Compound heterozygous mutations of two eIF2B genes in early childhood onset form of vanishing white matter disease

Background Diagnoses of vanishing white matter disease (VWMD) were difficult due to variable clinical features, severity, age of onset and wide range of mutations in eIF2G genes which cause VWMD. This study reported two novel mutations in eIF2B genes associated with VWMD to and expand our understanding of VWMD. Case presentation Relevant data from clinical diagnoses and genetic mutational analyses in two Chinese female patients with sporad ic VWMD were collected and analyzed. Protein structure/function was predicted. The identity of biological parents was confirmed based on variants called from the next-generation sequencing (NGS) data. Compound heterozygous mutations, c.254 T>A (p.Val85Glu), and c.597+2delT in the EIF2B2 gene, c.545C>T(p.Thr182Met) and c.1340C>T(p.Ser447Leu) in the EIF2B5 gene were detected in the two patients. Further phenotype investigation of both patients enables the diagnosis of the vanishing white matter disease .Three missense mutation c.254 T>A (p.Val85Glu) in the EIF2B2 gene, c.545C>T(p.Thr182Met) and c.1340C>T(p.Ser447Leu) in the EIF2B5 gene have been found and predicted to be deleterious. All the three mutation causes hydrophobicity and stability changes of proteins, and all the mutations were localized in conserved sequences. One novel mutation of c.1340C>T(p.Ser447Leu) in the EIF2B5 gene ,and two other known mutations. The iterative threading assembly refinement (I-TASSER) server generated three-dimensional (3D) atomic models based on protein sequences from the novel missense mutation of c.1340C>T(p.Ser447Leu) in the EIF2B5 gene, which showed that the protein structure changed . The novel mutation c.597+2delT in the EIF2B2 gene may cause the splice site to disappear. We also analyzed mutations in missense mutations that cause VWMD


KEYWORDS vanishing white matter disease (VWMD), compound heterozygous mutations, novel mutation
Conclusions This study expands the spectrum of genotypes and phenotypes of VWMD and provides new insights into the molecular mechanism of VWMD and aide the acute diagnosis and treatment of VWMD.

Background
Vanishing white matter disease (VWMD, OMIM#603896) is a rare neurologic disease inherited in an autosomal recessive manner. It has been proven that mutations in the genes encoding subunits of the eukaryotic translation initiation factor 2B are involved in the generation of VWMD [1]. The clinical phenotype of VWMD is closely linked to white matter demyelinating lesions and associated progressive neurologic deficits including spasticity, progressive cerebellar ataxia, epilepsy and cognitive impairment. Outside stress such as minor head trauma, infection with fever and acute fright might lead to rapid neurological deteriorations [2].The onset of symptoms begins in young children but may appear at or shortly after birth or later in life. Typical onset of VWMD is between the ages of 2-6 years but varies from prenatal to adulthood. Patients with early-infantile onset, especially before the age of 2 years, present a severe phenotype that commonly leads to progressive deterioration and early death [3]. Brain MRI reveals extensive white matter abnormalities, with the signal intensity of the abnormal white matter close to that of cerebrospinal fluid [4,5]. VWMD is caused by mutations in any of the five genes: EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5 [6]. Due to the complex clinical phenotype of VWMD, it is often challenging in clinical practice for acute diagnosis of VWMD. The progress in molecular analysis technology has provided new tools to aid in accurate diagnosis of VWMD. Here, we present two patients with VWMD who were precisely diagnosed by the technique of exome sequencing in combination with brain MRI and clinical features. The results of this study provided new insights into the molecular mechanism of VWMD.

Data analysis
The data analysis was applied as follows: Synonymous changes and single nucleotide polymorphisms (SNPs) with a minor allele frequency higher than 5% were removed (http://www.ncbi.nlm.nih.gov/projects/SNP).
Analysis was performed of the function of mutated genes and the association with the disease.

