EEF1D encodes the delta subunit of the eukaryotic translation elongation factor 1 complex, a molecular machine responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome during the elongation stage of protein synthesis. The protein functions as a guanine nucleotide exchange factor, facilitating the release of eEF1A from the ribosome so that translation can proceed. But EEF1D is a versatile gene, producing at least four distinct protein isoforms in humans. Three short isoforms — eEF1Bδ1, eEF1Bδ2, and eEF1Bδ3, comprising 281, 262, and 257 amino acids respectively — work as translational elongation factors in the cytoplasm. A fourth, much longer isoform called eEF1BδL, containing 647 amino acids, carries a nuclear localization signal at its N-terminus that transports it into the nucleus, where it serves as a transcription factor regulating the heat shock response. The dramatic length disparity between these polypeptides arises from a large coding exon that is excluded from the short isoform transcripts but included in the long isoform transcripts.

The clinical story of the two Iranian siblings is severe. The girl was born at term after an uneventful pregnancy with normal birth weight and length, but within her first year she developed microcephaly, with a head circumference below the 3rd percentile — severe microcephaly. At four months of age she began having generalized tonic seizures that recurred through infancy and early childhood. From birth she showed hypotonia, poor feeding, and suckling dysfunction requiring nasogastric feeding that was later transitioned to a gastrostomy. At one year she was nonverbal, unable to sit, crawl, or walk, and unresponsive to simple commands. She has remained wheelchair-bound with persistent axial hypotonia and global developmental delay. Ophthalmological examination revealed convergent strabismus and frequent blinking, while EEG demonstrated epileptiform discharges despite no active clinical seizures in recent years. Metabolic screening, including plasma amino acids, urine organic acids, and acylcarnitine profiling, was entirely normal.

Her younger brother followed a strikingly parallel course. Born at 37 weeks of gestation, he too developed progressive microcephaly within his first year, with a head circumference below the 3rd percentile. At 56 days of age he developed perioral cyanosis that prompted NICU admission, where clinicians identified laryngomalacia along with mild peripheral pulmonary stenosis, a small patent ductus arteriosus, and mild tricuspid regurgitation — none hemodynamically significant. He experienced neonatal-onset seizures, treated with a combination of levetiracetam, topiramate, and carbamazepine, occurring about twice a year until age four before gradually subsiding; he is now off antiepileptic medication. Unlike his sister, his EEG remained normal throughout follow-up. His MRI, however, revealed demyelination, white matter loss, and cerebellar atrophy. A comprehensive metabolic evaluation covering more than 70 metabolic disorders was negative. He remains non-ambulatory and non-verbal with failure to thrive, weighing just 7 kilograms at 19 months.

Whole exome sequencing of the family uncovered the molecular culprit: a biallelic splice junction loss variant, NM_001130053.5:c.1489-26_1493del, located at the splice junction of intron 7 of EEF1D. The variant was absent from gnomAD and Iranome population databases, and Sanger sequencing confirmed that both parents were heterozygous carriers while both children were homozygous — a classic autosomal recessive pattern in a consanguineous family. Under ACMG/AMP guidelines, the variant was classified as likely pathogenic based on PVS1, PP4, and PM2 criteria. Splicing prediction tools converged on a damaging mechanism: Human Splicing Finder predicted significant changes in auxiliary splicing sequences, including a minus-five alteration in ESE/ESS motif ratios, activation of a cryptic acceptor site, and likely splicing disruption through loss of the wild-type acceptor site. SpliceAI and Pangolin predictions strongly supported loss of the acceptor splice site with high confidence, and a new donor splice site was also predicted.

The new report joins a small but growing list. EEF1D was first proposed as a candidate gene for autosomal recessive intellectual disability in 2017, when Reuter and colleagues identified a homozygous frameshift variant, p.Glu24Serfs*26, in a Syrian family with three affected children presenting with intellectual disability, microcephaly, and short stature. A Turkish family followed, carrying a nonsense variant, p.Trp316Ter, associated with intellectual disability and developmental delay. Both of these variants specifically target the large exon present only in the transcripts of the eEF1BδL isoform, meaning they are unlikely to affect the shorter cytoplasmic isoforms. In 2023, Averdunk and colleagues identified two distinct homozygous truncating variants in separate consanguineous families from Turkey and Oman — c.1780T>C, p.Trp594Arg, and c.1905+1G>A — associated with severe intellectual disability, microcephaly, spasticity, and failure to thrive, with additional features including optic atrophy, poor feeding, and recurrent aspiration pneumonia. In 2024, Zhang and colleagues reported a Chinese patient with compound heterozygous variants. With the variant described in the present study, a total of six EEF1D variants have now been reported.

