intellectual disability: Understanding the potential mechanism of phenotypic variation

Epilepsy & Behavior 105 (2020) 106955

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EEF1A2 mutations in epileptic encephalopathy/intellectual disability: Understanding the potential mechanism of phenotypic variation Kexin Long a, Hua Wang d,e, Zhanyi Song f, Xiaomeng Yin c,⁎, Yaqin Wang b,⁎⁎ a

Institute of Medical Sciences, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China Department of Health Management Centre, the Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, China Department of Integrated Traditional Chinese and Western Medicine, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China d Maternal and Child Health Hospital of Hunan Province, Changsha, Hunan 410008, China e Key Laboratory of Birth Defects Research and Prevention, Changsha, Hunan 410008, China f Med Department of Pediatric Neurology, Chenzhou No.1 People's Hospital (Children's Hospital), Chenzhou, Hunan 423000, China b c

a r t i c l e

i n f o

Article history: Received 12 November 2019 Revised 27 January 2020 Accepted 27 January 2020 Available online xxxx Keywords: Lennox–Gastaut syndrome Epileptic encephalopathy Intellectual disability EEF1A2 De novo mutation

a b s t r a c t EEF1A2 encodes protein elongation factor 1-alpha 2, which is involved in Guanosine triphosphate (GTP)dependent binding of aminoacyl-transfer RNA (tRNA) to the A-site of ribosomes during protein biosynthesis and is highly expressed in the central nervous system. De novo mutations in EEF1A2 have been identified in patients with extensive neurological deficits, including intractable epilepsy, globe developmental delay, and severe intellectual disability. However, the mechanism underlying phenotype variation is unknown. Using nextgeneration sequencing, we identified a novel and a recurrent de novo mutation, c.294CN A; p.(Phe98Leu) and c.208GN A; p.(Gly70Ser), in patients with Lennox–Gastaut syndrome. The further systematic analysis revealed that all EEF1A2 mutations were associated with epilepsy and intellectual disability, suggesting its critical role in neurodevelopment. Missense mutations with severe molecular alteration in the t-RNA binding sites or GTP hydrolysis domain were associated with early-onset severe epilepsy, indicating that the clinical expression was potentially determined by the location of mutations and alteration of molecular effects. This study highlights the potential genotype–phenotype relationship in EEF1A2 and facilitates the evaluation of the pathogenicity of EEF1A2 mutations in clinical practice. © 2020 Elsevier Inc. All rights reserved.

1. Introduction The EEF1A2 gene (OMIM# 602959) encodes eukaryotic translation elongation factor 1 (eEF1A)-alpha 2, a protein belongs to the translation factor related (TRAFAC) class GTPase superfamily, and plays a key role in protein synthesis by delivering aminoacyl-tRNAs to the ribosome in a GTP-dependent process [1]. Elongation factor eEF1A has also been reported to have numerous noncanonical properties, such as apoptosis suppression and cytoskeletal regulation [2,3]. Compared with the other isoform of eEF1A (eEF1A1), eEF1A2 is unique in being tissue-specific, which is mainly expressed in neurons and muscle [4]. Deficiency of eEF1A2 due to homozygous deletion of the promoter and first exon in mice (called wasted, wst) leads to vacuolar degeneration of the anterior horn neurons in the spinal cord and the motor nuclei in the brain stem.

⁎ Correspondence to: X. Yin, Department of Integrated Traditional Chinese and Western Medicine, Xiangya Hospital, 87 of Xiangya Road, Changsha, Hunan 410008, China. ⁎⁎ Correspondence to: Y. Wang, Department of Health Management Centre, the Third Xiangya Hospital, Central South University, 138 Tongzipo Road, Changsha, Hunan 410013, China. E-mail addresses: [email protected] (X. Yin), [email protected] (Y. Wang).

https://doi.org/10.1016/j.yebeh.2020.106955 1525-5050/© 2020 Elsevier Inc. All rights reserved.

