Autosomal recessive intellectual disability caused by compound heterozygous variants of the EEF1D gene in a Chinese family

Abstract

Background

Intellectual disability is a prevalent neurodevelopmental disorder, with the majority of affected children exhibiting global developmental delay before the age of 5 years. In recent years, certain children have been found to carry homozygous variations of the EEF1D gene, leading to autosomal recessive intellectual disability. However, the pathogenicity of compound heterozygous variations in this gene remains largely unknown.

Methods

Trio whole-exome sequencing and copy number variation sequencing were done for the genetic etiological diagnosis of a 3-year and 11-month-old Chinese boy who presented with brachycephaly, severe to profound global developmental delay, and hypotonia in the lower limbs.

Results

In this case, compound heterozygous variants of the EEF1D gene were found in the child through trio whole-exome sequencing; one was a splice variant (NM_032378.6:c.1905+1G>A) inherited from his father, and the other was a nonsense variant (NM_032378.6:c.676C>T) inherited from his mother. The nonsense variant leads to the production of a premature termination (p.Gln226*). These variations have the ability to explain the clinical phenotypes of the child.

Conclusions

Our study expands the variation spectrum and provides compelling evidence for EEF1D as a candidate gene for autosomal recessive intellectual disability. However, due to the deficient number of reported cases, researchers need to further study EEF1D and supplement the clinical phenotypes and treatment measures.

BACKGROUND

Intellectual disability (ID) is a common disease in childhood that may result in physical and social dysfunction, with an incidence rate of approximately 1%–3% (Global Research on Developmental Disabilities Collaborators, 2018). Most children with ID are clinically identified before the age of 5 due to global developmental delay (GDD). The clinical etiological diagnosis of children with GDD/ID is challenging, given the heterogeneity of clinical phenotypes and the complex nature of its etiology (Srivastava & Schwartz, 2014). However, with the rapid development of gene testing technology, an increasing number of studies have found that genetic factors play an important role in the pathogenesis of GDD/ID in children (Gilissen et al., 2014; Wright et al., 2018). Currently, a total of 2615 genes associated with GDD/ID have been found (https://www.disgenet.org/). However, there are still new variations related to GDD/ID that need to be identified through high-throughput sequencing technology.

The EEF1D gene consists of 10 exons that encode a protein subunit of the type I translation elongation factor. Four protein isoforms can be produced by alternative splicing; a long isoform (eEF1BδL) composed of 647 amino acids, and three short isoforms (eEF1Bδ) composed of 281, 257, and 262 amino acids, respectively. The long isoform is associated with transcriptional activity (Hou et al., 2021), whereas the three short isoforms are necessary for maintaining the activity of the translation elongation factor eEF1A (Cristiano, 2022; Kaitsuka & Matsushita, 2015). The main difference between eEF1BδL and eEF1Bδ is the N-terminus of the long isoform, is the nuclear localization signal (NLS), which contains 367 amino acids, which is the functional domain for the EEF1D protein. Due to the NLS, eEF1BδL localizes to both the nucleus and cytoplasm and is highly expressed in the brain and testis, playing a crucial role in normal cerebral development (Kaitsuka et al., 2018). Conversely, the three short isoforms are widely expressed in the cytoplasm without tissue specificity (Kaitsuka et al., 2011). The complex formed by eEF1Bα, eEF1Bδ, and Valyl-tRNA synthetase converts inactive eEF1A-GDP to the active form, eEF1A-GTP; this is essential in promoting protein translation (Bondarchuk et al., 2022). Many studies have demonstrated that genetic variations leading to abnormal protein translations may impact cerebral development and contribute to GDD/ID (Hickman et al., 2022; Malone & Kaczmarek, 2022).

In this study, we identified a Chinese child with GDD who carried a compound heterozygous variant in the EEF1D gene (NM_032378.6:c.1905+1G>A, NM_032378.6:c.676C>T) by trio whole-exome sequencing (trio-WES) combined with copy number variation sequencing (CNV-seq). These variants were found to be causative of the child's phenotypes. To the best of our knowledge, this is the 11th reported case of GDD/ID associated with EEF1D variations and the first case reported in China.

METHODS Ethical compliance

This study was conducted in accordance with the approval of the ethics committee at the Third Affiliated Hospital of Zhengzhou University (2020, Medical Ethics No. 50). In addition, the necessity of the examination was explained to the patient's guardians, and we obtained the guardians' consent.

