Journal of Molecular Neuroscience https://doi.org/10.1007/s12031-021-01810-0 Identification of a Missense Variant in the EIF2B3 Gene Causing Vanishing White Matter Disease with Antenatal‑Onset but Mild Symptoms and Long‑Term Survival Mehdi Khorrami1 · Erfan Khorram1 · Omid Yaghini2 · Mojgan Rezaei3 Vida Yazdani6 · Maryam Riahinezhad7 · Majid Kheirollahi1 · Arash Hejazifar4 · Omid Iravani5 · Received: 15 October 2020 / Accepted: 2 February 2021 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021 Abstract Vanishing white matter disease (VWM) is a rare autosomal recessive leukodystrophy caused by a mutation in any of the five gene encoding subunits of the translation initiation factors eIF2B1 to eIF2B5. Whole-exome sequencing was performed on a 7-year-old boy with prenatal symptoms, including intrauterine-growth retardation, decreased movements, and oligohydramnios as well as mild intellectual disability, optic atrophy, macrocephaly, mild ataxia, and white matter lesions after birth. Analysis of WES data revealed a homozygous missense variant, c.C590T (p.Thr197Met) in the EIF2B3 gene (NM_0203650). The candidate variant was confirmed by Sanger sequencing and found to co-segregate with disease in family members. Pathogenicity analysis, 3D protein modeling, and stability assessment showed the deleterious effects of this nucleotide change. Previous studies suggest a direct relationship between the onset of symptoms and the progression rate and severity of the disease. All described cases of EIF2B deficiency with antenatal-onset led prenatal death; if they were born, they experienced clinical exacerbation, seizure, severe encephalopathy, and consequent infantile death (< 1 year). The patient of this study had never had seizure, which could be a potential explanation for the observed mild clinical picture, chronic state, and long-term survival until the age of seven. This study reported the first VWM due to EIF2B gene deficiency with antenatal-onset but mild symptoms and long-term survival. The result of this study showed that stressor factors, particularly seizure, could have a substantial role in poor prognosis and early neonatal death. Keywords EIF2B3 gene · Vanishing white matter disease · Leukodystrophy · Whole exome sequencing · Iran * Majid Kheirollahi mkheirollahi@med.mui.ac.ir Mehdi Khorrami mehdi.khorrami@resident.mui.ac.ir Erfan Khorram erfankhorram96@gmail.com Omid Yaghini yaghini@med.mui.ac Mojgan Rezaei mojadrian70@gmail.com Arash Hejazifar arash8268@gmail.com Omid Iravani om_iravani@yahoo.com Vida Yazdani vidayazdani1991@gmail.com 1 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran 2 Child Growth and Development Research Center, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran 3 Department of Biology, Faculty of Science, Yazd University, Yazd, Iran 4 Department of Biology, School of Sciences, The University of Isfahan, Isfahan, Iran 5 Legal Medicine Research Center, Legal Medicine Organization, Tehran, Iran 6 Department of Biology, Islamic Azad University, East Tehran Branch, Tehran, Iran 7 Department of Radiology, Isfahan University of Medical Sciences, Isfahan, Iran Maryam Riahinezhad maryam_riahinezhad@yahoo.com 13 Vol.:(0123456789) Journal of Molecular Neuroscience Introduction Leukodystrophies are a large, degenerative, highly heterogeneous group of inherited disorders, defined as the selective and primary involvement of the white matter in the central nervous system (CNS) regardless of the affected molecular pathway or white-matter component (van der Knaap and Bugiani 2017; Bugiani and van der Knaap 2017). These disorders show a broad spectrum of onset age from antenatal to senescence as well as a wide range of severity and clinical outcomes (van der Knaap et al. 2019; Hamilton et al. 2018). Generally, leukodystrophies are progressive and fatal, but instances of improvement, recovery, and long-time stability have also been described (Kevelam et al. 2016; Köhler et al. 2018; Lynch et al. 2017). Due to the nature of the disease and the high diversity of the