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Long QT syndrome (LQTS) is a familial autosomal dominant disease characterized by prolongation of the QT interval on the surface ECG, syncope, torsade de pointes and sudden cardiac death in young patients. Heritable LQTS is classified into different types (LQTS 1-12), and each type is linked to mutations in a specific gene. LQTS3 is associated with gain-of-function mutations in the SCN5A gene; the gene encodes the voltage-dependent cardiac sodium channel a-subunit that allows a large inward depolarizing current during phase 0 of the cardiac action potential. We identified and functionally characterized the novel SCN5A c.4416-4418delAAC (p.N1472del) mutation associated with LQTS. This deletion was engineered into the recombinant human heart sodium channel (hH1) and coexpressed with the human sodium channel ß-1 subunit (hß1) in the tsA201 cell line. The functional properties of the p.N1472del mutation were then determined by whole-cell patch clamp recording. The mutant channel had a decreased current density and the potential for half-maximal activation (V1/2) was shifted to more positive potential compared with wildtype (WT) channel; the slope factor (k) was higher in SCN5A-p.N1472del than in the WT channel. The mutant also exhibited a +12 mV depolarizing shift in the V1/2 of steady-state of inactivation compared with the WT, whereas WT and SCN5A-p.N1472del had similar slope factors. Moreover, the mutant had a slower recovery from inactivation experiments than WT. Finally, we tested if the mutant exhibited the increased persistent sodium current that is typical of SCN5A mutations associated with LQTS3. Current levels were persistently higher in SCN5Ap. N1472del than in the WT channel. In conclusion, our results demonstrate that p.N1472del mutation causes all the biophysical defects commonly associated with LQT (increased persistent current and positive shift in the voltage-dependence of inactivation) and is responsible for other functional alterations (positive shift in the voltage dependence of activation and a slower recovery from inactivation) which can be related to additional clinical defects.

Long QT syndrome (LQTS) is an inherited arrhythmic disorder associated with QT interval prolongation on ECG, syncope, torsade de pointes and sudden cardiac death in young patients. Heritable LQTS is classified in 12 types depending on mutations in a specific gene. In most cases, the mutations involve the genes encoding a-subunits of repolarizing K+ channels (KCNQ1 and KCNH2). Prolongation of the QT interval and action potential duration is associated with the decrease of outward K+ current in cardiomyocytes. The screening for LQTS causing mutations in a family from South Italy revealed two mutations in two genes: c.G1748A (p.R583H) in KCNQ1 and c.G323A (p.C108Y) in KCNH2. Interestingly, all subjects carrying just one of these two mutations did not show the LQT phenotype, whereas the only two subjects carrying both were clinically affected (QTc > 530ms). To analyze the genotype-phenotype association and to shed light on the pathogenetic defects responsible for these LQTS cases we characterized the biophysical features of the two mutant channels. We generated the KCNQ1 and KCNH2 mutants by site-directed mutagenesis. Each mutant was transiently expressed in CHO cells and functionally characterized by whole-cell patch clamp recordings. The biophysical studies showed that in the homozygous condition, mutation c.G323A led to a nonfunctional KCNH2 channel, whereas, in the heterozygous condition, mutant KCNH2 had significantly reduced current density at positive potentials and a negative shift in the voltage dependence of activation compared to the wild type. Furthermore, mutant KCNQ1-p.R583H had significantly reduced tail current density compared to the wild-type channel, but no significant changes in activating current density and voltage-dependence of activation. In conclusion, our results demonstrate that mutation KCNH2-p.C108Y induced a dominant negative effect in the heterozygous condition. Moreover, both KCNH2-p.C108Y and KCNQ1-p.R583H had some biophysical defects that could be combined and lead to the LQTS phenotype, as observed in the pedigree of the family reported herein.

