David W. Van Norstrand

Molecular and Functional Characterization of Novel Glycerol-3-Phosphate Dehydrogenase 1–Like Gene (GPD1-L) Mutations in Sudden Infant Death Syndrome

Background— Autopsy-negative sudden unexplained death, including sudden infant death syndrome, can be caused by cardiac channelopathies such as Brugada syndrome (BrS). Type 1 BrS, caused by mutations in the SCN5A-encoded sodium channel, accounts for ≈20% of BrS. Recently, a novel mutation in the glycerol-3-phosphate dehydrogenase 1–like gene (GPD1-L) disrupted trafficking of SCN5A in a multigenerational family with BrS. We hypothesized that mutations in GPD1-L may be responsible for some cases of sudden unexplained death/sudden infant death syndrome. Methods and Results— Using denaturing high-performance liquid chromatography and direct DNA sequencing, we performed comprehensive open-reading frame/splice site mutational analysis of GPD1-L on genomic DNA extracted from necropsy tissue of 83 unrelated cases of sudden unexplained death (26 females, 57 males; average age, 14.6±10.7 years; range, 1 month to 48 years). A putative, sudden unexplained death–associated GPD1-L missense mutation, E83K, was discovered in a 3-month-old white boy. Further mutational analysis was then performed on genomic DNA derived from a population-based cohort of 221 anonymous cases of sudden infant death syndrome (84 females, 137 males; average age, 3±2 months; range, 3 days to 12 months), revealing 2 additional mutations, I124V and R273C, in a 5-week-old white girl and a 1-month-old white boy, respectively. All mutations occurred in highly conserved residues and were absent in 600 reference alleles. Compared with wild-type GPD1-L, GPD1-L mutations coexpressed with SCN5A in heterologous HEK cells produced a significantly reduced sodium current (P<0.01). Adenovirus-mediated gene transfer of the E83K–GPD1-L mutation into neonatal mouse myocytes markedly attenuated the sodium current (P<0.01). These decreases in current density are consistent with sodium channel loss-of-function diseases like BrS. Conclusions— The present study is the first to report mutations in GPD1-L as a pathogenic cause for a small subset of sudden infant death syndrome via a secondary loss-of-function mechanism whereby perturbations in GPD1-L precipitate a marked decrease in the peak sodium current and a potentially lethal BrS-like proarrhythmic substrate.

Sudden Infant Death Syndrome-Associated Mutations in the Sodium Channel Beta Subunits

BACKGROUND Approximately 10% of sudden infant death syndrome (SIDS) cases may stem from potentially lethal cardiac channelopathies, with approximately half of channelopathic SIDS involving the Na(V)1.5 cardiac sodium channel. Recently, Na(V) beta subunits have been implicated in various cardiac arrhythmias. Thus, the 4 genes encoding Na(V) beta subunits represent plausible candidate genes for SIDS. OBJECTIVE This study sought to determine the spectrum, prevalence, and functional consequences of sodium channel beta-subunit mutations in a SIDS cohort. METHODS In this institutional review board-approved study, mutational analysis of the 4 beta-subunit genes, SCN1B to 4B, was performed using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing of DNA derived from 292 SIDS cases. Engineered mutations were coexpressed with SCN5A in HEK 293 cells and were whole-cell patch clamped. One of the putative SIDS-associated mutations was similarly studied in adenovirally transduced adult rat ventricular myocytes. RESULTS Three rare (absent in 200 to 800 reference alleles) missense mutations (beta3-V36M, beta3-V54G, and beta4-S206L) were identified in 3 of 292 SIDS cases. Compared with SCN5A+beta3-WT, beta3-V36M significantly decreased peak I(Na) and increased late I(Na), whereas beta3-V54G resulted in a marked loss of function. beta4-S206L accentuated late I(Na) and positively shifted the midpoint of inactivation compared with SCN5A+beta4-WT. In native cardiomyocytes, beta4-S206L accentuated late I(Na) and increased the ventricular action potential duration compared with beta4-WT. CONCLUSION This study provides the first molecular and functional evidence to implicate the Na(V) beta subunits in SIDS pathogenesis. Altered Na(V)1.5 sodium channel function due to beta-subunit mutations may account for the molecular pathogenic mechanism underlying approximately 1% of SIDS cases.

Overrepresentation of the proarrhythmic, sudden death predisposing sodium channel polymorphism S1103Y in a population-based cohort of African-American sudden infant death syndrome.

BACKGROUND The S1103Y-SCN5A polymorphism has been implicated as a proarrhythmic, sudden death predisposing risk factor in African Americans, including one postmortem investigation of African-American infants with sudden infant death syndrome (SIDS). OBJECTIVE The purpose of this study was to assess whether the relatively African-American-specific common polymorphism S1103Y in the SCN5A-encoded cardiac sodium channel is overrepresented in SIDS among African Americans. METHODS Seventy-one cases from a population-based cohort of unexplained infant deaths among African Americans (37 females and 34 males, average age 3 +/- 2 months, age range birth to 11 months) were submitted to the Mayo Clinic Windland Smith Rice Sudden Death Genomics Laboratory for postmortem genetic testing. Polymerase chain reaction and a restriction digest assay were performed to genotype this cohort for S1103Y. RESULTS Targeted mutational analysis of exon 18 in SCN5A of the African-American SIDS cohort (n = 71) revealed the S1103Y polymorphism in 16 (22.5%) of 71 African-American cases of SIDS compared to 135 (11.6%) of 1,161 ostensibly healthy adult African Americans (P = .01). CONCLUSION This study provides an independent assessment of the prevalence of S1103Y-SCN5A among African-American infants with sudden, unexpected, unexplained death prior to their first birthday. Further scrutiny and quantification of the risk apparently associated with S1103Y appear warranted.