Trio whole-exome sequencing

Genomic DNA was extracted from blood samples collected from the patient and his parents. The samples were sequenced using the IDT xGen Exome Research Panel capture library, followed by Illumina NovaSeq 6000 sequencing technology. The average sequencing depth reached 150×, Q30 > 90%. The next-generation sequencing (NGS) raw fastq data were aligned to the human reference genome (GRCh38/hg38) using the Burrows–Wheeler Aligner. Our called SNV data set was annotated by ANNOVAR. In addition, the data set was filtered to retain only high-quality rare SNVs with a probably damaging effect. We examined SNVs in coding or exon–intron junctions with a minor allele frequency (MAF) ≤0.01 in gnomAD, ExAC, and other public databases. Variants that fit the genetic patterns of the disease and deleterious mutations predicted by multiple software programs were screened out. The detected pathogenic variants associated with the clinical phenotype of the child were verified by Sanger sequencing.

Copy number variation sequencing

Using the TruSeq Library Building Kit, sequencing libraries were created after genomic DNA was fragmented. Using high-throughput sequencing technology, the libraries were sequenced (Illumina, San Diego, CA, USA). Using the Burrows–Wheeler technique, all sequences were aligned to the human reference genome hg38. The Database of Genomic Variants (http://dgv.tcag.ca) was used to select all putative CNVs found in WES data or CNV-seq. Public databases (Decipher, ClinVar, ClinGen, ISCA, and dbVar) and literature reviews were used to annotate the CNVs, and pathogenicity screening was carried out using OMIM, DECIPHER, Orphanet, and other databases. There were no structural variations or CNVs identified that could account for the patient's phenotype.

CASE PRESENTATION Patient and clinical presentation

The patient is a 3-year and 11-month-old boy who is the second child in a nonconsanguineous family. His birth history and maternal prenatal history were both normal. He has an 8-year-old sister who is developing regularly.

At the age of 1 year and 1 month old, the patient exhibited GDD with the inability to sit independently or grasp objects spontaneously. He also had hypotonia in the lower limbs. Additionally, he displayed brachycephaly (Figure 1a). The results of the Gesell developmental diagnostic scale at 1 year and 1 month showed significant developmental delays in various areas. The developmental quotient (DQ) for adaptive performance was 38.5, indicating severe delay. The language area DQ was also 38.5, indicating severe delay. The social behavior DQ was 37.7, indicating severe delay. The gross motor DQ was 44.6, indicating moderate delay. The fine motor DQ was 43.1, indicating moderate delay. After regular rehabilitation training in our hospital for 3 months, the patient did not show significant improvement in any aspect of his abilities. During this time, we excluded environmental factors and possible metabolic factors that may cause GDD/ID. Meanwhile, cerebral MRI revealed a slight thinning of the corpus callosum but did not show any abnormal signals that could explain the clinical features of the child (Figure 1b). After excluding the above three common causes that may be related to the occurrence of GDD/ID, we still failed to identify the patient's etiology, so we proceeded with trio-WES and CNV-seq to further investigate.

FIGURE 1. Craniofacial phenotypes and cranial MRI of proband. (a) He had brachycephaly. (b) The cerebral MRI showed a slight thinning of the corpus callosum but did not reveal any abnormal signals that could explain the phenotype of the patient.

At the age of 3 years and 11 month old, the boy continues to exhibit delayed developmental milestones. He raised his head at 6 months, sat independently at the age 2 years old, and walked at the age of 3 years old. Moreover, he currently has severe to profound ID. The results of the Gesell developmental diagnostic scale, conducted at 3 years and 11 month old, also indicate significant developmental delays across various domains. His DQ for adaptive performance is 30.7, indicating severe delay. Fine motor skills have a DQ of 28.2, indicating severe delay. His social behavior has a DQ of 29.7, indicating severe delay. In terms of gross motor skills, his DQ is 41.6, indicating moderate delay. In the language area, his DQ is 20.4, indicating profound delay. The clinical characteristics of this case and all previously reported cases are summarized in Table 1.

TABLE 1 Clinical characteristics of children with EEF1D variations.

