Molecular and functional characterization of novel glycerol-3-phosphate dehydrogenase 1 like gene (GPD1-L) mutations in sudden infant death syndrome

Abstract

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 approximately 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.

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.

Figure 2

Mutants–GPD1-L decreases I_Na in heterologous cells. Whole-cell I_Na 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 G_Na=[1+exp(V_1/2−V)/k]⁻¹, where the V_1/2 and k are the midpoint and slope factor, respectively, and G_Na=I_{Na (norm)}/(V−V_rev), where V_rev is the reversal potential and V is the membrane potential.

Figure 3

E83K–GPD1-L decreases I_Na in native cardiac myocytes. Whole-cell I_Na 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).