Compound heterozygous mutations P336L and I1660V in the human cardiac sodium channel associated with the Brugada syndrome

Abstract

Background: Loss-of-function mutations in SCN5A have been associated with the Brugada syndrome. We report the first Brugada syndrome family with compound heterozygous mutations in SCN5A. The proband inherited 1 mutation from each parent and transmitted 1 to each daughter.

Methods and results: The effects of the mutations on the function of the sodium channel were evaluated with heterologous expression in TSA201 cells, patch-clamp study, and confocal microscopy. Genetic analysis revealed that the proband carried 2 heterozygous missense mutations (P336L and I1660V) on separate alleles. He displayed a coved-type ST-segment elevation and a prolonged PR interval (280 ms). One daughter inherited P336L and exhibited a prolonged PR (210 ms). The other daughter inherited mutation I1660V and displayed a normal PR interval. Both daughters had a slightly elevated, upsloping ST-segment elevation. The parents had normal ECGs. Patch-clamp analysis showed that the P336L mutation reduced I(Na) by 85% relative to wild type. The I1660V mutation produced little measurable current, which was rescued by room temperature incubation for 48 hours. Sodium channel blockers also rescued the I1660V current, with mexiletine proving to be the most effective. Confocal immunofluorescence showed that I1660V channels conjugated to green fluorescent protein remained trapped in intracellular organelles.

Conclusions: Mutation P336L produced a reduction in cardiac I(Na), whereas I1660V abolished it. Only the proband carrying both mutations displayed the Brugada syndrome phenotype, whereas neither mutation alone produced the clinical phenotype. I1660V channels could be rescued pharmacologically and by incubation at room temperature. The present data highlight the role of compound heterozygosity in modulating the phenotypic expression and penetrance of Brugada syndrome.

Figure 1

Pedigree of the family (A) and 12-lead ECG recordings (B) of selected members of the Brugada syndrome family. Genetic analysis revealed that each parent carried a single but different heterozygous mutation. The father (I-1) carried a mutation that consisted of a substitution of the isoleucine for a valine at position 1660 (I1660V), whereas the mother (I-2) had a substitution of the proline at position 336 for a leucine (P336L). The proband in II-1 carried both heterozygous mutations P336L and I1660V, each inherited from a different parental allele (A), and displayed a significant ST-segment elevation on his ECG (B, II-1). In contrast, each daughter (III-1 and III-2) separately inherited only 1 mutation. Neither the parents of the proband nor the daughters exhibited a Brugada ECG. The paper speed of the ECG recordings was 25 mm/s.

Figure 2

Representative whole-cell current recordings for WT (A) and P336L mutant (B) in transfected TSA201 cells. Current recordings were obtained at test potentials between −100 and 0 mV in 5-mV increments from a holding potential of −120 mV. C, I-V relation for WT (n=11) and P336L (n=15) channels showing a reduction in current for P336L. D, Steady state–activation relation for WT and P336L. Chord conductance was determined with the ratio of current to the electromotive potential for the cells shown in C. Data were normalized and plotted against their test potential.

Figure 3

Representative steady state inactivation recordings for WT (A) and P336L (B) observed in response to the voltage-clamp protocol (top of figure). C, Steady state–inactivation relation for WT and P336L. Peak current was normalized to their respective maximum values and plotted against the conditioning potential.

Figure 4

Representative whole-cell current recordings for WT and I1660V mutant in transfected TSA201 cells (A). Current recordings were obtained at test potentials between −100 and 0 mV in 5-mV increments from a holding potential of −120 mV. Incubation of the cells at RT for 48 hours could rescue I1660V current but had no effect on WT currents (B). C, I-V relation for WT (n=6), I1660V (n=6), and RT-incubated I1660V (n=5) channels showing a rescue of current. D, Bar graph showing the magnitude of peak Na⁺ current for WT, I1660, and RT-incubated WT and I1660V channels.

Figure 5

Drug rescue of defective expression of mutant channels. A, Representative traces showing rescue of I1660V channels with mexiletine (300 μmol/L), quinidine (100 μmol/L), ranolazine (100 μmol/L), or ajmaline (100 μmol/L) treatment. Drugs were then washed out 30 minutes before the currents were recorded. B, I-V relation showing I1660V current rescued after incubation with the various drugs for 48 hours. C, Bar graph showing the magnitude of peak Na⁺ current for WT drug-rescued I1660V current. All incubations were done at 37°C.

Figure 6

Representative confocal XYZ scans showing localization of Na⁺ channels in TSA201 cells. WT channels conjugated to GFP marked staining in both the periphery and the center of the cell, which suggests that WT channels are manufactured in the cell center and trafficked to the cell membrane (A). In contrast, I1660V channels showed staining localized in the perinuclear region of the cell, which suggests that mutant channels are manufactured but remain trapped within the cell (B). These trapped I1660V channels can be rescued by RT incubation (C).