Clinical features	Our study	Averdunk et al. (2023)	Ugur Iseri et al. (2019)	Reuter et al. (2017)
Age	3 years and 11 months	5, 6, 10, 22 years	12, 8, 4 years	NA
Sex	Male	4 females	3 females	1 female, 2 males
Variation	c.1905+1G>A, p.?; c.676C>T, p.Gln226*	c.1905+1G>A, p.?; c.1780T>C, p.Trp594Arg	C.948G>A, p.Trp316*	C.69del, p.Glu24Serfs*26
Term labor	+	NA	+	NA
Brachycephaly	+	−	−	−
Microcephaly	−	+	+	+
Hypertelorism	−	NA	+	NA
Strabismus	−	+	NA	NA
Nystagmus	−	+	NA	NA
Micrognathia	−	NA	+	NA
Heterochromia iris	−	NA	NA	+
Optic atrophy	−	3 children	NA	NA
Abnormality of retinal pigmentation	−	1 child	NA	NA
Global developmental delay	+	+	+	+
Intellectual developmental delay	+	+	+	+
Age of walking alone	3 years	−	−	NA
No language expression	+	+	+	1 child was reported
Seizure	−	1 child	+	1 child was not presence
Short stature	−	+	NA	+
Hypomyotonia	+	Before 1 year	+	−
Muscle spasm	−	After 1 year	NA	NA
Active patellar tendon reflex	−	NA	+	NA
Cerebral MRI	Corpus callosum is slightly thin	Hypoplastic corpus callosum, abnormal myelination. 2 children with arachnoid cyst	2 children with thin corpus callosum	NA
Treatment	Physical therapy, occupational therapy, speech therapy, conductive education, sensory integration	NA	NA	NA
Outcome	Survive	1 child died of recurrent aspiration pneumonia and pulmonary infection	Survive	Survive

Note: NA is not available, “+” is presence, “−” is not presence.

Genetic analysis

Finally, trio-WES found genetic variations that explained the phenotype of the child, and CNV-seq did not detect copy number variations that may explain the occurrence of GDD. Trio-WES detection revealed that the proband carried a compound heterozygous variant of EEF1D on chromosome 8. The specific variant, NM_032378. 6: c.1905+1G>A is a splicing variation that was inherited from his father and was documented in the ClinVar database. Splicing software predicted this variation to be pathogenic, with a dbscSNV_ADA_SCORE of 1, and a dbscSNV_RF_SCORE of 0.938. The frequency of this variation in the population was found to be 0.000003989 in the Genome Aggregation Database (gnomAD), but it was not found in the Exome Aggregation Consortium (ExAC) database or the 1000 Genomes database. The patient also inherited a nonsense variant, NM_032378.6: c.676C>T (p.Gln226*), from his mother, which is predicted to result in a premature termination. This variant was not documented in the ClinVar database, and its occurrence frequency was not found in the ExAC, gnomAD, or 1000 Genomes databases (Figure 2a,b). The 226th amino acid and the whole protein encoded by the EEF1D gene have excellent homology in mammals (Figure 2c). The c.676C>T (p.Gln226*) variation disrupts the N-terminal region of eEF1BδL, whereas the c.1905+1G>A variation disrupts the guanine exchange factor (GEF) domain of eEF1Bδ.

FIGURE 2. The identification of EEF1D variants in our proband. (a) The pedigree of the family. The affected proband was highlighted by a filled symbol with an arrow. (b) The Sanger sequencing for this family. The compound heterozygous variants were identified, and the variants were shown in the red boxes. (c) The EEF1D protein was conserved in multiple species, including humans, chimpanzees, mice, cows, and cats. (d) The four protein isoforms were produced through selective splicing of the EEF1D gene mRNA sequence and their functional domain. DBD, DNA-binding domain; GEF, guanine nucleotide exchange factor; NLS, nuclear localization signal; PBD, protein-binding domain.

The four protein isoforms and their functional domains were produced through selective splicing of the EEF1D gene mRNA sequence and are shown in Figure 2d.

DISCUSSION AND CONCLUSIONS

The first case of ID associated with EEF1D gene was reported in 2017 (Reuter et al., 2017). Since then, a total of 10 cases have been reported, with a male-to-female ratio of 4:1 and ages ranging from 4 to 22 years old. The primary clinical features observed in these cases were GDD/ID (10/10), including severe ID (5/5), microcephaly (10/10), no language expression (8/8), inability to walk alone (7/7), epilepsy (4/5), short stature (7/7), hypotonia (3/7), and hypertonia (4/7). Some children had micrognathia (3/3), hypertelorism (3/3), strabismus/nystagmus (4/4), optic atrophy (3/3), abnormal iris color (1/1), abnormality of retinal pigmentation (1/1), feeding difficulties in infancy (2/2), and other symptoms. The frequency of seizures ranged from twice per year to once per month, and EEG abnormalities were observed (2/2). In the most severe case, the child died at the age of 5 years due to recurring aspiration pneumonia and lung infection. Cerebral MRI showed agenesis of the corpus callosum/thin corpus callosum (4/4) or arachnoid cyst (2/2) (Averdunk et al., 2023; Reuter et al., 2017; Ugur Iseri et al., 2019). The child described in this study shares severe to profound GDD, no language expression, and hypotonia with previously reported cases. The differences in this child are his ability to walk without the walker device and his brachycephaly. However, he has difficulty performing physical activities such as running, jumping, and walking up and down stairs.

