Homozygous EEF1A2 mutation causes dilated cardiomyopathy, failure to thrive, global developmental delay, epilepsy and early death

Abstract

Eukaryotic elongation factor 1A (EEF1A), is encoded by two distinct isoforms, EEF1A1 and EEF1A2; whereas EEF1A1 is expressed almost ubiquitously, EEF1A2 expression is limited such that it is only detectable in skeletal muscle, heart, brain and spinal cord. Currently, the role of EEF1A2 in normal cardiac development and function is unclear. There have been several reports linking de novo dominant EEF1A2 mutations to neurological issues in humans. We report a pair of siblings carrying a homozygous missense mutation p.P333L in EEF1A2 who exhibited global developmental delay, failure to thrive, dilated cardiomyopathy and epilepsy, ultimately leading to death in early childhood. A third sibling also died of a similar presentation, but DNA was unavailable to confirm the mutation. Functional genomic analysis was performed in S. cerevisiae and zebrafish. In S. cerevisiae, there was no evidence for a dominant-negative effect. Previously identified putative de novo mutations failed to complement yeast strains lacking the EEF1A ortholog showing a major growth defect. In contrast, the introduction of the mutation seen in our family led to a milder growth defect. To evaluate its function in zebrafish, we knocked down eef1a2 expression using translation blocking and splice-site interfering morpholinos. EEF1A2-deficient zebrafish had skeletal muscle weakness, cardiac failure and small heads. Human EEF1A2 wild-type mRNA successfully rescued the morphant phenotype, but mutant RNA did not. Overall, EEF1A2 appears to be critical for normal heart function in humans, and its deficiency results in clinical abnormalities in neurologic function as well as in skeletal and cardiac muscle defects.

Figure 1

Genetic findings in a family affected by a EEF1A2 mutation. (A) Pedigree of the family carrying the EEF1A2 mutation. (B, C) Weight records for proband (II:1)(B) and affected sibling (II:2)(C). (D) Sanger sequencing chromatograms for the proband (a), affected sibling (b), mother (c) and father (d). (E) Conservation of proline residue at amino acid 333 of EEF1A2 across various species. (F–H) Molecular modeling showing the conformational change induced in EEF1A2 by a p.P333L substitution. P333 and L333 are shown in stick with purple and red colors, respectively. The wild-type is colored in green, and the mutant is colored in blue. The potential binding partner is colored in grey.

Figure 2

Effect of the expression of TEF2 variants mimicking human EEF1A2 mutations in Saccharomyces cerevisiae. (A) Western blot showing cellular levels of TEF2-V5 and its variants expressed under the GPD promoter in p416GPD plasmid. (B) tef2 single mutant show a rapamycin sensitivity phenotype that is complemented by the expression of V5-TEF2 or P331L variant, but not G70S variant. (C) In the plasmid shuffling experiments shown above, tef1tef2 strain is transformed with both pTEF2 TRP1 and p416GPD URA3 expressing either TEF2-V5 or its variants G70S and P331L. Plating on FAA medium select for the colonies that have lost pTEF2 TRP1 and only have p416GPD derived plasmids. The figure shows that missense mutation p.G70S lead to the loss of function of Tef2 (EEF1A2) while p.P331L variant still remains some functionality.

Figure 3

Morpholino-based knockdown of eef1a2 in developing zebrafish. (A) eef1a2 qRT-PCR and standard RT-PCR demonstrating an 8-fold reduction of mRNA in MO-injected (5 ng) zebrafish embryos. (B) Western blot analysis of Eef1a2 protein levels in WT and MO-injected (5 ng) zebrafish embryos using an anti-EEF1A2 antibody. Membranes were restained with tubulin to demonstrate adequate loading of protein in lanes. (C) Dorsal curvature in developing zebrafish after injection of 5 ng of MO ranges from mild to severe. (D) Dose-dependent response to MO injection at 5 ng, 7.5 ng, and 10 ng. (E) Light microscopy of living zebrafish hearts and heads. (F) Differences in heart rate between control and MO-injected zebrafish embryos at 2 dpf. (G) Rescue experiments in MO-injected zebrafish embryos showing the ability of the WT human EEF1A2 mRNA to salvage WT phenotype with fewer affected zebrafish while embryos injected with p.P333L human EEF1A2 mRNA were not rescued and developed a phenotype similar to that of the MO-injected zebrafish.