Mutation confirmed by Sanger method sequencing
The PCR product was sequenced using a ABI 3730XL and analyzed with DNASTAR software in order to confirm the mutations discovered in the patients

Protein function prediction for the missense mutation
To predict the effect of amino acid substitutions, we performed in silico analysis using the PROVEAN (http://sift.jcvi.org) and.The variant is predicted to be deleterious if the PROVEAN score is≤2.5, and neutral if the score is >2.5.

Protein hydrophobicity analysis
We use the software ProtScale (https://web.expasy.org/protscale/) and the software

Protein stability analysis
To predict the stability of the protein after missense mutation, we use the software (http://mupro.proteomics.ics.uci.edu).The result is represented by the value detal delta G.
If the stability is reduced, it is represented by DECREASE stability.

Protein structure prediction on the I-TASSER server for the nonsense mutation and intron splicing mutation
The I-TASSER Suite pipeline consists of 4 general steps:threading template identification, iterative structure assembly simulation, model selection and refinement, and structurebased function annotation. The server is available at http://zhanglab.ccmb.med.

Ethical considerations
This study was approved by the Ethics Committee of Shanghai Children's Hospital.
Informed consent was obtained from the parents of the patient for using their blood samples for genetic analysis. The study complied with Chinese bioethics laws and the Helsinki declaration. Table 1 Case A.

Clinical data
In 2016, a 4-year-old Chinese girl, presenting with mild walking difficulty caused by minor head trauma was referred to the Neurology Department of the Shanghai Children's Hospital for treatment. She was born of nonconsanguineous parents at 39 weeks gestation with birth weight 3kg and birth length 50cm. She was reported to by the parents be normal, except she had a mild mental development delay. Ataxia or spasticity had also been observed by her parents. Neither electroencephalogram test nor Electrocardiogram revealed abnormalities The results of the brain magnetic resonance imaging indicated extensive white matter damage, with major damage appearing around the area of the dentate nucleus and globus pallidus (Fig. 1a, was unremarkable with alanine aminotransferase at 26 U/L (normal range 5-40 U/L) and aspartate aminotransferase at 33 U L (normal range 8-40 U/L). Her pulmonary, urological, cardiac, and renal functions and her levels of glucose and lactate were normal. She could walk with help, but her language ability was poor.
Magnetic resonance imaging (MRI) of the brain revealed that the symmetrical diffuse signal in the cerebral white matter was close to the cerebrospinal fluid on T1-weighted, T2-weighted and flair images (Fig. 1). No significant results were reported from screening for metabolic errors, including analyzing the amino acids level in the blood or urinary organic acid analysis. The girl was G2P1 with a birth weight of 3190 g. No history of genetic disorder has been found in her family.

Identification of Novel Mutations in EIF2B5 and EIF2B2
In case A, two missense heterozygous variants were identified as compound heterozygous in the EIF2B5 gene, c.545C>T(p.T182M) at exon 4 and c.1340C>T(p.S447L) at exon 9, which were inherited from the mother and the father respectively(see Figure 3 for more detail). One missense heterozygous variant and one canonical splicing variant in the EIF2B2 gene, c.254 T>A (p.V85E) at exon 2 and c.597+2del T at exon 4 inherited from the parents, were found in the compound heterozygous state in case B (Fig. 2). Pedigree of the 2 sporadic patients (Fig. 3).

Protein function prediction of the gene mutations
The PROVEAN scores of the two missense mutations c.545C>T (p.T182M) and c.1340C>T