To date, all reported pathogenic variations of EEF1D gene have been found in a homozygous state. Among these variations, there were 3 cases of nonsense variations (C.948G>A, p.Trp316*), 3 cases of frameshift variations (C.69del, p.Glu24Serfs*26), 2 cases of missense variations (c.1780T>C, p.Trp594Arg), and 2 cases of splice variations (c.1905+1G>A, p.?) (Averdunk et al., 2023; Reuter et al., 2017; Ugur Iseri et al., 2019). In this particular patient, a compound heterozygous variation (c.1905+1G>A, c.676C>T) was identified. Notably, the c.676C>T (p.Gln226*) variation is reported for the first time (Figure 2d). It should be mentioned that the c.676C>T (p.Gln226*) variation only affects the long isoform, resulting in the production of premature termination. The long isoform can promote the transcription of genes containing the heat shock response element (HSE). This further contributes to the heat shock response (HSR). It was also discovered that the N-terminal region of eEF1BδL is indispensable for transcriptional activity (Kaitsuka & Matsushita, 2015). Additionally, it can also promote the expression of heme oxygenase 1 (HO-1) and participate in the cellular stress response (Kaitsuka et al., 2011). HO-1 has a protective effect on the nervous vasculature and nervous system, and an imbalance of HO-1 homeostasis in vivo may lead to the impairment of cognitive function (Choi & Kim, 2022). eEF1BδL-knockout mice were found to have seizures, poor motor abilities, and abnormal cerebral development, which may be linked to the HSR and cellular stress (Kaitsuka et al., 2018). These phenotypes in the mice model closely resemble those observed in humans, further supporting the association between loss of eEF1BδL function and disease. The c.676C>T (p.Gln226*) variation, which produces a premature termination, primarily affects the N-terminal domain, impairs normal nervous system development, and contributes to the observed phenotypes.

The short eEF1Bδ isoform is a subunit of the translation elongation factor eEF1A complex that is responsible for the translocation of eEF1A on the ribosome, which is important in promoting protein translation (Bondarchuk et al., 2022). Any factors that cause damage to the protein translation process may be harmful to cerebral development (Park et al., 2022). Variations in eEF1A may result in clinical phenotypes such as ID, seizure, and autism (McLachlan et al., 2019), which closely resemble the phenotypes caused by EEF1D variations. It was confirmed that the C-terminal of eEF1Bδ contains crucial functional domains, including the GEF domain, which enables the eEF1Bδ trimer to maintain eEF1A activity and promote protein translation (Bondarchuk et al., 2022). The c.1905+1G>A variation may impair the GEF domain, which further disrupts the protein translation process and results in impaired cerebral function and clinical phenotypes. It is worth noting that, in comparison to individuals with homozygous c.1905+1G>A variation, the phenotype of our patient was differing. While both exhibit GDD/ID and hypotonia, our patient presents with brachycephaly and has the ability to walk independently. However, our patient does not have feeding difficulties, recurrent aspiration, respiratory tract infections, or spasticity in the lower limbs (Averdunk et al., 2023).

The World BioBank has highlighted that the first 1000 days after fertilization are critical for child development (Jamison et al., 2018). In this report, we also emphasize the significance of early diagnosis of GDD/ID, especially in infants and young children. Genetic testing can allow for a clear diagnosis, guide treatment, and determine prognosis, thereby preventing the birth of children with the same condition within the family. There have been limited reports of ID related to EEF1D gene variations. More studies on the EEF1D gene are still needed, which can provide more clinical cases and functional experiments that can be used to find the basis for clinical diagnoses and treatments. A precise diagnosis serves as the foundation of precision medicine, and we believe that precision medicine will soon address the majority of hereditary diseases, alleviating the burden on both society and families.

AUTHOR CONTRIBUTIONS

Dengna Zhu conceived and supervised the experiments and revised the draft. Jiamei Zhang, Hongxing Liu drafted the draft. Mingmei Wang and Yiran Xu collected the clinical data. Yang Fan analyzed the data of trio-WES and CNV-seq. All authors reviewed the results and approved the final manuscript.

ACKNOWLEDGMENTS

We are particularly grateful to the patient's parents for granting permission to publish this clinical report and also thank Cipher Gene for their sequencing knowledge support. This work was supported by the Joint Open Research Fund of Henan Key Laboratory of Child Brain Injury and Henan Pediatric Clinical Research Center (KFKT2021102).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

The data sets used in this study are available from the corresponding author when required.

ETHICS STATEMENT

This study was approved by the Ethics Committee of the Third Affiliated Hospital of Zhengzhou University (2020, Medical Ethics No. 50). The informed consent of the child's family was obtained simultaneously.