What makes the new variant mechanistically distinctive is its position. While the Reuter and Ugur Iseri variants are loss-of-function changes affecting the alternatively spliced exon of eEF1BδL, the Iranian variant is the first homozygous splice junction variant at intron 7, predicted to cause a frameshift deletion and severe neurodevelopmental disorder. The disease mechanism remains speculative, but mouse models offer compelling clues. Researchers who initially identified the eEF1BδL isoform created mice with a targeted deletion of the large EEF1D exon, effectively knocking out eEF1BδL. These animals appeared to develop normally until fear conditioning tests, after which they exhibited reduced brain weight, degeneration in the hippocampus and midbrain, thinner cortical layers, and in some cases audiogenic seizures. Earlier work had shown that targeted suppression of eEF1BδL impacts cellular survival during heat stress. Under heat stress, expression of the shorter eEF1Bδ isoforms decreases while levels of the longer eEF1BδL isoform increase, suggesting its specific involvement in the cellular stress response and implying a critical role in brain function, particularly in response to external stimuli.

Cross-species evidence underscores just how fundamental this gene is. Data from the DISCO single-cell omics database show that EEF1D is widely expressed in most or all regions of the human brain, spanning astrocytes, oligodendrocytes, excitatory neurons, inhibitory neurons, and microglia. In C. elegans, the orthologue eef-1B.1 is broadly expressed in neurons of the central nervous system, and its downregulation through RNA interference causes embryonic lethality, indicating the gene is crucial for development. Single-cell RNA sequencing in Drosophila melanogaster shows the fly orthologue broadly expressed in neurons and glial cells of the central nervous system, and mouse single-cell data reveal Eef1d expression throughout the body, including neuronal cells of the brain. Allen Institute gene expression data show the mouse orthologue is mainly expressed in the cerebellum, hippocampus, and midbrain — regions tied to motor coordination, cognition, and learning. Moreover, a loss-of-function allele generated by the International Mouse Phenotyping Consortium causes severe deficits in embryonic development culminating in preweaning lethality when homozygous, while heterozygous mutations are associated with multisystem developmental conditions.

Phenotypically, the new report both confirms and extends the clinical spectrum. All previously reported individuals, along with the two Iranian siblings, presented with developmental delay or intellectual disability — 13 of 13 affected cases across studies. Twelve of 13 showed microcephaly, 12 of 13 lacked language expression, and 8 of 13 experienced seizures. The Iranian patients also exhibited short stature and muscular hypotonia. While none of the individuals from the present study or from the Turkish and Averdunk studies could sit, stand, or walk independently, one individual in the Reuter study achieved assisted walking by age 15. Features such as optic atrophy, poor feeding, and recurrent aspiration pneumonia were exclusively reported by Averdunk and colleagues. The present study identified unique features for the first time, including intermittent fever, laryngomalacia attributable to hypotonia, frequent blinking, and axial hypotonia. Notably, the c.1905+1G>A variant has now recurred in two unrelated Omani families, raising the possibility of a founder effect in the Omani population that would require haplotype analysis to confirm.

The findings carry practical weight for families and clinicians alike. Variants in several genes encoding subunits of the translation elongation factor 1 complex have now been linked to intellectual disability and neurodevelopmental disorders, demonstrating that the elongation stage of translation is essential for neurodevelopment — and that genes once considered housekeeping molecules can harbor non-canonical functions, as shown for translation-related genes such as YARS1. The researchers suggest that potential alternative functions of EEF1D should be explored as possible contributors to disease pathogenesis, and that additional functional studies are needed to elucidate how mutant EEF1D affects both the long and short protein isoforms. For now, the report demonstrates that whole exome sequencing can be a valuable diagnostic tool for affected individuals with a suspected genetic neurodevelopmental disorder, and it shows that disease-causing variants in EEF1D produce a diverse range of neurodevelopmental presentations, including autosomal recessive intellectual disability. How EEF1D dysfunction disrupts brain development — through impaired translation, a compromised heat shock response, or something not yet imagined — remains the central question for future work.

Subject of Research: A novel biallelic EEF1D splice junction loss variant identified in Iranian siblings with a severe neurodevelopmental disorder

Article Title: EEF1D‐Associated Neurodevelopmental Disorders: Evidence From Iranian Siblings With a Biallelic Splice Junction Loss Variant

Article References: Ganji, M., Kaki, A., Mohammadi, M. F., Behroozi, J., Kahani, S. M., Rezaei, B., Fayazi, A., Mohammadi, P., Bereshneh, A. H., & Garshasbi, M. (2026). EEF1D ‐Associated Neurodevelopmental Disorders: Evidence From Iranian Siblings With a Biallelic Splice Junction Loss Variant. Molecular Genetics & Genomic Medicine, 14(10), Article e70287. https://doi.org/10.1002/mgg3.70287

Image Credits: AI Generated

DOI: 10.1002/mgg3.70287

Keywords: EEF1D, neurodevelopmental disorder, whole exome sequencing, splice junction variant, intellectual disability, microcephaly, autosomal recessive, eEF1BδL, heat shock response, translation elongation factor, consanguinity, NEDTCHAL