subtypes, there are different classifications of leukodystrophies that have evolved based on the knowledge of their time. A comprehensive classification has been proposed (van der Knaap and Bugiani 2017) with the following categories: myelin defect (hypomyelination, dysmyelination, demyelination, and myelinolytic diseases), astrocytopathies (i.e., vanishing white matter), leukoaxonopathies, microgliopathies, and leuko-vasculopathies (van der Knaap and Bugiani 2017). Childhood ataxia with central hypomyelination, also known as vanishing white matter disease (VWM), is one of the most prevalent (the exact prevalence is not known) hereditary childhood leukoencephalopathies; however, it may affect people of any age, including neonates and adults. It is characterized by neurological deterioration, ataxia, spasticity, optic atrophy, some organ involvement, and MRI findings (Alsalem et al. 2012). The phenotypic manifestations show a wide spectrum of onset age, including antenatal/congenital onset, infantileonset (< 1 year), early childhood-onset (1 to 4 years), late childhood/juvenile-onset (4 to 18 years), and adult-onset (onset ≥ 18 years) (Hamilton et al. 2018). Generally, there is a direct relation between the onset age and disease severity and the progression state. While the prenatal/ congenital form is lethal, patients with later-onsets have normal or mildly delayed early developmental milestones, followed by progressive chronic or subacute course (Wei et al. 2019). In early-onset forms, motor deterioration is the first and chief clinical presentation. Adult-onset forms show variability in disease severity regardless of the onset age, but cognitive impairment and behavior problems are generally the superior manifestations (Güngör et al. 2020; Accogli et al. 2019; Scali et al. 2006). The antenatal or congenital form has the lowest frequency, is highly progressive, and leads to prenatal or neonatal death (within the first year of life) (Wu et al. 2009). In the antenatal-onset form, the clinical manifestation includes oligohydramnios, 13 decreased fetal movement, intrauterine growth retardation during pregnancy, irritability, hypotonia, lethargy, hepatosplenomegaly, vomiting, seizures, contractures, cataract, microcephaly, apnea, and severe encephalopathy after birth (Van Der Knaap et al. 2003; Trimouille et al. 2020; Song et al. 2017). In the early infantile form of VWM, also known as Cree leukoencephalopathy (onset age < 1 year), patients present severe encephalopathy, multiple neurological symptoms, sudden loss of motor skills, and rapid deterioration entailing early death within a few months (Woody et al. 2015). Usually, in the early childhood-onset form (onset age 1 to 4 years), children reach early age-anticipated milestones, while in some cases, mild neurodevelopmental delay is observed. Progression rate is variable, ranging from a rapid decline and death within 5 years after the onset to chronic manifestations and death decades after the disease diagnosis. Cases who survive for 2 years since the disease onset may develop macrocephaly. In later childhood/juvenile-onset form (onset age 4 to 18 years), the course of the disease is chronic with long-term survival, although it can be highly severe and lead to the death in the affected individuals only a few months after diagnosis. In the adult-onset form, there are frequent descriptions of behavioral problems associated with cognitive impairment prior to the onset of neurological symptoms (Hamilton et al. 2018; van der Knaap et al. 2006). The clinical picture is relatively benign and consists of acute, transient neurologic symptoms, extreme headache, and amenorrhea (Ho et al. 2020). VWM is caused by biallelic pathogenic variants in any of the five genes encoding different subunits of the conserved eukaryotic translation initiation factors B (EIF2B1 (alpha), EIF2B2 (beta), EIF2B3 (gamma), EIF2B4 (delta), and EIF2B5 (epsilon)) (Van Der Knaap et al. 2002). All variant types may occur in the EIF2B genes; however, mutations that cause a