Vatta, Matteo

Background: Genes encoding sodium channel interacting proteins (ChIPs) have been identified as novel long QT syndrome (LQTS)-susceptibility genes. Approximately 25% of patients with LQTS remain genotype negative. ChIPs comprising the sodium channel macromolecular complex represent attractive candidates for disease susceptibility. Previously, in human jejunal circular smooth muscle, we demonstrated that syntrophin γ2 regulates sodium channel gating by a PDZ-domain interaction involving the C-terminus. In the heart, syntrophin α is more highly expressed than γ2, therefore, syntrophin α was evaluated as a possible LQTS-susceptibility ChIP. Methods: Comprehensive open reading frame/splice mutational analysis of syntrophin α was performed using PCR, DHPLC, and direct DNA sequencing on a cohort of 50 unrelated patients with LQT1–10 negative/phenotype positive LQTS (34 females; average age at diagnosis 26 ± 16 years; average QTc 531 ± 60.4 ms; QTc range 480 –759 ms). Mutations were engineered by site-directed mutagenesis and transiently expressed in HEK293 cells containing the stably-expressed SCN5A- encoded sodium channel α subunit (hNaV1.5). Results: Analysis of syntrophin α, encoded by SNTA1 , revealed 3 missense mutations in 2 patients (4%). A female diagnosed with LQTS at 13 years (QTc, 480 ms) with family history of autopsy-negative unexplained sudden death, was compound heterozygous for P74L/A257G while a male, diagnosed with LQTS at 18 years following syncopal episodes (QTc, 529 ms), hosted the A390V mutation. These mutations were absent in 600 reference alleles and A257G and A390V involved highly conserved residues. Functional studies revealed a marked leftward shift in sodium current activation for A257G and a significant rightward shift in channel inactivation for A390V-SNTA1. Both kinetic perturbations precipitated a ``gain-of-function” increase in the sodium channel window current. Conclusion: We provide the sentinel report of SNTA1 as a novel LQTS-susceptibility gene with a putative pathogenic mechanism involving a secondary ``gain-of-function” in the NaV1.5 sodium channel. Thus, CAV3-LQTS (LQT9), SCN4B-LQTS (LQT10), and now SNTA1-LQTS represent functional homologues with LQT3-like perturbations.

Background— Sudden infant death syndrome (SIDS) is a leading cause of death during the first 6 months after birth. About 5% to 10% of SIDS may stem from cardiac channelopathies such as long-QT syndrome. We recently implicated mutations in α1-syntrophin (SNTA1) as a novel cause of long-QT syndrome, whereby mutant SNTA1 released inhibition of associated neuronal nitric oxide synthase by the plasma membrane Ca-ATPase PMCA4b, causing increased peak and late sodium current ( I Na ) via S-nitrosylation of the cardiac sodium channel. This study determined the prevalence and functional properties of SIDS-associated SNTA1 mutations. Methods and Results— Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing of SNTA1 ’s open reading frame, 6 rare (absent in 800 reference alleles) missense mutations (G54R, P56S, T262P, S287R, T372M, and G460S) were identified in 8 (≈3%) of 292 SIDS cases. These mutations were engineered using polymerase chain reaction–based overlap extension and were coexpressed heterologously with SCN5A, neuronal nitric oxide synthase, and PMCA4b in HEK293 cells. I Na was recorded using the whole-cell method. A significant 1.4- to 1.5-fold increase in peak I Na and 2.3- to 2.7-fold increase in late I Na compared with controls was evident for S287R-, T372M-, and G460S-SNTA1 and was reversed by a neuronal nitric oxide synthase inhibitor. These 3 mutations also caused a significant depolarizing shift in channel inactivation, thereby increasing the overlap of the activation and inactivation curves to increase window current. Conclusions— Abnormal biophysical phenotypes implicate mutations in SNTA1 as a novel pathogenic mechanism for the subset of channelopathic SIDS. Functional studies are essential to distinguish pathogenic perturbations in channel interacting proteins such as α1-syntrophin from similarly rare but innocuous ones.

Mutations in 11 genes that encode ion channels or their associated proteins cause inherited long QT syndrome (LQTS) and account for ≈75–80% of cases (LQT1–11). Direct sequencing of SNTA1 , the gene encoding α1-syntrophin, was performed in a cohort of LQTS patients that were negative for mutations in the 11 known LQTS-susceptibility genes. A missense mutation (A390V-SNTA1) was found in a patient with recurrent syncope and markedly prolonged QT interval (QTc, 530 ms). SNTA1 links neuronal nitric oxide synthase (nNOS) to the nNOS inhibitor plasma membrane Ca-ATPase subtype 4b (PMCA4b); SNTA1 also is known to associate with the cardiac sodium channel SCN5A. By using a GST-fusion protein of the C terminus of SCN5A, we showed that WT-SNTA1 interacted with SCN5A, nNOS, and PMCA4b. In contrast, A390V-SNTA1 selectively disrupted association of PMCA4b with this complex and increased direct nitrosylation of SCN5A. A390V-SNTA1 expressed with SCN5A, nNOS, and PMCA4b in heterologous cells increased peak and late sodium current compared with WT-SNTA1, and the increase was partially inhibited by NOS blockers. Expression of A390V-SNTA1 in cardiac myocytes also increased late sodium current. We conclude that the A390V mutation disrupted binding with PMCA4b, released inhibition of nNOS, caused S -nitrosylation of SCN5A, and was associated with increased late sodium current, which is the characteristic biophysical dysfunction for sodium-channel-mediated LQTS (LQT3). These results establish an SNTA1-based nNOS complex attached to SCN5A as a key regulator of sodium current and suggest that SNTA1 be considered a rare LQTS-susceptibility gene.