Channelopathies are diseases caused by deranged functioning of ion channel subunits or the proteins that regulate them. Long QT and Brugada syndrome are included in this group. In particular, long QT syndrome (LQTS) is a familial autosomal dominant disease characterized by prolongation of the QT interval on the surface ECG, syncope, torsade de pointes and sudden cardiac death in young patients. Each type of heritable LQTS (LQTS 1-12) is linked to mutations in a specific gene. Mutations occur more frequently in the cardiac ion channel coding genes (SCN5A, KCNH2,KCNQ1) and ancillary β-subunits (KCNE1 and KCNE2). Differently, BrS is an inherited cardiac disease characterized by ST segment elevation in the right precordial leads (V1 to V3), susceptibility to ventricular tachyarrhythmia and sudden cardiac death, typically during rest or sleep. BrS is inherited as an autosomal dominant trait and its prevalence in Caucasians is 5/1000. The disorder is linked to mutations in the SCN5A gene. Our project was designed to functionally characterize the novel mutations found in genes related to LQTS and BrS to better understand the pathogenesis of pathological phenotypes . To this aim, we firs amplified by PCR all coding exons, 5’ and 3’ UTR of the SCN5A, KCNQ1, KCNH2, KCNE1 and KCNE2 genes and analyzed them by dHPLC and automatic sequencing. The mutants were generated by QuickChange site-directed mutagenesis. Mutants were transiently transfected in mammalian cells for in vitro analysis. We characterized the LQT3 associated p.ΔN1472 mutation that we found in SCN5A gene. The electrophysiological studies demonstrated that the hH1 mutation had a shift in the voltage-dependence of inactivation, a positive shift in the voltage dependence of activation and a slower recovery from inactivation compared to WT channel. Moreover, the persistent current levels were much higher in SCN5A-p.ΔN1472 than in the WT channel. We also studied mutations KCNH2-p.C108Y and KCNQ1-p.R583H. Interestingly, only subjects carrying both mutations manifested severe LQTS. The biophysical studies showed that in the homozygous condition, KCNH2-p.C108Y, led to a non-functional KCNH2 channel, whereas, in the heterozygous condition, mutant KCNH2 had a significantly reduced current density and a negative shift in the voltage dependence of activation compared to the WT. Furthermore, mutant KCNQ1-p.R583H had a significantly reduced tail current density compared to the WT channel, but no significant changes in activating current density and in voltage-dependence of activation. In conclusion, we demonstrate that the SCN5A-p.ΔN1472 and KCNH2-p.C108Y mutants exhibit characteristic biophysical properties causing LQTS; whereas KCNQ1-p.R583H, in combination with KCNE1-WT, does not exhibit striking biophysical defects, but in combination with mutant KCNH2 it results in a more severe phenotype. Our results allow to better understand the pathogenesis of LQTS phenotype and to increase the knowledge of ion channel behavior in the pathological conditions.

Congenital long QT syndrome (LQTS) is associated with high genetic and allelic heterogeneity. In some cases, more than one genetic variant is identified in the same (compound heterozygosity) or different (digenic heterozygosity) genes, and subjects with multiple pathogenic mutations may have a more severe disease. Standard-of-care clinical genetic testing for this and other arrhythmia susceptibility syndromes improves the identification of complex genotypes. Therefore, it is important to distinguish between pathogenic mutations and benign rare variants. We identified four genetic variants (KCNQ1-p.R583H, KCNH2-p.C108Y, KCNH2-p.K897T, and KCNE1-p.G38S) in an LQTS family. On the basis of in silico analysis, clinical data from our family, and the evidence from previous studies, we analyzed two mutated channels, KCNQ1-p.R583H and KCNH2-p.C108Y, using the whole-cell patch clamp technique. We found that KCNQ1-p.R583H was not associated with a severe functional impairment, whereas KCNH2-p.C108Y, a novel variant, encoded a non-functional channel that exerts dominant-negative effects on the wild-type. Notably, the common variants KCNH2-p.K897T and KCNE1-p.G38S were previously reported to produce more severe phenotypes when combined with disease-causing alleles. Our results indicate that the novel KCNH2-C108Y variant can be a pathogenic LQTS mutation, whereas KCNQ1-p.R583H, KCNH2-p.K897T, and KCNE1-p.G38S could be LQTS modifiers.

Isolated familial pseudohyperkalemia is a dominant red cell trait characterized by cold-induced ‘passive leak’ of red cell potassium ions into plasma. The causative gene of this condition is ABCB6, which encodes an erythrocyte membrane ABC transporter protein bearing the Langereis blood group antigen system. In this study analyzing three new families, we report the first functional characterization of ABCB6 mutants, including the homozygous mutation V454A, heterozygous mutation R276W, and compound heterozygous mutations R276W and R723Q (in trans). All these mutations are annotated in public databases, suggesting that familial pseudohyperkalemia could be common in the general population. Indeed, we identified variant R276W in one of 327 random blood donors (0.3%). Four weeks’ storage of heterozygous R276W blood cells resulted in massive loss of potassium compared to that from healthy control red blood cells. Moreover, measurement of cation flux demonstrated greater loss of potassium or rubidium ions from HEK-293 cells expressing ABCB6 mutants than from cells expressing wild-type ABCB6. The R276W/R723Q mutations elicited greater cellular potassium ion efflux than did the other mutants tested. In conclusion, ABCB6 missense mutations in red blood cells from subjects with familial pseudohyperkalemia show elevated potassium ion efflux. The prevalence of such individuals in the blood donor population is moderate. The fact that storage of blood from these subjects leads to significantly increased levels of potassium in the plasma could have serious clinical implications for neonates and infants receiving large-volume transfusions of whole blood. Genetic tests for familial pseudohyperkalemia could be added to blood donor pre-screening. Further study of ABCB6 function and trafficking could be informative for the study of other pathologies of red blood cell hydration.