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Molecular and Functional Characterization of Novel
Glycerol-3-Phosphate Dehydrogenase 1–Like Gene (GPD1-L)
Mutations in Sudden Infant Death Syndrome
David W. Van Norstrand, BS;* Carmen R. Valdivia, MD;* David J. Tester, BS;
Kazuo Ueda, MD, PhD; Barry London, MD, PhD;
Jonathan C. Makielski, MD; Michael J. Ackerman, MD, PhD
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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. (Circulation.
2007;116:000-000.)
Key Words: arrhythmia 䡲 death, sudden 䡲 genetics 䡲 ion channels 䡲 pediatrics
A
n estimated 500 to 1000 people in the United States die
of sudden cardiac arrest every day. Most of these deaths
occur in elderly individuals as a result of ischemic heart
disease. However, each year, several thousand people ⬍40
years of age die suddenly, representing a disproportionate
loss to the community and a number of lost life-years that
rival ischemic heart disease–precipitated sudden deaths.1,2
For two thirds of the cases, a medicolegal examination is able
to determine the cause of death, commonly attributing it to
entities like hypertrophic cardiomyopathy, arrhythmogenic
right ventricular cardiomyopathy, coronary artery anomalies,
Clinical Perspective p ●●●
ruptured aortic aneurysms, or aortic valve stenosis.3–5 However, recent studies have indicated that as many as one third
of sudden deaths in the young remain unexplained after a
complete autopsy; such cases are labeled autopsy-negative
sudden unexplained death (SUD).3,4,6,7 In addition, 60% to
Received March 22, 2007; accepted August 31, 2007.
From the Department of Molecular Pharmacology and Experimental Therapeutics (D.W.V., D.J.T., M.J.A.), the Department of Medicine/Division of
Cardiovascular Diseases (M.J.A.), and the Department of Pediatric and Adolescent Medicine/Division of Pediatric Cardiology (M.J.A.), Mayo Clinic,
Rochester, Minn; the Departments of Medicine and Physiology (C.R.V., K.U., J.C.M.), University of Wisconsin, Madison; and the Cardiovascular
Institute, University of Pittsburgh, Pittsburgh, Pa (B.L.).
The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Child
Health and Development, the National Heart, Lung, and Blood Institute, or the National Institutes of Health.
*Dr Van Norstrand and Dr Valdivia contributed equally to this work.
Correspondence to Michael J. Ackerman, MD, PhD, Mayo Clinic Windland Smith Rice Sudden Death Genomics Laboratory, Guggenheim 501, Mayo
Clinic, Rochester, MN 55905. E-mail [email protected]
© 2007 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIRCULATIONAHA.107.704627
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2
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November 13, 2007
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80% of sudden deaths in children ⬍1 year of age are reported
as autopsy-negative sudden infant death syndrome (SIDS).8,9
Underlying molecular aberrations in cardiac ion channels
implicated in heritable arrhythmia syndromes such as
long-QT syndrome (LQTS), catecholaminergic polymorphic
ventricular tachycardia, and Brugada syndrome (BrS) account
for a significant proportion of SUD,10 –14 as well as for 10%
to 15% of SIDS.15,16 In addition, the Southeast Asian phenomenon of sudden unexplained nocturnal death syndrome,
the most common cause of natural death in young Asians, has
been shown recently to be phenotypically, genotypically, and
functionally the same disorder as BrS.17 “Gain-of-function”
mutations in the SCN5A-encoded sodium channel are associated with LQT3,18 –20 whereas “loss-of-function” mutations in
SCN5A result in type 1 BrS (BrS1). Given that SCN5A
mutations account for only 20% of BrS,21 the vast majority of
BrS remains pathogenetically unknown. Recently, a genomewide linkage study involving a large pedigree with BrS
phenotype revealed that the glycerol-3-phosphate dehydrogenase 1–like gene (GPD1-L) is associated with BrS and that
the BrS2-associated GPD1-L mutation yields a “secondary”
loss of function of the cardiac sodium channel.22
Given the recent implication of GPD1-L in the pathogenesis of BrS, a disease of known pathogenetic association with
sudden death, we hypothesized that mutations in GPD1-L
may confer risk for a lethal ventricular arrhythmia and thus be
responsible for some cases of autopsy-negative SUD and
SIDS. In this study, we sought to determine the spectrum and
prevalence of GPD1-L mutations in a cohort of 83 unrelated
SUD cases. The identification of a GPD1-L mutation in an
SUD victim ⬍1 year of age prompted us to explore our
population-based cohort of SIDS (n⫽221) to formally determine the spectrum and prevalence of GPD1-L mutations in
SIDS.