Consequently, eEF1A2-depleted mice exhibited decreased severe neurodegeneration, muscle wasting, and death by 28 days [5]. In humans, there is also evidence linking de novo dominant EEF1A2 mutations to extensive neurological deficits, including intractable epilepsy, globe developmental delay, severe intellectual disability, hypotonia, and autistic behavior [4,6–17]. Mutation of EEF1A2 was initially identified in a patient with severe intellectual disability, accompany with early-onset epilepsy and autistic features [6]. However, later studies identified mutations in patients with a more severe form of epileptic encephalopathy, and those patients were found to have poor responses to drug therapies. In addition, homozygous missense mutation of EEF1A2 was recently detected in patients with dilated cardiomyopathy, failure to thrive, global developmental delay, epilepsy, and early death [15]. Therefore, it is unknown whether phenotype variation correlates with EEF1A2 mutation genotypes. In this study, in order to determine the genetic cause of two Chinese families with Lennox–Gastaut syndrome (LGS) and intellectual disability, we combined copy number variants analysis, whole exome sequencing (WES), and variant-interpretation guidelines and ultimately identified a novel de novo heterozygous mutation [c.294C N A; p. (Phe98Leu)] and a recurrent mutation [c.208G NA; p.(Gly70Ser)] in

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K. Long et al. / Epilepsy & Behavior 105 (2020) 106955

EEF1A2 gene. We further systematically reviewed all EEF1A2 mutations and analyzed their molecular heterogeneity, aiming to define the genotype–phenotype correlation and the role of EEF1A2 mutations in epileptic encephalopathy and intellectual disability.

were added to obtain the total damaging ranking scores, which were used for the subsequent analysis of the genotype–phenotype relationship. 3. Results

2. Materials and methods 3.1. Patient information 2.1. Standard protocol approvals and patient consents The study was approved by the ethical review board of Xiangya Hospital, Central South University, Changsha, China. In this study, 2 Chinese Han families with male patients presenting epileptic encephalopathy fulfilling the diagnosis of LGS were included. Epileptic seizures and epilepsy were diagnosed and classified according to the criteria of the Commission on Classification and Terminology of the International League Against Epilepsy (ILAE) [18–21]. The legal guardians of the participants provided written informed consent for genetic investigation. Patients and parental genomic deoxyribonucleic acid (DNA) were obtained from peripheral blood leukocytes according to standard procedures as previously described [22]. 2.2. Single nucleotide polymorphism (SNP) array analysis and whole exome sequencing For the patient of the two families, genome-wide copy number variations (CNVs) analysis was performed with Illumina HumanCytoSNP-12 BeadChip. Data from the images were analyzed using cnvPartition Plugin v3.1.6 for GenomeStudio (Illumina). Pathological or clinical significance CNVs were screened according to the following criteria: (1) associated with known epilepsy-associated syndromes, (2) overlapping with or interrupt known epilepsy genes or hotspots, (3) relatively large size (more than 1 Mb), (4) rare CNVs, and (5) de novo [23]. Whole exome sequencing was performed in patient–parent trio by using the SureSelectXT Human All Exon Kit (V5; Agilent Technologies, Santa Clara, CA) and sequenced on the Illumina HiSeq X system (Illumina, San Diego, CA) with 150 bp paired-end reads. Exome data processing, variant calling, and variant annotation were performed as previously described [22,24]. We then analyzed the variants under de novo, autosomal recessive inherited and X-linked inherited models. Autosomal dominant inherited variations were also considered. The candidate variants were further confirmed using Sanger resequencing in all available family members. 2.3. Variant-level evaluation of the damaging effect We obtained predictive scores and pathogenicity consequences of missense variants from the VarCards database [25], which contained 23 in silico missense algorithms, including SIFT [26,27], PolyPhen2_HDIV [28], PolyPhen2_HVAR [28], LRT [29], MutationTaster [30], MutationAssessor [31], FATHMM [25], PROVEAN [25], MetaSVM [32], MetaLR [32], VEST3 [33], M-CAP [34], CADD [35], GERP++ [36], DANN [37], fathmm-MKL [38], Eigen [39], GenoCanyon [40], fitCons [41], PhyloP [42], PhastCons [43], SiPhy [44], and REVEL [45]. The proportion of algorithms predicted to be deleterious through those 23 tools was defined as damaging scores, which was sorted in ascending order. ReVe was a combination of the REVEL and VEST3 methods and consistently showed well performance in the evaluation of pathogenicity [46]. The predicted pathogenicity scores of ReVe were also directly downloaded from the VarCards database and sorted in the abovementioned ranking method. Grantham score was a prediction of the effect of substitutions between amino acids based on chemical properties and evolutionary sense [47]. SNAP2 was a neural networkbased classifier predicting the impact of single amino acid substitutions on protein function [48]. Higher Grantham scores and SNAP2 scores were considered more deleterious and were given higher rankings. The ranking scores for the above four parts of pathogenicity assessment