Connexin43 Mutation Causes Heterogeneous Gap Junction Loss and Sudden Infant Death

Background— An estimated 10% to 15% of sudden infant death syndrome (SIDS) cases may stem from channelopathy-mediated lethal arrhythmias. Loss of the GJA1-encoded gap junction channel protein connexin43 is known to underlie formation of lethal arrhythmias. GJA1 mutations have been associated with cardiac diseases, including atrial fibrillation. Therefore, GJA1 is a plausible candidate gene for premature sudden death. Methods and Results— GJA1 open reading frame mutational analysis was performed with polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing on DNA from 292 SIDS cases. Immunofluorescence and dual whole-cell patch-clamp studies were performed to determine the functionality of mutant gap junctions. Immunostaining for gap junction proteins was performed on SIDS-associated paraffin-embedded cardiac tissue. Two rare, novel missense mutations, E42K and S272P, were detected in 2 of 292 SIDS cases, a 2-month-old white boy and a 3-month-old white girl, respectively. Analysis of the E42K victims parental DNA demonstrated a de novo mutation. Both mutations involved highly conserved residues and were absent in >1000 ethnically matched reference alleles. Immunofluorescence demonstrated no trafficking abnormalities for either mutation, and S272P demonstrated wild-type junctional conductance. However, junctional conductance measurements for the E42K mutation demonstrated a loss of function not rescued by wild type. Moreover, the E42K victims cardiac tissue demonstrated a mosaic immunostaining pattern for connexin43 protein. Conclusions— This study provides the first molecular and functional evidence implicating a GJA1 mutation as a novel pathogenic substrate for SIDS. E42K-connexin43 demonstrated a trafficking-independent reduction in junctional coupling in vitro and a mosaic pattern of mutational DNA distribution in deceased cardiac tissue, suggesting a novel mechanism of connexin43-associated sudden death.

α1-Syntrophin Mutations Identified in Sudden Infant Death Syndrome Cause an Increase in Late Cardiac Sodium Current

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 &agr;1-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 (INa) 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 SNTA1s 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. INa was recorded using the whole-cell method. A significant 1.4- to 1.5-fold increase in peak INa and 2.3- to 2.7-fold increase in late INa 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 &agr;1-syntrophin from similarly rare but innocuous ones.

Genomic risk factors in sudden infant death syndrome

Sudden infant death syndrome (SIDS) is a major contributor to postneonatal infant death, and is the third leading cause of infant mortality in the USA. While public health efforts have reduced these deaths in recent years, the pathogenesis of SIDS remains unclear. Epidemiological data on SIDS-related deaths have suggested genetic factors, and many studies have attempted to identify SIDS-associated genes. This has resulted in a large body of literature implicating various genes and their encoded proteins and signaling pathways in numerous cohorts of various sizes and ethnicities. This review has undertaken a systematic evaluation of these studies, identifying the pathways that have been implicated in these studies, including central nervous system pathways, cardiac channelopathies, immune dysfunction, metabolism/energy pathways, and nicotine response. This review also explores how new genomic techniques will aid in advancing our knowledge of the genomic risk factors associated with SIDS, including SNPs and copy number variation. Last, this review explores how the current information can be applied to aid in our assessment of the at risk infant population.

Sudden infant death syndrome: Do ion channels play a role?

It has been more than 30 years since an association between sudden infant death syndrome (SIDS) and abnormal cardiac repolarization was first postulated.1,2 Enormous advances in our clinical understanding of heritable arrhythmia syndromes (aka “the cardiac channelopathies”), our scientific understanding of ion channels and how mutant ion channels impart their proarrhythmic phenotype to the patient, and our laboratory techniques, which include high-throughput mutational analysis, have given us the tools to begin to dissect a complex, multifactorial disease such as SIDS. Now, a clearer picture of how cardiac channelopathies can create the pathogenic substrate for sudden death during the first year of life as well as the prevalence of channelopathic SIDS among autopsy negative sudden infant deaths is emerging. This review highlights the substantial progress that has been made over the last decade, in particular highlighting the most recent findings that solidify the role that ion channels play in SIDS.

α1-Syntrophin Mutations Identified in Sudden Infant Death Syndrome Cause an Increase in Late Cardiac Sodium CurrentCLINICAL PERSPECTIVE

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 &agr;1-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 (INa) 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 SNTA1s 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. INa was recorded using the whole-cell method. A significant 1.4- to 1.5-fold increase in peak INa and 2.3- to 2.7-fold increase in late INa 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 &agr;1-syntrophin from similarly rare but innocuous ones.