Methods
Medical Examiner/Coroner–Referred
Cases of SUD/SIDS
From September 1998 to January 2006, 83 medical examiner/
coroner cases of SUD (26 females, 57 males; average age,
14.6⫾10.7 years; range, 1 month to 48 years) from 57 medical
examiner offices were referred to the Mayo Clinic Windland Smith
Rice Sudden Death Genomics Laboratory for a cardiac channel
molecular autopsy. Seven of these decedents were ⬍1 year of age
and would be classified technically as SIDS. By definition, to be
accepted as a case of SUD, the death had to be sudden, unexpected,
and unexplained after the conclusion of a comprehensive medicolegal autopsy. Decedents with a premortem diagnosis of a cardiac
channelopathy either in himself or herself or in a family member
were excluded from this study.
This study was approved by the Mayo Foundation Institutional
Review Board. Although informed consent is waived for investigations involving decedents, written informed consent from the decedent’s parents or appropriate next of kin was obtained.
Population-Based Cohort of SIDS
Between January 1996 and December 2000, 221 cases from a
population-based cohort of unexplained infant deaths (84 females,
137 males; average age, 3⫾2 months; range, 3 days to 12 months)
were submitted for postmortem genetic testing. SIDS was defined as
for SUD, with the additional requirement of occurring before 1 year
of age. Infants whose death was due to asphyxia or specific disease
were excluded.
The present study was approved by the Mayo Foundation Institutional Review Board as an anonymous study. As such, only limited
medical information was available, including sex, ethnicity, and age
at death. Time of day, medication use, and position at death were not
available. By definition, the infant’s medical history and family
history were negative.
GPD1-L Mutational Analysis
DNA was extracted from autopsy blood with the Puregene DNA
Isolation Kit (Gentra, Minneapolis, Minn) or from frozen necropsy
tissue with the Qiagen DNeasy Tissue Kit (Qiagen, Inc, Valencia,
Calif). The entire coding region (8 translated exons) of GPD1-L was
examined with polymerase chain reaction, denaturing highperformance liquid chromatography, and direct DNA sequencing as
previously described.23 Primer sequences, polymerase chain reaction
conditions, and denaturing high-performance liquid chromatography
conditions are available on request.
To be considered a possible SUD/SIDS-predisposing mutation,
the genetic variant had to (1) be a nonsynonymous variant (synonymous single-nucleotide polymorphisms were excluded from consideration), (2) involve a highly conserved residue, (3) be absent
among 600 reference alleles from 200 healthy white and 100 healthy
black control subjects, and (4) have resulted in a functionally altered,
proarrhythmic cellular phenotype. Control genomic DNA was obtained from the Human Genetic Cell Repository sponsored by the
National Institute of General Medical Sciences and the Coriell
Institute for Medical Research (Camden, NJ). None of these control
alleles were obtained from infants.