Protein stability analysis
The protein after c.545C>T(p.T182M) mutation in EIF2B5 predicted both value and sign of energy change using SVM and sequence information only (Recommended) delta delta G = phenotypes of VWMD are ataxia, spasticity, and epilepsy in some patients [9]. Progressive rarefaction and cystic degeneration occurring in the white matter of the brain are the typical features of VWMD. Magnetic resonance imaging (MRI) of the brain shows symmetrically and diffusely abnormal white matter, with the signal intensity in the abnormal white matter is close to that of cerebrospinal fluid on T1-weighted, T2-weighted and fluid-attenuated inversion recovery [5]. Eline M. C et al [10]. performed a longitudinal multicenter study of 296 genetically diagnosed VWM patients and found that the median age of the first onset was 3 years old, with 60% of patients developing symptoms before the age of 4 years old. The nature of the first signs varied for different ages of onset.
Overall, motor problems were the most common presenting sign, especially in children.
Adolescent and adult-onset patients are more likely to show cognitive problems early after disease onset. The earlier the onset age, the higher the mortality rate, especially the age of onset is less than 1 year old. Absence of stress-provoked episodes and absence of seizures predicted more favorable outcome. In our study, the age of onset of patient B was 7 months, and combined with epilepsy and difficulty walking. The age of onset of patient A was 4 years old, no seizures, clinical symptoms were lighter than patient B.
VWM can be related to mutations in any of the five genes (EIF2B1-5), encoding the five subunits of eukaryotic translation initiation factor eIF2B (eIF2Bα, β, γ, δ, and ε). eIF2B assumes a task in the initiation of mRNA translation. Under stress conditions, protein synthesis is inhibited via eIF2B inactivation [11]. EIF2B5 is the most frequently mutated gene; mutations in EIF2B5 are detected in 57% of the patients. Mutation rates of the other genes are EIF2B4 (17%), EIF2B2 (15%), EIF2B3 (7%), and EIF2B1 (4%) respectively [12].  Identifying the genetic basis of VWMD in these two patients allows us to provide genetic counseling for their families and give them the option of prenatal testing, as in patient B whose mother has delivered a normal boy after prenatal testing. The neurological deterioration of these two patients was caused by febrile infections and minor head trauma.
There is no cure for this devasting disease. Physical therapy and rehabilitation for motor dysfunction and antiepileptic drugs for seizures control are suggested in clinical practice.
In addition, to avoid possible deterioration from outside attack, patients are advised to for vaccinations, low-dose of maintenance antibiotics during winter, antibiotics for minor infections, antipyretics for fever, and helmet when outside to minimize the impact of brain trauma. But scientists have been searching for ways to treat this disease. Zhou L et al [22] fibroblasted from two VWMD children were reprogrammed into iPSCs by using a virusfree nonintegrating episomal vector system. They discovered the astrocyte abnormalities, whether it can inhibit astrocyte abnormalities to treat VWMD, provides us with a new perspective. Mutations in eIF2B genes cause VWMD, a fatal leukodystrophy that can manifest following physical trauma or illness, conditions that activate the integrated stress response (ISR) [23]. Hodgson RE et al [24]. provided evidence that the composition and function of mammalian eIF2B bodies are regulated by the ISR and the drugs that control it. Abbink TEM et al [25] found the compound ISRIB (for "ISR inhibitor") was administered to VWMD mice to increase eIF2B activity. In brains of VWMD compared to wild-type mice we observed the most prominent changes in translation concerning ISR mRNAs;their expression levels correlated with disease severity.

Conclusion
We have reported here two VWMD cases with compound heterozygous mutations, c.

Ethics approval and consent to participate
The study procedure was permitted by the Ethics Committee of Children's Hospital of Shanghai, Shanghai Jiao Tong University,and informed written consent was obtained from the patient's guardian.

Consent for publication
A signed informed consent was obtained from the patient's guardian for publication of this case report and accompanying neuroimages.

Availability of data and materials
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interest.

Funding
This work was supported by grants from the National Natural Science Foundation of China      Pedigree of the 2 sporadic patients.

Figure 4
The 3-dimensional atomic model based on protein sequence of EIF2B5 from widetype generated by iterative threading assembly refinement server.

Figure 5
The 3-dimensional atomic model based on protein sequence from the missense mutation of c.1340C>T(p.S447L) in EIF2B5 generated by the iterative threading assembly refinement server.  EIF2B5 protein domain and distribution of fourteen pathogenic missense mutation sites causing vanishing white matter disease on the domain. p.T182M and p.S447L are missense mutations we found in our case, and p.S447L is a newly discovered mutation that has not been reported before.

Supplementary Files
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