3.1.1. Patient 1 The male patient in family 1, aged 3 years, was the second child of nonconsanguineous healthy Chinese parents with unremarkable family history. There is no history of spontaneous abortion and stillbirth. The boy was born at 38 weeks by cesarean section with no pregnancy or delivery complications. His birth weight was 3600 g (+1.74 standard deviation [SD]). Length was 50.5 cm (+0.33 SD), and head circumference was 36.0 cm (+1.21 SD). At the age of 2.4 months, he started to have a series of head drop, lasting 1–2 s. In the beginning, the attacks lasted 1–2 min and occurred one time during 4–5 days; the frequency increased to 7–8 times per day. At 2 years of age, he developed generalized tonic, tonic–clonic, and atypical absence seizures, and her electroencephalogram (EEG) showed a slow background pattern with diffuse, slow spike–wave complexes. Valproic acid was prescribed together with topiramate, however with little effect on the symptoms. On evaluation, he showed acquired microcephaly after clusters of seizures with a head circumference of 43 cm (− 3.86 SD) at 2 years of age. Motor milestones, like grasping objects, sitting, and walking, were not yet achieved until 2 years old, and the ability of speech was absent. He had axial hypotonia with less eye contact. Metabolic analyses in urine and patterns for amino acids in plasma did not show any abnormalities. The routine hematological and chemical examination was normal. Brain magnetic resonance imaging (MRI) at the age of 4 months showed mildly dilated cerebrospinal fluid spaces, slight white matter immaturity, without major brain malformations. Visual evoked potential and auditory evoked potentials were normal. A LGS was diagnosed according to the following: childhood intractable seizures with multiple seizure types (of which tonic and atypical absence predominate) and having an EEG that demonstrates a slow spike–wave pattern of about 2 to 2.5 Hz (Fig. 1A). 3.1.2. Patient 2 The male patient 2, aged 8 years, was the second pregnancy of a 35year-old woman with a healthy brother. No history of family seizures, spontaneous abortion, and stillbirth were investigated. He was delivered by cesarean section at 41+3 weeks secondary to the uterine scar. Apgar scores were 81 and 105 with no delivery complications. Birth weight was 2900 g (− 3.06 SD), and occipitalfrontal circumference (OFC) was 33 cm (− 1.15 SD). The seizures occurred at 4 months of age, with a nodding head which then evolved to eye deviation and arm extension. At 3.5 years old, there were new seizure types, mostly myoclonic and tonic, with head deviation and stiffness of arms and legs, at a frequency of 10 times a day. Atypical absence was also been noted. Valproic acid was prescribed together with Topamax later, but they showed little effect on the symptoms. Developmental delay, hypotonia with acquired microcephaly (≤−3 SD), no ability of walking, and no language were shown as neurological presentations. A comprehensive metabolic investigation was initiated but largely uninformative. Brain MRI was normal. Electroencephalogram at 8 years old showed a pattern of LGS with diffuse spike–wave bursts in the slow background (Fig. 1B). 3.2. Identification of de novo mutations in EEF1A2 causative for LGS First, in the genome-wide CNVs analysis, no significant clinical or pathological CNV was observed in patient 1. Then, WES was performed; after sequencing data analyses and variants filtering, we identified a novel de novo variant [NM_001958.3: c.294C N A; p.(Phe98Leu)] of

K. Long et al. / Epilepsy & Behavior 105 (2020) 106955 Fig. 1. Electroencephalogram (EEG) features of the patients with LGS. A. EEG of patient 1 at 2 years of age. Left, EEG shows slow background activity with medium to high amplitude sharp-slow waves discharges around 2–3 Hz, predominating on the right temporal area (arrow). Right, EEG tracing shows a sequence of diffuse fast, low-voltage spikes (from the purple mark), lasting about 3 s, followed by diffuse postictal slowing. B. EEG of patient 2 at 8 years of age. Left, EEG shows atypical absence with high amplitude slow spike–waves discharges around 2 Hz, diffuse and irregular, conspicuously in both frontal areas. Right, ictal EEG features of tonic seizures with a paroxysm of diffuse low-voltage spike–waves around 20–26 Hz. Clinical onset of seizure corresponds with a brown mark. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 3

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K. Long et al. / Epilepsy & Behavior 105 (2020) 106955

EEF1A2 in patient 1, which encodes the elongation factor 1-alpha 2 (Fig. 2A, B). On the one hand, EEF1A2 gene was reported to be associated with epileptic encephalopathies with the identification of at least 12 de novo missense variants in 21 sporadic cases (Table 1). The G70S, E122K residues were two hot spots with variants at these residues accounting

for 42.9% (9/21) of reported cases. Moreover, the EEF1A2-related epileptic phenotype ranged from West syndrome to unspecified epileptic encephalopathy including LGS in two patients. According to the guideline for evaluation of the pathogenic potential of genes with de novo variants in epileptic encephalopathies [49], these findings highlighted