Plasmid Constructions of Conventional
Mammalian Expression Vectors and
Bicistronic Adenovirus Shuttle Vectors
The full-length coding sequence of GPD1-L (GenBank accession
No. BC028726) was subcloned into pIRES2EGFP (Clontech Laboratories, Palo Alto, Calif) to generate pGPD1-L–IRES2EGFP. For
transduction into primary cardiac myocytes, the bicistronic adenovirus shuttle vector of GPD1-L and green fluorescent protein (GFP)
was generated. GPD1-L cDNA was subcloned into an entry vector of
pENTR1A-IRES2EGFP, which was created by incorporation of the
multicloning sites, an internal ribosome entry site, and GFP coding
region into pENTR1A (Invitrogen, Carlsbad, Calif). A recombination reaction between pAd/CMV/V5-DEST (Invitrogen) generated
pAdGPD1-L–IRES2EGFP. The E83K–, I124V–, and R273C–
GPD1-L mutants were incorporated into the wild-type (WT) vector
by site-directed mutagenesis. All clones were sequenced to confirm
integrity.
Transfection Into HEK Cells and
Neonatal Cardiomyocytes
WT–GPD1-L and GPD1-L mutant constructs in pIRES2EGFP were
transfected into HEK cells, along with the SCN5A-expressing vector
(GenBank accession No. AB158469) using FuGENE6 reagent according to the manufacturer’s procedure. Neonatal ventricular myocytes were isolated from the hearts of 1- to 2-day-old mice through
collagenase digestion (Worthington Biochemical Corp, Freehold,
NJ) in calcium- and magnesium-free Hanks’ balanced salt solution
(pH 7.4). The isolated cells were washed 3 times with PBS and
plated in medium 199 (Gibco-BRL, Gaithersburg, Md) supplemented with (in mmol/L) 5 creatine, 2 L-carnitine, and 5 taurine and
0.2% BSA at a field density of 10 000 cells/cm2 on 35-mm culture
dishes precoated with laminin (Sigma, St Louis, Mo). After 1 hour,
the media was changed to remove the nonadherent cells and then
infected with adenovirus shuttle vectors of the WT–GPD1-L and
E83K–GPD1-L mutant.
Electrophysiological Measurements
Macroscopic sodium currents were measured 48 hours after transfection with the standard whole-cell patch clamp method at room
Van Norstrand et al
Table.
GPD1-L Mutation and SIDS
3
Summary of Kinetic Parameters for SCN5A Alone, With WT GPD1-L and With Mutant GPD1-L
Peak INa
Activation
Inactivation
pA/pF
n
V1/2, mV
k
n
V1/2, mV
k
n
SCN5A⫹WT–GPD1-L
⫺355⫾43
14
⫺41⫾1
4.4⫾0.2
11
⫺73⫾1
5.0⫾0.1
8
SCN5A⫹E83K–GPD1-L
⫺148⫾29*
9
⫺41⫾2
4.4⫾0.3
9
⫺74⫾1
5.1⫾0.3
7
SCN5A⫹I124V–GPD1-L
⫺153⫾36*
6
⫺38⫾1
4.5⫾0.3
5
⫺75⫾2
5.0⫾0.2
5
SCN5A⫹R273C–GPD1-L
⫺137⫾27*
6
⫺33⫾2*
5.0⫾0.4
5
⫺74⫾3
5.3⫾0.5
5
SCN5A
⫺319⫾55
11
⫺41⫾1
4.5⫾0.3
7
⫺77⫾2
5.6⫾0.3
7
Samples
The fitted kinetic parameters and INa density from the experiments (n) were averaged and are reported as mean⫾SEM. pIRES2EGFP, WT–GPD1-L,
and GPD1-L mutant were transiently expressed in a mammalian cell line, along with SCN5A. The fit parameters were obtained from fitting the
individual experiments with the Boltzmann fits. All parameters were analyzed by 1-way ANOVA across pIRES2EGFP and WT–, E83K–, I124V–, and
R273C–GPD1-L.
*P⬍0.01 vs WT–GPD1-L.
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temperature (22°C to 24°C). The extracellular solution contained the
following: 140 mmol/L NaCl, 4 mmol/L KCl, 1.8 mmol/L CaCl,
0.75 mmol/L MgCl, and 5 mmol/L Hepes and was adjusted to pH 7.4
with NaOH. The pipette (intracellular) solution contained the following: 120 mmol/L CsF, 20 mmol/L CsCl, 2 mmol/L EGTA, and
5 mmol/L Hepes and was adjusted to pH 7.4 with CsOH. Electrodes
used were manufactured from borosilicate glass using a puller (P-87,
Sutter Instrument Co, Novato, Calif) and were heat polished with a
microforge (MF-83, Narishige, Tokyo, Japan). The electrode resistances ranged from 1 to 2 mol/L⍀. Cells were mounted on an
inverted microscope (Nikon Instruments Inc, Melville, NY) in a
Faraday cage and were perfused continuously with the bath solution.
