transmembrane, and linker in KCNH2. These correspond to estimated predictive values ranging from 0% in the interdomain linker of SCN5A to 100% in the transmembrane/linker/pore regions of KCNH2. The estimated predictive value is also high in the linker, pore, transmembrane, and C terminus of KCNQ1 and the transmembrane/linker of SCN5A.

Conclusions: Distinguishing pathogenic mutations from rare variants is of critical importance in the interpretation of genetic testing in LQTS. Mutation type, mutation location, and ethnic-specific

Background: should be viewed as variants of uncertain significance and prompt further investigation to clarify the likelihood of disease causation. However, mutations in regions such as the transmembrane, linker, and pore of KCNQ1 and KCNH2 may be defined confidently as high-probability LQTS-causing mutations. These findings will have implications for other genetic disorders involving mutational analysis.

Figure 1. Topological Depiction of All Protein-Altering Mutations

White circles represent case mutations. Black circles depict rare (each observed only once) genetic variants observed among the > 1300 healthy volunteers while black squares depict the genetic variants/polymorphisms observed more than once among the controls. All 155 distinct mutations, both radical and missense types, are included. Shaded regions: (1) SAD (subunits assembly domain); (2) PAS; (3) PAC; (4) cNBD (cyclic nucleotide binding domain).

Figure 2. Mutation Rates Across Gene-Encoded Regions

Depicted are the relative frequencies of mutations of all types (2a) and missense only (2b) in cases compared with controls. Note the relatively higher rate of mutations of all types across most regions in cases than controls. Furthermore, in most regions, there is a relatively higher frequency of missense mutations in cases than controls. The exceptions are the N-terminus and inter-domain linker (IDL) regions of SCN5A. N = N-terminus. T/L/P = Transmembrane/Linker/Pore. C = C-terminus. IDL = Inter-domain linker. T/L = Transmembrane/Linker.

Figure 2. Mutation Rates Across Gene-Encoded Regions

Depicted are the relative frequencies of mutations of all types (2a) and missense only (2b) in cases compared with controls. Note the relatively higher rate of mutations of all types across most regions in cases than controls. Furthermore, in most regions, there is a relatively higher frequency of missense mutations in cases than controls. The exceptions are the N-terminus and inter-domain linker (IDL) regions of SCN5A. N = N-terminus. T/L/P = Transmembrane/Linker/Pore. C = C-terminus. IDL = Inter-domain linker. T/L = Transmembrane/Linker.

Figure 3. Estimated Predictive Values for KCNQ1

Estimated predictive values in clinically definite cases for a missense mutation occurring in each major structure-function domain in the KCNQ1-encoded Kv7.1 potassium channel. The 95% confidence intervals are provided in parentheses. White circles represent case mutations. Black circles depict rare genetic variants observed only once among healthy volunteers while black squares depict the genetic variants/polymorphisms observed more than once among the controls. Shaded region (1) represents the SAD (subunits assembly domain).

Figure 3. Estimated Predictive Values for KCNQ1

Estimated predictive values in clinically definite cases for a missense mutation occurring in each major structure-function domain in the KCNQ1-encoded Kv7.1 potassium channel. The 95% confidence intervals are provided in parentheses. White circles represent case mutations. Black circles depict rare genetic variants observed only once among healthy volunteers while black squares depict the genetic variants/polymorphisms observed more than once among the controls. Shaded region (1) represents the SAD (subunits assembly domain).

Figure 4. Estimated Predictive Values for KCNH2

Estimated predictive values in clinically definite cases for a missense mutation occurring in each major region in the KCNH2-encoded Kv11.1 potassium channel. The 95% confidence intervals are provided in parentheses. White circles represent case mutations. Black circles depict rare genetic variants observed only once among healthy volunteers while black squares depict the genetic variants/polymorphisms observed more than once among the controls. Shaded regions (2) and (3) are the PAS and PAC specialized regions, respectively, and shaded region (4) represents the specialized cNBD (cyclic nucleotide binding domain).

Figure 4. Estimated Predictive Values for KCNH2

Estimated predictive values in clinically definite cases for a missense mutation occurring in each major region in the KCNH2-encoded Kv11.1 potassium channel. The 95% confidence intervals are provided in parentheses. White circles represent case mutations. Black circles depict rare genetic variants observed only once among healthy volunteers while black squares depict the genetic variants/polymorphisms observed more than once among the controls. Shaded regions (2) and (3) are the PAS and PAC specialized regions, respectively, and shaded region (4) represents the specialized cNBD (cyclic nucleotide binding domain).

Figure 5. Estimated Predictive Values for SCN5A

Estimated predictive values in clinically definite cases for a missense mutation occurring in each major region in the SCN5A-encoded Nav1.5 sodium channel. The 95% confidence intervals are provided in parentheses. White circles represent case mutations. Black circles depict rare genetic variants observed only once among healthy volunteers while black squares depict the genetic variants/polymorphisms observed more than once among the controls.