K. Long et al. / Epilepsy & Behavior 105 (2020) 106955

the possible pathogenic role of EEF1A2 gene in epileptic encephalopathy. On the other hand, the identified mutation, located in highly conserved residue, was absent from 8000 control chromosomes of Chinese origin, the publicly available databases (the Genome Aggregation Database, 6500 Exomes Sequencing Project and 1000 Genomes Project), and was consistently predicted pathogenic by in silico prediction tools (SIFT, PolyPhen-2, and MutationTaster) (Fig. 2C). The F98L mutation is the same amino acid change as a previously established pathogenic variant (c.292TN C, p.(Phe98Leu)) supporting its pathogenicity. According to the standards and guidelines for the interpretation of sequence variants by American College of Medical Genetics and Genomics (ACMG) [50], the identified novel variant was categorized to be “pathogenic” (PS1 PS2 PM1 PM2 PP2 PP3 PP4), which was the main cause of epileptic encephalopathy and intellectual disability in patient 1. In patient 2, the genome-wide CNVs analysis was also normal, and by WES, we discovered a recurrent de novo heterozygous mutation [NM_001958.3: c.208G NA; p.(Gly70Ser)], which was validated by Sanger sequencing (Fig. 2A, B, C). In fact, as a variant hotspot, the de novo c.208G N A; p.(Gly70Ser) mutation in EEF1A2 has been reported in at least five patients with epileptic encephalopathy [4,6,8,9,14]. According to the standards and guidelines for the interpretation of sequence variants by ACMG [50], the identified variant was categorized to be “pathogenic” (PS1 PS2 PM1 PM2 PP2 PP3 PP4). 3.3. Genotype–phenotype relationship in EEF1A2 To explore the factors determining phenotypic variations, we systematically reviewed EEF1A2 mutations and analyzed the potential correlations between genotypes and phenotypes. To date, at least 13 mutations have been identified in 24 sporadic cases, including two cases in this study (Table 1) [4,6–11,13–17]. Mutations identified in each case involved missense changes in highly conserved residues although functional studies were not performed. All mutations were associated with epilepsy and intellectual disability. And the most common types of seizures were myoclonic and epileptic spasms, occurring in up to 43.5% and 34.8% of cases, respectively. Most of the mutations were related to refractory seizures with severe intellectual disability, suggesting a potential genotype–phenotype correlation. To achieve the variant-level evaluation of damaging effect, we obtained damaging scores of EEF1A2 mutations from the VarCards database, which were the proportion of tools predicted to be deleterious through a total of 23 in silico missense algorithms. Moreover, we also gained ReVe, Grantham, and SNAP2 scores for further supplementary assessment, which were not included in the 23 tools (Table 2). Then, the above four parts of pathogenicity scores were sorted in ascending order, respectively. A higher pathogenicity score was considered more deleterious and given higher rankings. The total damaging ranking scores were the summation of ranking scores for the above four parts of pathogenicity assessment and were used for the subsequent analysis of the genotype–phenotype relationship (Table 2). Five missense mutations (G70S, I71L, L246P, D252H, R266W) located in the t-RNA binding domain, which plays an essential role in the translation of amino-acylated tRNA in protein synthesis, were associated with severe epilepsy (Fig. 2D, Table 1). These mutations were closed to or overlapped with eEF1B binding site, through which eEF1A and the subunits of eEF1B make the eEF1 complex, participating in translation [4,51]. Among them, G70S, R266W, D252H, and L246P were the top four mutations in the pathogenicity consequences with