complete loss of protein are rare and reported only in a compound heterozygous state with a missense mutation, proposing that these variants are incompatible with life (Van der Knaap et al. 2010).Furthermore, there is a report of an atypical form of CACH/VWM with biallelic pathogenic variant diagnosed based on MRI findings but no neurological symptoms after long-term follow-up (Fontenelle et al. 2008). The eIF2B protein complex is a guanine nucleotide exchange factor that activates the eukaryotic initiation factor 2 (eIF2) through catalyzing the exchange of GDP for GTP. The eIF2-GTP transfers the initiator methionyl-transfer RNA (Met-tRNAiMet) to the 40S ribosomal subunit and subsequently initiates the translation (Leegwater et al. 2001; Pavitt and Proud 2009). It has been reported that stress factors such as febrile seizure, head trauma, infection, extreme fright, fever, or surgical events could induce major episodes of neurological Journal of Molecular Neuroscience deterioration. The eIF2B protein complex plays a key role in the regulation of cellular response in various physical, chemical, and oxidative stressor conditions; this might be a possible explanation for episodic aggravation of the neurologic symptom in patients with EIF2B defects under stress conditions (Van Der Knaap et al. 2002). In this study, using whole-exome sequencing (WES), we reported a homozygous missense variant in the EIF2B3 gene in a 7-year-old boy presenting with mild intellectual disability, optic atrophy, macrocephaly, mild ataxia, and white matter lesions on the brain imaging. In spite of the antenatal-onset of symptoms, the described patient, manifested mild presentation, and long-term survival. Genomic DNA Extraction Blood sample (5 ml) was collected in ethylenediaminetetraacetic acid (EDTA)-containing tubes from the patient (IV-1), parents (III-1 and III-2), and the healthy sibs (IV-2) (Fig. 1a). Highquality genomic DNA was extracted using Prime Prep Genomic DNA Extraction kit (QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instruction. The quality and quantity of the extracted DNA was determined by agarose gel electrophoresis and Nanodrop 2000 spectrophotometer (Nonodrop 2000 Thermo Scientific, USA). Whole‑Exome Sequencing and Pathogenicity Assessment This study was approved by the Institutional Review Board (IRB) of Isfahan University of Medical Science (IR.MUI. MED.REC.1398.186). Written informed consent was obtained to conduct the study and for the publication of results from legal guardians (parents). An accurate and complete questionnaire was completed about family history, environmental risks, maternal complications during pregnancy, health history, developmental milestones, and disease progression. WES was performed in the IV:1 (Fig. 1a), and interpretation of results was performed according to the American College of Medical Genetics and Genomics (ACMG) guidelines (Richards et al. 2015). The DNA sample has been sent to Macrogen (South Korea) (https​://www.macro​gen.com/) for WES. The paired-end DNA sequencing was performed using SureSelect Human All Exon V7 (Agilent Technologies, Inc., Santa Clara, CA, USA) by Novogene Co., Ltd., Hong Kong. Captured DNA fragments were sequenced using Novaseq 4000 platform (Illumina, San Diego, CA, USA) with coverage of 100× mean depth (Illumine Inc., San Diego, CA, USA). Briefly, the variant analysis was done via mapping the FASTQ to the reference genome (UCSC hg19) using the BurrowsWheeler Alignment software (http://bio-bwa.sourc​eforg​e. net/) with default parameters. Variants were called with Genome Analysis Tool Kit software (https​://gatk. broad​insti​tute.org/) and annotated using Annovar software. Variants with a minor allele frequency (MAF) > 0.01 were filtered in databases such as exome aggregation consortium (ExAC) (http://exac.broad​insti​tute.org), 1000 genomes project Fig. 1  Pedigree of the studied family and the detected mutations. a Pedigree chart of the family with relevant genotype. b The cytosine nucleotide at position 590 was a highly