Voltage clamp data were generated with an Axopatch 200B amplifier
with series-resistant compensation and pClamp software (9.2 Axon
Instruments, Foster City, Calif). Membrane current data were digitalized at 100 kHz, low-pass filtered at 5 kHz, and then normalized
to membrane capacitance.
Activation was measured by clamp steps of ⫺120 to 70 mV in
10-mV increments from a holding potential of ⫺140 mV and fitted
to the Boltzmann function GNa⫽[1⫹exp(V1/2⫺V)/k]⫺1, where V1/2
and k are the midpoint and slope factor, respectively, and GNa⫽
INa (norm)/(V⫺Vrev), where Vrev is the reversal potential and V is the
membrane potential. Steady-state inactivation was measured using a
two-step protocol with a holding potential of ⫺140 mV. The first
step was a 1-second conditioning pulse from ⫺150 to 0 mV in
10-mV increments. The second step to 0 mV recorded the available
current after the conditioning pulse. The line represents a fit to the
Boltzmann function: INa⫽INa-max[1⫹exp(Vc⫺V1/2)/k]⫺1, where Vc is
the membrane potential. The numbers and fit parameters are given in
the Table. An indication of adequate voltage control was the graded
response of the peak current-voltage relationship.
Statistical Analysis
All data points are shown as the mean value, and the bars represent
the SEM. Determinations of statistical significance were performed
with Student t test for comparisons of 2 means or with ANOVA for
comparisons of multiple means. A value of P⬍0.05 was considered
statistically significant. With exact binomial confidence intervals
used for an allele frequency, absence of the variants of interest in at
least 600 reference alleles indicates with a 95% confidence interval
that the true allelic frequency is ⬍1%, which satisfies the genetic
distinction between annotation as a mutation rather than a
polymorphism.
The authors had full access to and take full responsibility for the
integrity of the data. All authors have read and agree to the
manuscript as written.
Results
Mutation Analysis
Overall, mutational analysis of GPD1-L revealed a putative
pathogenic mutation in 1 of 83 cases (⬇1%) of SUD. This
GPD1-L mutation–positive decedent was among the 7 cases
who died before 1 year of age (ie, SIDS cases). Therefore, we
examined our population-based cohort of SIDS to further
investigate the spectrum and prevalence of GPD1-L mutations in SIDS. Here, 2 of 221 SIDS cases (⬇1%) hosted novel
missense mutations in GPD1-L.
Figure 1 details the molecular characterization of the 3
missense mutations. An abnormal denaturing highperformance liquid chromatography elution profile (Figure
1A) and subsequent DNA sequencing (Figure 1B) led to the
identification of a nucleotide substitution (307 G3 A) producing a lysine (K) for glutamic acid (E) substitution (E83K)
in a 3-month– old white boy. A nucleotide substitution
(nucleotide 430 A3 G) causing an I124V (isoleucine, I, to
valine, V) missense mutation was identified in a 5-week– old
white girl. Similarly, a nucleotide substitution (nucleotide
877 C3 T) yielded an R273C (arginine, R, to cysteine, C)
missense mutation in a 1-month– old white boy. All of these
missense mutations were absent in 600 reference alleles and
involved highly conserved residues across a variety of species
(Figure 1C). Figure 1D depicts the linear topology of
GPD1-L and location of the mutations.