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damaging ranking scores more than 34 (Table 2). In particular, the hotspot mutation G70S (6/24 patients), located in the t-RNA binding domain I, had a significant relationship with an early seizure onset age in five patients, ranging from 10 weeks to 4 months old, and refractory epileptic encephalopathy. In relative terms, the t-RNA binding domain II mutations, D252H and R266W, tended to be related with relatively variable onset age of intractable seizure, ranging from infant to 8 years old, accompanied with severe developmental delay and intellectual disability. The R266W mutation identified in a patient with seizure onset during infancy, leading to a substitution of exposed positive-charged arginine (ARG) residue with uncharged tryptophan (TRP), was predicted to disrupt the original hydrogen bond and wild-type salt bridge between ARG-266 and GLU-268 [52]. Likewise, the other three missense mutations (D91N, A92T, F98L) located in the GTP hydrolysis sites domain, where overlapped with eEF1B binding site, were also associated with refractory epilepsy with relatively early onset age, ranging from 1 month to 2 years old (Fig. 2D, Table 1). Especially, D91N and F98L were with damaging ranking scores more than 32, indicating the obvious detrimental effect on protein structure and/or function. The D91N mutation was predicted to replace buried negative-charged aspartic acid (ASP) residue with uncharged asparagine (ASN) and disrupt a salt bridge formed by ASP-91 and HIS95. All side-chain/side-chain and side-chain/main-chain H-bonds formed by ASP-91 residue could be influenced to some degree by this substitution as well. In addition, two missense mutations (E122K, E124K) located in the domain I except for the above regions, which was closed to GTP/ guanosine diphosphate (GDP)-binding site (Fig. 2D). As one of the mutation hotspots, the E122K mutation resulted in the severe phenotype of epileptic encephalopathy with onset age ranging from 2 to 10 months old in 4 patients and relative mild epileptic phenotype in only one 4 years old age patient. Similarly, the mutation E124K caused the seizure with onset age at 3 months old. However, three patients with E122K or E124K mutation had a relatively favorable outcomes of seizures after treatment, indicating that these two mutations with damaging ranking scores from 17 to 25 may have relatively moderate deleterious effects. The structural changes of E122K and E124K mutations included residue charge switches, possible disruptions of Hbonds, and salt bridges. In contrast, the two missense mutations (P333L, R423C) located in the actin-binding sites in domain III led to relatively various phenotypes (Fig. 2D, Table 1). The first homozygous mutation, P333L, was found in two siblings who exhibited global developmental delay, failure to thrive, dilated cardiomyopathy, and epilepsy onset at 7 and 12 months old, respectively. Though with heterozygous P333L mutation, which had 20 damaging ranking scores, neither parents of the two siblings show encephalopathy, intellectual disability, nor other obvious neurodevelopmental abnormalities. In structural analysis, the mutant has a more stable domain II with more defined β-sheets compared with the wild-type [15]. Additionally, two patients with R423C mutation, which had a relatively lower ranking score, revealed the onset of seizures in infancy with movement disorders, such as involuntary movements and spastic palsy. Accordingly, residue charge switches and possible influence on H-bonds were noted in structural changes prediction. All these results supported that these two mutations might impair the protein function and express the clinical phenotype in a respectively distinct manner needing to be further explored.

Fig. 2. De novo mutations in EEF1A2 identified in patients with LGS. A. Pedigree of two nonconsanguineous families with LGS showing cosegregation of the EEF1A2 mutation with the disease. The genotype is indicated under each individual (+ sign, wild-type [WT] allele; −sign, mutant allele). B. Sanger sequencing analysis confirmed two de novo missense mutations, c.294C NA (p.F98L) and c.208GNA (p.G70S), in affected individuals, respectively. C. F98L and G70S are both highly conserved across several species. D. Schematic representation of the eEF1A2 protein. The mutations identified in the families we report are indicated in red. G70S is located in the t-RNA binding sites domain, and F98L is in the GTP hydrolysis sites domain. Mutations listed below the gene are associated with refractory epileptic encephalopathy onset at an early age (before infantile period), whereas those listed above are associated with either relatively later onset or favorable outcome of epilepsy. Thereinto, the mutations with severe molecular alteration (damaging range scores more than 30) are marked in bold. The mutations that may be with controlled seizures are indicated in blue and that without specific onset age are indicated with an asterisk. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Table 1 Overview of phenotypes observed in patients with mutations in EEF1A2. Mutation

Seizure onset/types

Seizure prognosis

ID/developmental delay

Behavior problems

Hypotonia

Facial Acquired dysmorphism microcephaly

Other

Reference

Trio 91

22 y/F

c.208GNA; p. (Gly70Ser)/de novo

NR

+, global developmental delay

−

−

−

de Ligt et al. [6]

14 y/M

c.208GNA; p. (Gly70Ser)/de novo

+, autistic and aggressive behaviors NR

+

Trio B

+

NR

+

Increased ankle tone; incoordination, gait instability

Veeramah et al. [8]

Lam case 1

3 y/F

c.208GNA; p. (Gly70Ser)/de novo

Intractable +, global developmental delay, absent speech

−

+

+

Lam et al. [4]

NR/F

c.208GNA; p. (Gly70Ser)/de novo

NR

NR

NR

NR

NR

−, no microcephaly at 9 m NR

Dysphagia

2008D06721

−

de Kovel et al. [9]

Patient 34

NR

c.208GNA; p. (Gly70Ser)/de novo

NR

+

+

NR

+

NR

−

Helbig et al. [14]

