Loss-of-function mutations in the cardiac calcium channel underlie a new clinical entity characterized by ST-segment elevation, short QT intervals, and sudden cardiac death

Abstract

Background: Cardiac ion channelopathies are responsible for an ever-increasing number and diversity of familial cardiac arrhythmia syndromes. We describe a new clinical entity that consists of an ST-segment elevation in the right precordial ECG leads, a shorter-than-normal QT interval, and a history of sudden cardiac death.

Methods and results: Eighty-two consecutive probands with Brugada syndrome were screened for ion channel gene mutations with direct sequencing. Site-directed mutagenesis was performed, and CHO-K1 cells were cotransfected with cDNAs encoding wild-type or mutant CACNB2b (Ca(v beta2b)), CACNA2D1 (Ca(v alpha2delta1)), and CACNA1C tagged with enhanced yellow fluorescent protein (Ca(v)1.2). Whole-cell patch-clamp studies were performed after 48 to 72 hours. Three probands displaying ST-segment elevation and corrected QT intervals < or = 360 ms had mutations in genes encoding the cardiac L-type calcium channel. Corrected QT ranged from 330 to 370 ms among probands and clinically affected family members. Rate adaptation of QT interval was reduced. Quinidine normalized the QT interval and prevented stimulation-induced ventricular tachycardia. Genetic and heterologous expression studies revealed loss-of-function missense mutations in CACNA1C (A39V and G490R) and CACNB2 (S481L) encoding the alpha1- and beta2b-subunits of the L-type calcium channel. Confocal microscopy revealed a defect in trafficking of A39V Ca(v)1.2 channels but normal trafficking of channels containing G490R Ca(v)1.2 or S481L Ca(v beta2b)-subunits.

Conclusions: This is the first report of loss-of-function mutations in genes encoding the cardiac L-type calcium channel to be associated with a familial sudden cardiac death syndrome in which a Brugada syndrome phenotype is combined with shorter-than-normal QT intervals.

Figure 1

A, Twelve-lead ECG of patient 1 before and after ajmaline recorded with V₁ and V₂ displaced superiorly 2 intercostal spaces. B, Pedigree of family of patient 1 (III-6, arrow: proband). Arrows in ECGs depict prominent (type I) ST-segment elevation. C, Twelve-lead ECG of patient 2 before and after ajmaline (1 mg/kg) and at baseline with V₁ and V₂ displaced superiorly 2 intercostal spaces to unmask a type 1 ST-segment elevation. D, Twelve-lead ECG of patient 3 with V₁ through V₃ in normal position and displaced superiorly 2 intercostal spaces.

Figure 2

Heart rate dependence of QT interval of patient 2. Plotted are the QT_peak and QT_end (QT) intervals as a function of heart rate recorded during bicycle ergometry exercise test.

Figure 3

DNA sequence analysis. A, Heterozygous C1442T transition in exon 13 of CACNB2b predicting replacement of serine by leucine at position 481. B, Cartoon of Ca_vβ2b (cytoplasmic) showing the location of the mutation and the position of the β-subunit interaction domain (BID) segment. C, Left, Heterozygous C to T transition (arrow) at position 116 in exon 2 of CACNA1C allele in patient 2 predicting a substitution of a valine for alanine at position 39; Right, heterozygous A to G transition (arrow) at position 1468 in exon 10 of CACNA1C allele in patient 1 predicting replacement of glycine by arginine at position 490. D, Predicted topology of Ca_v1.2 showing the location of the mutations. Sschematic modified from Splawski et al, with permission from Elsevier. Copyright 2004. The loop between domains I and II contains a conserved motif named “AID” (α-subunit interaction domain) that binds to the β-subunit segment called BID.

Figure 4

Representative whole-cell Ca²⁺ currents recorded from CHO cells transfected with WT CACNA1C (A) or A39V (B) and G490R (C) mutant CACNA1C and S481L mutant CACNB2b (D). Currents were elicited with the pulse protocol illustrated in the inset above panel B. E, Current-voltage (I-V) relationship for WT (n=5), A39V (n=7), and G490R (n=10) Ca_v1.2 channels (exon 8A variant). F, I-V relationship for WT (n=10), A39V (n=12), and G490R (n=25) Ca_v1.2 channels (exon 8 variant). *P< 0.05 compared with G490R. #P< 0.05 compared with A39V. G, I-V relationship for WT (n=10), S481L (n37) Ca_vβ2b channels. *P< 0.05 compared with S481L. H through K, Representative confocal XYZ scans showing localization of EYFP-tagged Ca_v1.2 channels in CHO cells. H, Cell expressing WT Ca_v1.2 channels showed marked peripheral and cytoplasmic fluorescence. I, Cells expressing A39V Ca_v1.2 channels showed fluorescence localized in the perinuclear region of the cell. J, Cell expressing G490R Ca_v1.2 channels exhibit a fluorescence pattern similar to WT, which suggests that trafficking is not impaired. K, Cells expressing S481L Ca_vβ2b channels exhibit a fluorescence pattern similar to WT, which suggests that trafficking is not impaired.