Although the family history of the E83K victim included a
history of poorly documented cardiac arrhythmias, no diagnosis of any arrhythmia syndrome, including BrS, had been
assigned to any family members before the victim’s sudden
death. Although we assume on the basis of the family history
that this mutation may be heritable rather than sporadic, we
were unable to acquire DNA from the infant’s parents
because they declined participation. Because of the blinded,
anonymous nature of the SIDS population-based cohort, it
was not possible to obtain further information on the I124Vpositive and R273C-positive decedents and their families.
Functional Characterization of SIDS-Associated
Mutations in GPD1-L
HEK cells were transiently cotransfected with SCN5A and
GPD1-L. Representative current traces for SCN5A coexpressed with WT–, E83K–, I124V–, or R273C–GPD1-L are
shown in Figure 2A, with typical INa for a step depolarization
from ⫺140 mV to various potentials. Coexpression of WT–
GPD1-L caused robust peak INa density. However, consistent
with a BrS1-like loss-of-function phenotype, INa density was
reduced significantly with the GPD1-L mutants (Figure 2 and
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Figure 1. Identification of GPD1-L mutations in SIDS. Depicted are the denaturing high-performance liquid chromatography profiles
(A; normal, top, and abnormal, bottom) and DNA sequencing chromatograms for E83K–, I124V–, and R273C–GPD1-L (B). Note that
the reverse complement is shown for the I124V-positive sequence chromatogram. C, Sequence homologies are compared for all
mutations. D, Localization of the 3 SIDS-associated mutations on the linear topology of GPD1-L. Predicted protein domains are
also indicated.
the Table). Peak INa-voltage relationships (Figure 2B) show
an 8-mV positive shift in the midpoint of the activation
relationship for R273C but no differences for the other
mutations (the Table). Inactivation kinetics were not affected
by the mutations (the Table). Unlike LQT3 gain-of-function
SCN5A mutations, increased late INa was absent.
When WT–GPD1-L and E83K–GPD1-L were transfected
into a stable SCN5A-expressing HEK293 cell line, similar
decreases in INa density were obtained (⫺1137⫾186 versus
⫺357⫾50 pA/pF, WT–GPD1-L [n⫽4] versus E83K–
GPD1-L [n⫽7], respectively; P⬍0.001). To assess the effects
in a more native myocardial cell environment, neonatal
cardiomyocytes were transduced for 48 hours with adenovirus containing the WT– or the E83K–GPD1-L-IRES2EGFP
recombinants. Only beating GFP-positive heart cells were
selected. Representative current traces (Figure 3A and 3B)
and summary data from 8 cells from 2 infections (Figure 3C)
also show dramatically reduced INa density in native cardiomyocytes as a result of mutant E83K–GPD1-L.
Discussion
Sodium channel–interacting proteins (ChIPs) have been implicated recently in the pathogenesis of arrhythmias and
sudden death, including SIDS. In particular, mutations in 2
SCN5A-associated ChIPs, namely caveolin-3 (CAV3)24 and
the sodium channel ␤-4 subunit (SCN4B),25 represent novel
LQTS-susceptibility genes, and CAV3 mutations have been
reported in black SIDS cases.26 In addition, a mutation in
SCN5A that disrupts sodium channel association with the
cytoskeletal protein ankyrin-G results in a BrS-like loss of
function.27 Here, we detail 3 novel SIDS-associated mutations (E83K, I124V, and R273C) in a novel sodium ChIP,
GPD1-L. Whether these mutations are familial or sporadic is
indeterminate. In the case of E83K, a family history of
unspecified cardiac arrhythmias remains unexplored because
of nonparticipation. The anonymous nature of the SIDS
population-based cohort prevented further investigation.