Lam case 2

9 y/M

−

NR

+

−

Drachycephaly

Lam et al. [4]

Lam case 3

14 y/F

c.211ANC; p. (Ile71Leu)/de novo c.271GNA; p. (Asp91Asn)/de novo

4 m/myoclonic, absences, grand mal insults 10 w/epileptic spasms, then myoclonic, myoclonic/tonic, atonic and GTCS 2 m/myoclonic, tonic–clonic, absence seizures Early infancy/epileptic encephalopathy Childhood/epileptic encephalopathy, Lennox–Gastaut syndrome +

−

+

+

−

Brachycephaly, reduced Lam et al. [4] bone density with fractures

Patient 17

6 y/F

NR

NR

−

Lopes et al. [13]

9 y/F

+

+

−

Reduced bone density

Lam et al. [4]

Patient 2

12 y/F

c.364GNA; p. (Glu122Lys)/de novo

Infancy/focal, myoclonic, tonic, tonic–clonic 4 m/epileptic spasms

+, like Rett syndrome −

+

Lam case 4

+, global developmental delay, Intractable +, global developmental delay, absent speech Controlled

+, severe ID, motor delay, absent speech

NR

+

6 y/F

c.364GNA; p. (Glu122Lys)/de novo

10 w/epileptic spasms

Controlled

+, severe ID, nonverbal, gross motor delay

+

+

Talipes varus, ataxic gait, sleep disorder, mild atrophy of cerebrum Unsteady gait

Inui case 1

2 y/F

c.364GNA; p. (Glu122Lys)/de novo

10 m/myoclonic, atypical absence

NR

+

Syndactyly, internal strabismus

Inui et al. [10]

Inui case 2

2 y/M

c.364GNA; p. (Glu122Lys)/de novo

NR

+

NR

Progressive cereberal atrophy

Inui et al. [10]

De Rinaldis case

13 y/M

c.364GNA; p. (Glu122Lys)/de novo

8 m/myoclonus, myoclonic-atonic seizure 4 y/myoclonic absences

− Intractable +, severe ID, global developmental delay, absent speech − Intractable +, severe ID, global developmental delay, absent speech Controlled +, severe ID, absent speech +, autistic and self-injury behaviors

+, small head circumference at birth +, head circumference b2nd centile at 5 years NR

Nakajima et al. [7]

Lam case 5

+, autistic and self-injury behaviors −

−

+

+

De Rinaldis et al. [17]

Lam case 6

10 y/F

c.370GNA; p. (Glu124Lys)/de novo

3 m/myoclonic seizures, absences

Controlled

−

−

−

−

Failure to thrive, ataxic gait, sleep disorder, periventricular hyperintensities Gait immature but essentially normal

Lance case

17 y/M

c.737T NC; p. (Leu246Pro)/hetera

+

Controlled

+, autistic behavior, bipolar disorder, aggression

+

+

−

Lance et al. [16]

Patient 1

8 y/F

c.754GNC; p. (Asp252His)/de novo

8 y/generalized tonic

NR

+

+

+, small head circumference

Choreoathetotic movements, strabismus, hypothyroidism, asthma, gastroesophageal reflux, kidney dysfunction Mild atrophy of cerebrum

c.274GNA; p. (Ala92Thr)/de novo c.292T NC; p. (Phe98Leu)/de novo

2 y/epileptic spasms, atypical absences 1 m/epilepsy

Intractable +, severe ID, episodic regression, absent speech

NR

+, global developmental delay, absent speech Intractable +, global developmental delay, absent speech NR

+, mild developmental delay, significant delays in language +, developmental delay, significant delays in language

+, severe ID, absent speech +, autistic behavior

Lam et al. [4]

Lam et al. [4]

Nakajima et al. [7]

K. Long et al. / Epilepsy & Behavior 105 (2020) 106955

Age/sex

This study c.208GNA; p. (Gly70Ser)/de novo 8 y/M Our patient 2

c.294CNA; p. (Phe98Leu)/de novo 3 y/M Our patient 1

c.1267C NT; p. (Arg423Cys)/de novo NR/F Ostrander case 6

M: male; F: female; y: years; m: months; w: weeks; (+): features presented; (−): features absent; NR, not reported; ID, intellectual disability; GTCS, generalized tonic–clonic seizures. a Biological parental samples were not available.