conserved EIF2B3 gene in multiple-species alignment. c The result of the co-segregation study using Sanger sequencing was consisting with the autosomal recessive nature of the vanishing white matter disease. The proband (IV:1) was homozygous for pathogenic variant (C590T; p. Thr197 Met). Parents and healthy brother were heterozygote for discussing variant Materials and Methods Subjects and Phenotype Investigation 13 Journal of Molecular Neuroscience phase 3 database (https​://www.inter​natio​nalge​nome.org/), dbSNP version 147, HGMD (http://www.hgmd.cf.ac.uk/ ac/index​.php), exome sequencing project (ESP) (https​://evs. gs.washi​ngton​.edu/), and Iranome (http://www.irano​me.ir/) to downstream analysis according to their chromosomal location, mode of inheritance, functional consequences, inheritance pattern, and clinical presentation. Computational predictive tools such as MutationTaster (http://www.mutat​ionta​ster.org/), PROVEAN (http://prove​an.jcvi.org/index​.php), SIFT Indel (http://sift-dna.org/), DDIG Indel (http://spark​s-lab.org/ddig/), and PANTHER (http://www.panth​erdb.org/) were used to predict the pathogenicity of the detected variant. Co‑segregation Analysis via Sanger Sequencing In order to verify the candidate variant, specific primers (F:5′-ACT​TAC​CAG​TGC​CTA​CTA​CCTG -3′ and R: 5′-CAC​ CTA​GAT​AAG​TTT​CCG​CCTTG—3′) flanking region of the detected variant were designed. The PCR product was subsequently visualized using 1% agarose gel, and bidirectional sequencing was carried out by an ABI 3130 sequencer (Applied Biosystems-USA). The result was compared with the EIF2B3 gene reference sequence (NM_020365). Protein Structure Analysis and Stability Assessment 3D structure of the eIF2B-EIF complex taken from the Protein data bank (PDB ID:6o9z) and used for investigating the structural effect of the candidate variant. The process of mutagenesis, visualizing, and structural analysis was carried out through UCSF ChimeraX version 1.1(http://www.rbvi. ucsf.edu/chimer​ ax). In addition, different stability prediction servers such as mCSM (http://biosi ​ g .unime ​ l b.edu.au/ mcsm_ppi2/), DUET (http://biosi​g.unime​lb.edu.au/duet/), and I-Mutant2.0 (http://gpcr2​.bioco​mp.unibo​.it/~emidi​o/IMutan​t2.0/I-Mutan​t2.0_Detai​ls.html) were used to explore the effect of the mutation on the stability of the protein. Results Clinical Description The proband (IV:1) (Fig. 1a) is a 7-year-old boy presented with neurodevelopmental delay, optic atrophy, macrocephaly, and mild ataxia (Supplementary Fig. S1). Parents were first cousins with no relevant medical history. Pregnancy was uneventful until the sixth month where intrauterine growth retardation, and decreased fetal movements were detected. The delivery was cesarean section due to oligohydramnios. Birth weight, height, and head circumference were 1150 g, 39 cm, and 28.5 cm respectively, and hospitalized due to poor feeding and 13 severe vomiting for 20 days. Developmental milestones were normal up to 14 months, after which motor development stopped and regressed, and the subject showed delayed speech and abnormal gait. Currently, at the age of 7 years, the neurological examination revealed spasticity, hyporeflexia of four limbs, mild to moderate intellectual disability, mild broad-based gait ataxia, macrocephaly, low weight, short stature, mild ptosis, telecanthus (also present in his father), clinodactyly, pes planus, and constipation. Ophthalmology evaluation revealed pigmentary changes in the posterior pole of the retina, optic atrophy, horizontal nystagmus, and astigmatism. Moreover, unilateral cryptorchidism was present, but the testis and epididymis had a normal size and parenchymal echogenicity. The EEG was mildly abnormal, and the subject had never experienced a seizure. MRI of the child’s brain and spine had been performed at the age of 6 showed symmetrical abnormal signal intensity as high on T2W, FLAIR, and low on T1W involving posterior fossa (as pons, midbrain), deep white matter in periventricular, subcortical, and putamen