In 2002, Weiss et al,28 using linkage analysis in a large
multigenerational family, discovered a novel BrS locus
(BrS2) on chromosome 3, distinct from the BrS1/LQT3
locus. Subsequent candidate gene analysis identified a novel
missense mutation, A280V, in GPD1-L that was shown to
cosegregate with the BrS phenotype.22,28 Although little is
known about GPD1-L, it is expressed in cardiac tissue and
colocalizes with the SCN5A-encoded cardiac sodium channel
at the plasma membrane.22
Van Norstrand et al
GPD1-L Mutation and SIDS
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Figure 2. Mutants–GPD1-L decreases INa in heterologous cells. Whole-cell INa traces were elicited by step depolarization of 24-ms
duration to ⫺20 mV from a holding potential of ⫺140 mV and normalized to cell capacitance. A, WT–GPD1-L and mutants–GPD1-L
were coexpressed transiently with SCN5A in HEK293 cell (P⬍0.01). B, The current-voltage relationship displays the summary data of
the current densities (n⫽5 to 9) fitted to the Boltzmann functions GNa⫽[1⫹exp(V1/2⫺V)/k]⫺1, where the V1/2 and k are the midpoint and
slope factor, respectively, and GNa⫽ INa (norm)/(V⫺Vrev), where Vrev is the reversal potential and V is the membrane potential.
Overexpression studies of the identified BrS2-associated
GPD1-L mutant with the SCN5A-encoded cardiac sodium
channel confirmed a BrS phenotype of reduction in peak
sodium current and normal inactivation kinetics, suggesting a
loss of function of the sodium channel. Surface membrane
expression of the sodium channel in the presence of mutant
GPD1-L also was shown to be reduced, suggesting that this
particular GPD1-L mutation disrupts trafficking and localization of the sodium channel to the membrane.22
Here, we provide molecular and functional evidence detailing GPD1-L as a novel susceptibility gene for SIDS. After
elucidation of the E83K–, I124V–, and R273C–GPD1-L
mutations in highly conserved residues that were absent in
600 reference alleles, a marked (⬇60%) reduction in peak INa
was demonstrated not only in heterologous expression systems for all 3 mutants but also in native cardiomyocytes
(E83K–GPD1-L). The demonstration of surface membrane
colocalization with SCN5A raises the possibility of a direct
interaction with the sodium channel. Alternatively, mutant
GPD1-L may exert its effects via a number of possible
indirect mechanisms. For instance, GPD1-L may interact
with any of a number of known sodium ChIPs such as the
cytoskeletal protein ankyrin-G, scaffolding proteins such as
the syntrophins, the caveolae-associated caveolin-3, or sodium channel degradation machinery proteins such as
Nedd4 –2.24,29 In addition, GPD1-L may affect protein kinase
A– dependent phosphorylation of the channel, which blocks
retention signals in the endoplasmic reticulum.30,31 Third,
oxidation state has been shown to play a role in ion channel
function and the arrhythmogenesis of atrial fibrillation.32,33
Thus, modulation via NAD⫹/NADH levels within the cell
may contribute to what is already an increasingly complex
picture of cardiac sodium channel regulation.
A functionally altered “SCN5A-centric” view of SIDS
pathogenesis is beginning to emerge for the subset of SIDS
cases stemming from heritable arrhythmia syndromes. Perturbed SCN5A channels resulting from primary mutations or
rare genetic variants have now been implicated as a pathogenic mechanism for as much as 6.5% of white SIDS
cases,15,16 a surprisingly high number considering that SCN5A
mutations account for only 5% to 10% of LQTS.15 Additionally, the common sodium channel polymorphism, S1103Y,
previously associated with increased risk of arrhythmia and
sudden cardiac death in blacks,34,35 has been demonstrated to
confer increased risk of SIDS in black infants.36 Moreover,
functional perturbations in the cardiac sodium channel via
mutations in the sodium ChIP caveolin-3 have been identified
in 6% of black SIDS cases.26
Although these SIDS-associated mutations have been reported most commonly as gain-of-function LQT3-like mutations, evidence increasingly suggests that loss-of-function
mutations in SCN5A also can result in sudden death–
predisposing clinical phenotypes, including BrS and sick
sinus syndrome. Here, we report the discovery of novel
mutations in the sodium ChIP GPD1-L in 3 SIDS victims.