−

+ + − Intractable +, global developmental delay, absent speech

−

This study −

+ + − Intractable +, global developmental delay, absent speech

Mildly dilated cerebrospinal fluid spaces

Ostrander et al. [11] NR NR − NR +, global developmental delay NR

−

Died at 4.5 y/F 5 y/M Case II:2

Lam case 7

Died at 29 c.998CNT; p m/M (Pro333Leu)/homo Case II:1

c.998CNT; p (Pro333Leu)/homo c.1267C NT; p. (Arg423Cys)/de novo

12 m/febrile, tonic–clonic 4 m/epileptic spasms, myoclonic, myoclonic-tonic, tonic, tonic–clonic b1 m/migrating partial seizures, myoclonic, flexor spasm 2.4 m/epileptic spasms, tonic, tonic–clonic, atypical absence 4 m/epileptic spasms, myoclonic, tonic, atypical absence

NR

+, global developmental delay, absent speech Intractable +, global developmental delay, absent speech

NR

+

−

Dysphagia, quadriparetic spastic cerebral palsy

Lam et al. [4]

Cao et al. [15]

Dilated cardiomyopathy, hepatomegaly Multiple food allergies, choreic movements

Cao et al. [15] Dilated cardiomyopathy NR

+ −, tight heel cords with hypertonia −, + hypertonic + + NR Intractable +, regression, global developmental delay, only words

+ NR + NR

Infancy/focal epilepsy, temporal lobe epilepsy 7 m/absences, tonic–clonic, eye deviation, febrile c.796CNT; p. (Arg266Trp)/de novo NR Patient 35

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4. Discussion

+

at birth NR

−

Helbig et al. [14]

K. Long et al. / Epilepsy & Behavior 105 (2020) 106955

In this study, a novel de novo heterozygous mutation [c.292TNC, p. (Phe98Leu)] and a recurrent mutation [c.208G NA; p.(Gly70Ser)] in EEF1A2 were identified in two patients with LGS and intellectual disability. The present study highlights the pathogenic role of EEF1A2 in epileptic encephalopathies and intellectual disability. The further systematical analysis revealed a potential genotype–phenotype correlation in patients with EEF1A2 mutations. The main common feature of our two patients is the specific progression from infantile spasms to LGS. According to previous studies, infantile spasms and hypsarrhythmia could gradually progress to LGS with slow spike–waves, which accounted for about 20% of all cases with LGS. It also noted that those who had infantile spasms and evolved to LGS tended to have a poor outcome [53]. As various epileptic encephalopathies share overlapping features and may evolve from one to another, it is important to investigate the underlying genetic etiology. The identification of EEF1A2 mutations in patients with LGS who evolve from infantile spasms may aid clinicians in predicting the prognosis of such patients. To date, at least 13 mutations in EEF1A2 have been identified, all of which were missense variants resided in essentially functional domains. All reported patients had epilepsy with varying severity, developmental delay, and intellectual disability. Almost all missense mutations in the t-RNA binding sites of domain I with severe molecular alteration were associated with an earlier onset age of refractory epileptic encephalopathy, typically observed in the most common mutation at residue G70 in the first t-RNA binding sites. The G70S mutation could cause multiple types of seizures, from myoclonic, tonic–clonic, to epileptic spasms and absence. Consistent with the severity of the phenotype in the human patients, G70S mutation was demonstrated that it could result in nonfunctional eEF1A2 protein and was unable to facilitate protein synthesis or protect mice from neurodegeneration in G70S/− mice [54]. Likewise, most of the mutations located in the GTP hydrolysis sites domain with severe deleterious effects were also associated with refractory early-onset epilepsy. The GTP hydrolysis on eEF1A2 could lead to the steady positioning of the aminoacyl-tRNA in the A site, which subsequently triggers the transpeptidation reaction [55]. On the other hand, the majority of mutations located in the domain II t-RNA binding sites resulted in intractable seizures with relatively variable onset age. All those suggested that the t-RNA binding domains and GTP hydrolysis domain play a crucial role in the protein function and the damaging of the corresponding function could contribute to protein synthesis impairments and apoptosis of neuronal cells, further involving in the pathogenesis of serious phenotype. The clinical expression was potentially determined by the location of mutations and alteration of molecular effects, supporting the significance of molecular subregional stratification in evaluating the pathogenicity of variants [56]. However, mutations located in the domain I except for the above region, which were predicted to have relatively moderate deleterious effects, might lead to early-onset severe seizures with relatively favorable outcomes after antiepileptic drugs treatment in some patients. According to the found in yeast, E122K equivalent mutation could result in translational infidelity, which was supported as a possible mechanism under disease phenotype. In addition, mutations located in the actin-binding sites in domain III with moderate or less deleterious effects could result in relatively various phenotypes. Among them, the P333L mutation was pathogenic in homozygosity, which may stabilize the dynamic of domain II and change the binding affinity of EEF1A2 to its binding partners [15]. And the R423C mutation was buried at the interdomain junction of the protein and was thus likely to have consequences for the protein structure or conformation, which contribute to the infancy onset of seizures with movement disorders. All those suggested that the location of mutations is possibly one of the determinants of clinical severity. Further investigations with more patients will be