nuclei (Fig. 2). Whole‑Exome Sequencing Identified a Missense Mutation c.C590T; p.T197M in EIF2B3 Analysis of the WES on patient IV-1, revealed a total of 76,932 variants, 2237 of which occurred in coding regions with MAF > 0.01. Mean depth of coverage was 100× for more than 94% of the sequences. Subsequent data filtering specified a missense variant: c.C590T; p.Thr197Met in EIF2B3 gene (MIM_606273, NM_020365, and ENSG00000070785) (Exon 6) on chromosome 1 which matched the observed clinical manifestation and was selected for further analyses. The detected variant substituted a threonine residue with methionine in the position 197. The variant was present in the heterozygous state in some population databases with very low frequency such as ExAC_ALL (0.0000714) and gnomAD_exome_ALL (0.0000531), and it was submitted in dbSNP (rs749213552). c.C590T was not reported in the homozygote state or in association with phenotypic presentation and was not submitted in ClinVar database. In addition, this variant was not reported in Iranom project, in which 800 healthy individuals from eight major ethnic groups in Iran went through WES. No other pathogenic exonic, intronic, synonymous, and compound heterozygous mutations were found, which could match the clinical presentation of the studied patient. The candidate variant was predicted to be deleterious with different pathogenicity predictive tools (Table 1). The detected alteration changed a highly conserved residue of EIF2B3 among multiple-species alignment (Fig. 1b), and the phred-scaled combined annotation dependent depletion score of this variant was T:26.0. Journal of Molecular Neuroscience Fig. 2  T2W (a–c) shows symmetrical diffusely high signal intensity involving periventricular white matter to the subcortical. Also, high signal intensity on pons at corticospinal tract (a) and putamen was observed. T1W (d–f) shows white matter volume loss, low signal intensity in corticospinal tract of pons and putamen nuclei. FLAIR (g–i) shows symmetrically diffusely high signal intensity in periventricular white matter, high signal intensity in pons and putamen 13 Journal of Molecular Neuroscience Table 1  Protein prediction analyses of the missense variant in EIF2B3 gene Protein prediction algorithm PolyPhen-2 SIFT FATHMM PROVEAN REVEL Mutation tester c.C590T; PT197M Probably damaging Deleterious Damaging Damaging Likely disease causing Disease causing Familial Segregation was Consistent with the Autosomal Recessive Nature of the VWD Using specific primers, 980 bp of EIF2B3 gene containing c.C590T was amplified and sequenced in all family members and found to be co-segregating with the phenotype. Parents (III-1 and III-2) and the younger healthy sibling (IV-2) were heterozygous, and the affected member of the family (IV-1) harbored the pathogenic variant in the homozygous state (Fig. 1c). In Silico Analysis Revealed Conformational Change and Decreased Stability The structural analysis determined that the altered residue, Threonine 197, located in the interface region between subunit γ and subunit ε (Catalytic sub complex of eIF2B protein complex) which are encoded by EIF2B3 and EIF2B5 genes respectively. As a result, threonine substituted with methionine, which has a larger side-chain and probably would clash with phenylalanine at residue 231 of subunit ε and thus cause a conformational change (Fig. 3). In addition, stability analysis with different servers revealed a decrease in ΔΔG of mutant structure (Table 2). Discussion VWM is an extremely rare autosomal recessive leukodystrophy with progressive episodic neurologic deterioration first recognized in 1992 (Schiffmann et al. 1994). Patients are generally diagnosed with MRI finding and neurologic signs including optic atrophy, cerebellar ataxia, spasticity, and intellectual disability (Woody et al. 2015). This condition has heterogeneous nature and is caused by a defect in any of the five gene-encoding different subunits of the conserved eukaryotic initiation translation factors EIF2B (EIF2B1, EIF2B2, EIF2B3, EIF2B4, EIF2B5) (Scheper et al. 2006). More than half of the mutations (57%) occur in the EIF2B5 gene. While eIF2B protein is expressed ubiquitously, it is still not obvious why CNS seems to be more susceptible to its dysfunction (Bugiani et al. 2010). Previous studies have suggested that some aspects of the genotype-phenotype correlation and similar phenotypic expression, onset age, and prognosis of VWM within families harboring the 13 same mutation have been reported; however, variability has been described, especially in less severe cases. The severity of neurologic deficit correlates with the onset age. In antenatal, congenital, and early infantile forms, the course of the disease is very severe and leads to early death. On the contrary, in patients with an onset age of 4 or more, the severity of the disease varies significantly from rapid decline and subsequent death to mild presentation and long-term survival (Takano et al. 2015). Certain conditions, as fever, seizure, major surgical events, or head trauma induce major episodes of neurological deterioration. Some patients recover relatively after a while, but in the majority of affected members, the symptoms do not return to the pre-exacerbated state (Abbink et al. 2018). In this study, WES was performed on a 7-year-old boy with prenatal symptoms, including oligohydramnios, decreased fetal movement, and postnatal presentation, including mild intellectual disability, optic atrophy, mild ataxia, and white matter lesions. WES data analysis revealed a homozygous missense variant, c.C590T; p.Thr197Met in EIF2B3 gene, which co-segregated with the phenotype within the family. Based on the structural analysis, the mutation (p. Thr197Met) led to the substitution of threonine with methionine which has a longer side-chain and clashes with phenylalanine at residue 231 in the subunit gamma (catalytic subunit); this probably results in a conformational change in the structure, and subsequently, the function of the protein. Moreover, stability analysis revealed that mutant structure has ΔΔG < 0, meaning that the altered protein had a lower stability than the wild type. According to the literature, previous studies have suggested a correlation between the onset of symptoms and disease severity, and all described cases (seven patients) of EIF2B defects with antenatal-onset (intrauterine-growth retardation, decreased movements, and oligohydramnios) either died before birth or experienced clinical exacerbation, seizure, severe encephalopathy, and subsequent infantile death (< 1 year) (Table 3) (Van Der Knaap et al. 2003; Trimouille et al. 2020). In cases with earlier onset, the importance of the role of stressors is not well understood. The patient of this study had never had seizure, which could be a potential explanation for the observed mild clinical picture, chronic state, and long-term survival until the age of 7. Contrary to different progression rates and disease severities, primary clinical manifestations in the studied patient were Journal of Molecular Neuroscience Fig. 3  Three-dimensional (3D) structure and mutagenesis analysis of eIF2B-eIF complex. a The mutation occurred in the interface region of the ε (encoded bye IF2B5 gene) and γ (encoded bye IF2B3 gene) subunits (catalytic site) (pink box). b In the native form, threonine at Table 2  Stability analysis of mutant protein. the mutant structure has ΔΔG* < 0 which indicate the destabilizing effect of c.C590T; p.Thr197Met mutation residue 197 has not clashed with other amino acids. c In mutant form, substituted methionine due to the longer side-chain has clashed with Phenylalanine 231 from subunit ε and consequently makes a conformational change in the ε subunit Stability predic- ΔΔG(ΔGm-ΔGw) tion server Prediction outcome Address I-Mutant2.0 −0.46 kcal/mol Destabilizing DUET mCSM −0.026 kcal/mol −0.143 kcal/mol Destabilizing Destabilizing (http://gpcr2​.bioco​mp.unibo​.it/~emidi​o/IMutan​t2.0/I-Mutan​t2.0_Detai​ls.html http://biosi​g.unime​lb.edu.au/duet/ http://biosi​g.unime​lb.edu.au/mcsm_ppi2/ m mutant, w wild type 13 13 8m Age of death 4.5 m + NA NA + NA + NA + + + + + NA NA Female 5m + NA NA + NA + NA + + + + + NA NA Female 3 14 Alive at 7 years old NA NA NA NA NA NA NA NA – – + NA + + Male 1 Current c. 590 C > T EIF2B3 * Myoclonic convulsions WG weeks of gestation, TOP termination of the pregnancy, NA Not available Ref 14 + Feeding difficulties Other NA NA Hyperreflexia of extremities pancreatitis + Hypotonia NA + Cataract Abnormal ovaries + Seizures + + oligohydramnios Hepatosplenomegaly + Decreased movements + NA Microcephaly at birth NA NA Intrauterine growth retardation Hypoplastic kidneys Female Sex respiratory failure 1 Sib 2 c. 599G > T/ c. 871C > T 1 Mutation Family EIF2B2 Gene 16 8.5 m NA NA NA NA NA + + + NA + NA + + NA 1 2 c.97A > G 8m NA NA NA NA NA + + + NA + NA + + NA 2 Table 3  Clinical summary and results of genetic testing of the present cases and previously reported cases 14 3.5 m + NA NA NA + NA + + NA + + NA + + Male 1 3 c.1447C > T EIF2B4 4m + NA NA NA + NA + + NA + + NA + + Female 2 c.1172C > A 10 m + slight + NA NA NA NA NA + +* + NA + + Female 1 4 NA NA – NA NA NA NA NA – NA NA NA + + Female 2 15 TOP in 27 WG TOP in 32 WG NA NA – NA NA NA NA NA – NA NA NA + + Female 1 5 c.468C > G/c.1165G > A EIF2B5 Journal of Molecular Neuroscience Journal of Molecular Neuroscience similar to the previously reported cases. Furthermore, we observed clinodactyly, pes planus, and constipation which were not previously reported in VWM; however, their etiology might not be related to EIF2B3. In conclusion, this study reported the first VWM case caused by EIF2B deficiency with antenatal-onset but mild symptoms and long-term survival. The results showed that stressor factors, such as high temperatures, head trauma, and especially seizure could have a substantial role in poor prognosis and early neonatal death; therefore, they must be managed and/or avoided as much as possible in patients with VWM. Patients should be under close observation, and precautions should be taken to prevent fever, infection, major surgical events, head trauma, excessive fright, and distraction. Low doses of antibiotics, prophylactic anticonvulsant drugs, and vaccination could be useful in disease management. Supplementary Information The online version contains supplementary material available at https​://doi.org/10.1007/s1203​1-021-01810​-0. Acknowledgements We would like to express our special thanks to our patients, their family, medical genetics lab GENEAZMA, and Isfahan University of Medical Science for their collaboration. Author Contribution MK contributed in data analysis and interpretation, drafted and revised the manuscript. EK contributed in manuscripts writing and laboratory works. MR contributed in data collection and laboratory works. AH: contributed in data analysis and interpretation. OI and OY supervised the clinical evaluation. MR contributed in clinical evaluation. VY contributed in data collection and manuscript revision. MAK designed and supervised the study. All of the authors read and approved the final manuscript to be published and agreed to be responsible for the accuracy of the data and details. Funding The project was supported by deputies of research of Isfahan University of Medical Sciences [194068]. Data Availability Data are available upon reasonable request. Declarations Ethics Approval The study was approved by the Ethical Committee of the Isfahan University of Medical Science (IR.MUI.MED. REC.1398.186). Consent to Participate Written informed consent was obtained from the legal guardian of patients (parents). Consent for Publication Written informed consent for publication of clinical details and images (Fig. 2 and Supplementary Fig. 1) was obtained from the patient legal guardian. Conflict of Interest The authors declare that they have no conflict of interest. References Abbink TE, Wisse LE, Wang X, Proud CG (2018) Role of Eukaryotic initiation factor eIF2B in vanishing white matter disease. 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