The 3 mutations in GPD1-L precipitate a marked loss of
function with respect to the peak sodium current akin to that
described as the mechanism for primary BrS-causing mutations in the cardiac sodium channel. Given the functional data
for these mutations and that reported by London et al,22 we
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Figure 3. E83K–GPD1-L decreases INa in native cardiac myocytes. Whole-cell INa traces were elicited by step depolarization of 24-ms
duration to ⫺20 mV from a holding potential of ⫺140 mV and normalized to cell capacitance. Adenoviral recombinants of (A) WT- and
(B) E83K-GPD1-L-IRES2EGFP were used to transduce neonatal cardiomyocytes for 24 hours (P⬍0.01). C, Summary data of the
sodium current densities. *Significant decrease in sodium current density vs WT–GPD1-L (P⬍0.01).
hypothesize that these mutations conferred the principal
substrate for a lethal ventricular arrhythmia during infancy.
Furthermore, in addition to BrS, loss-of-function mutations in
SCN5A can result in sick sinus syndrome and bradycardia,
providing an alternative arrhythmogenic mechanism.37–39
Presently, it appears that ⬇5% to 10% of SIDS is precipitated by disruption of the sodium channel macromolecular
complex via mutations in either its pore-forming ␣ subunit
(SCN5A) or in 2 of its ChIPs, caveolin-3 and now GPD1-L.
Genes encoding other ChIPs warrant further scrutiny as novel
candidate genes for unexplained sudden death during infancy.
Acknowledgments
We gratefully acknowledge the medical examiners, coroners, and
forensic pathologists from across the United States for their referrals
of SUD and SIDS cases. We also thank Jing Wang (University of
Wisconsin) for technical assistance in the myocyte experiments.
Sources of Funding
Dr Ackerman’s research program is supported by the Mayo Clinic
Windland Smith Rice Comprehensive Sudden Cardiac Death Program, the CJ Foundation for SIDS, the Dr Scholl Foundation, the
Hannah Wernke Memorial Foundation, the American Heart Association (Established Investigator Award), and the National Institutes of
Health (HD42569). The project described was supported by National
Institute of Child Health and Development Grant No. R01HD042569
(Dr Ackerman) and National Heart, Lung, and Blood Institute Grant
No. R01HL71092 (Dr Makielski).
Disclosures
None.
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CLINICAL PERSPECTIVE
Sudden infant death syndrome (SIDS) claims ⬎2000 seemingly healthy infants each year in the United States and is
considered a multifactorial syndrome, with both environmental and genetic factors converging on the vulnerable infant
during the first year of life. Among the sources underlying this vulnerability, cardiac channelopathies, particularly
congenital long-QT syndrome, are believed to contribute to ⬇5% to 10% of SIDS. Perturbations in the cardiac sodium
channel represent the most common channelopathic cause of SIDS. Although “gain-of-function” mutations in the
SCN5A-encoded pore-forming ␣ subunit cause type 3 long-QT syndrome, loss-of-function mutations cause another
potentially lethal channelopathy known as Brugada syndrome. Beyond this ␣ subunit, the sodium channel is increasingly
appreciated as a macromolecular complex. For this reason, sodium channel–interacting proteins represent viable candidates
in the pathogenesis of SIDS. Here, we detail the discovery of the new Brugada syndrome–susceptibility gene, GPD1-L, in
a population-based cohort of SIDS. Three novel missense mutations in GPD1-L were discovered and are shown to cause
a marked reduction in the cardiac sodium current not only in a standard in vitro heterologous expression system but also
in native cardiocytes. The molecular/cellular phenotype was consistent with a loss-of-function sodium channel disease like
Brugada syndrome, providing probable cause and manner (ventricular fibrillation secondary to a Brugada syndrome–like
mechanism) for ⬇1% of this SIDS cohort.
Molecular and Functional Characterization of Novel Glycerol-3-Phosphate Dehydrogenase
1 −Like Gene (GPD1-L) Mutations in Sudden Infant Death Syndrome
David W. Van Norstrand, Carmen R. Valdivia, David J. Tester, Kazuo Ueda, Barry London,
Jonathan C. Makielski and Michael J. Ackerman
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Circulation. published online October 29, 2007;
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