8

K. Long et al. / Epilepsy & Behavior 105 (2020) 106955

Table 2 Quantitative evaluation of the damaging effects of mutations in EEF1A2. DNA bases changes

Amino acids change

D:A algorithmsa

Damaging scoresb/rankingc

ReVe scores/ranging

Grantham scores/ranging

SNAP2 scores/ranging

Total damaging ranging scoresd

c.208GNA c.211ANC c.271GNA c.274GNA c.292TNC c.294CNA c.364GNA c.370GNA c.737TNC c.754GNC c.796CNT c.998CNT c.1267C NT

G70S I71L D91N A92T F98L F98L E122K E124K L246P D252H R266W P333L R423C

22:23 19:23 22:23 18:23 21:23 21:23 19:23 18:23 20:23 21:23 18:23 19:23 12:23

0.96 (12) 0.83 (5) 0.96 (12) 0.78 (2) 0.91 (9) 0.91 (9) 0.83 (5) 0.78 (2) 0.87 (8) 0.91 (9) 0.78 (2) 0.83 (5) 0.52 (1)

0.971 (12) 0.84 (5) 0.77 (3) 0.777 (4) 0.979 (13) 0.97 (11) 0.922 (7) 0.89 (6) 0.957 (10) 0.934 (8) 0.946 (9) 0.728 (2) 0.726 (1)

56 (5) 5 (1) 23 (4) 58 (8) 22 (2) 22 (2) 56 (5) 56 (5) 98 (10) 81 (9) 101 (12) 98 (10) 180 (13)

32 (7) 22 (5) 86 (13) 4 (2) 67 (10) 67 (10) 49 (8) 12 (4) 24 (6) 58 (9) 81 (12) 8 (3) −25 (1)

36 17 32 16 34 32 25 17 34 35 35 20 16

a b c d

Number of algorithms predicted to be deleterious: total in silico algorithms in VarCards. Proportion of algorithms predicted to be deleterious through a total of 23 tools in VarCards. Ranking scores of pathogenicity scores were showed in brackets. The total damaging ranking scores were the summation of ranking scores for the above four parts of pathogenicity assessment.

necessary to elucidate the genotype–funotype–phenotype relationship of EEF1A2-related neurodevelopmental disorder.

5. Conclusions In conclusion, we identified a novel and a recurrent de novo mutations in EEF1A2 that were a causative factor for LGS in two respective families, confirming its pathogenic role in epileptic encephalopathy and intellectual disability. A further systematic review of EEF1A2 mutations revealed that the onset age and severity of epilepsy were potentially related to the affected functional domains and the severity of the molecule effects. This study highlights the role of the location and molecular effects of mutations in phenotype expression and is expected to facilitate the evaluation of the pathogenicity of EEF1A2 mutations in clinical or genetic counseling. Further investigations with more patients will be necessary to elucidate the pathogenic mechanism and genotype–funotype–phenotype relationship of EEF1A2-related neurodevelopmental disorder. Acknowledgments The authors are indebted to all the patients and family members for their generous participation in this work. This work was supported by the National Natural Science Foundation of China (grant numbers: 81601119), the New Xiangya Talent Project of the Third Xiangya Hospital, Central South University (grant numbers: JY201515), the Hunan Provincial Science and Technology Department, China (grant numbers: 2015TP2029), National Bureau of traditional Chinese medicine—Inheritance Studio of National famous traditional Chinese medicine expert for Jiabang Li (project numbers (2018): 134), and the Youth Foundation of Xiangya Hospital, Central South University (grant numbers: 2018Q013). Declaration of competing interest The authors have declared that no conflict of interest exists. References [1] Soares DC, Barlow PN, Newbery HJ, Porteous DJ, Abbott CM. Structural models of human eEF1A1 and eEF1A2 reveal two distinct surface clusters of sequence variation and potential differences in phosphorylation. PLoS One 2009;4: e6315. [2] Potter M, Bernstein A, Lee JM. The wst gene regulates multiple forms of thymocyte apoptosis. Cell Immunol 1998;188:111–7. [3] Abbott CM, Newbery HJ, Squires CE, Brownstein D, Griffiths LA, Soares DC. eEF1A2 and neuronal degeneration. Biochem Soc Trans 2009;37:1293–7.

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