*Corresponding author: Dr. Anne Fogli, INSERM UMR384, Faculté de Médecine, 28 place Henri Dunant, 63000 Clermont-Ferrand. Phone: 33 4 73 44 86 57. Fax: 33 4 73 17 83 83. E-mail: firstname.lastname@example.org
The CACH/VWM syndrome is an autosomal recessive leukodystrophy characterized by a broad spectrum of clinical presentations and by diffuse cavitary degeneration of the white matter on MRI. Mutations responsible for this disorder are missense or frameshift mutations occurring in the five genes (EIF2B1-5) that encode the translation eukaryotic initiation factor eIF2B. We found that a patient with infantile CACH/VWM carries a mutation in the acceptor splice site of EIF2B5 exon 6. In lymphoblastoid cells of the patient, we detected an abnormal EIF2B5 transcript in which exon 6 was absent, however, the predicted protein product lacking part of the non-catalytic domain encoded by exon 6 was not detected. The eIF2B GEF activity was severely decreased. These data support the importance of the non-catalytic domain of the eIF2Bɛ subunit in the eIF2B complex formation and activity.
The eIF2B factor is involved in the translation initiation process by converting the eIF2 factor from an inactive GDP-bound form to an active GTP-bound form through its nucleotide guanine exchange activity (GEF activity). The active eIF2-GTP can be integrated to the pre-initiation 43S complex which is a key regulator of protein synthesis particularly under cellular stress such as endoplasmic reticulum (ER) stress or viral infection. To date, the large majority of the mutations described in the five genes EIF2B1-5 are missense or frameshift mutations leading to instability of the enzymatic eIF2B complex and/or to an impairment of interaction with eIF2 subunits (Richardson et al., 2004). This results in a decrease in the eIF2B GEF activity which correlates with the severity of the disease (Fogli et al., 2004b). Premature terminations have been observed in nine CACH/VWM families, but none in homozygozity, suggesting that the complete loss of eIF2B factor's function is lethal for humans (Fogli & Boespflug-Tanguy, 2006; Pronk et al., 2006; Scali et al., 2006).
We report here the clinical phenotype and functional analysis of an acceptor splice site mutation of exon 6 (c.766-1G>A) in the EIF2B5 gene in two affected siblings presenting with an infantile form of CACH/VWM. This paternally transmitted mutation is associated with the classically mild c.338G>A mutation (p.Arg113His) in the maternal allele. In the lymphoblastoid cells of one of the patients, an abnormal EIF2B5 transcript in which the entire exon 6 is absent was identified whereas the corresponding eIF2Bɛ protein product was not detected by Western analysis and the eIF2B GEF activity was severely decreased. These results suggest a rapid degradation of the exon 6 deficient subunit or its low incorporation in the eIF2B complex leading to its instability and decreased enzymatic activity.
Materials and Methods
The two affected sisters of this non consanguineous French family (G569) present with a classical infantile form of CACH/VWM disease. After an initial normal psychomotor development (walking age at 14 months) both developed progressive gait difficulties between 3 and 4 years of age related to a cerebello-spastic syndrome. Loss of autonomous walking was observed between 11 and 13 years of age. The oldest one (G569-3) had a rapid neurological deterioration at 13 years of age after status epilepticus without any provoking factor and death occurred at 26 years of age. For her younger sister (G569-4), frequent seizures had been observed for 10 years, a rapid neurological degradation occurred at 17 years old after bladder surgery leading to death at 18. Cerebral MRI of patient G569-3 performed at 25 years of age showed a typical diffuse abnormal signal of the cerebral hemispheric white matter with a CSF like signal intensity on T1 and T2 weighted images and a cystic breakdown on fluid-attenuated inversion recovery (FLAIR) images (Figure 1). For the patient G569-4, a computerized axial tomography (CAT) scan of the head performed at 6 years of age showed extensive hypo-density of the cerebral white matter. No other imaging studies were available.
Proteins, Genomic DNA and RNA Analyses
Genetic analyses were performed after informed consent was obtained from the parents. Samples were available only for the index patient G569-3 and her two parents (G569-1 and G569-2). Total genomic DNA was extracted from lymphoblastoid cell lines prepared from each subject using the Nucleon kit (Amersham, Buckinghamshire, UK). The exons and flanking intronic regions of the EIF2B5 gene were amplified by PCR and sequenced as previously described (Fogli et al., 2004a).
Total RNA was extracted from lymphoblastoid cell lines using the RNeasy® Mini Kit (Qiagen GmbH, Hilden, Germany) and cDNA was obtained by RT-PCR amplification using the Superscript III™ Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. A 690 bp cDNA fragment was amplified by PCR using a forward primer in exon 2 and a reverse primer in exon 7 (forward: 5′-TGCCACAGGTGTACAGGAAA-3′; reverse: 5′-ATGACGTCAGCACAGACAGC-3′). PCR was performed in a final volume of 25μl containing 100 ng cDNA, 1 pmole of each primer, 5 mM each of dNTP, 10X buffer, 1 mM MgCl2, 1 unit of DNA polymerase (AmpliTaq Gold, Applied Biosystems, Foster City, USA). Initial denaturation at 95°C for 12 min was followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 62°C for 30 seconds and extension at 72°C for 45 seconds. A final extension step was performed at 72°C for 10 minutes. PCR products were resolved on a 2% agarose gel. After single-band gel excision and purification using the DNA Gel Extraction Kit (Millipore Corporation, Bedford, USA), each of the amplified fragments was directly sequenced in both forward and reverse directions.
Proteins were extracted from lymphoblastoid cells lines using the RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100, 0,1% SDS, 50 mM NaF, 2 mM Na3VO4, 25 mM β-glycerophosphate, 1 mM PMSF, 1X protease cocktail inhibitor P8340 from Sigma). The lysates were centrifuged at 10 000 g for 10 min and the resulting supernatants quantified using the classical Bradford method (Biorad, Hercules, USA). For Western Blotting, 200 μg of total proteins were loaded per well on a 6% SDS-PAGE gel to electrophoresis and transferred on nitrocellulose membrane (Millipore). After 2h of saturation with TBST supplemented with 5% skimmed milk powder (Regilait, France), the membrane was incubated with the anti-eIF2Bɛ antibody (epitope outside the coding region of exon 6) at a 1:500 dilution (#3595, Cell Signaling Technology, Danvers, USA) and with an anti-tubulin antibody at a 1:1000 dilution (T3526, Sigma) overnight at 4°C. After incubation with a secondary anti-Rabbit antibody HRP-conjugated at 1:1500 dilution (#7074, Cell Signaling Technology) and anti-Mouse antibody HRP-conjugated at 1:2000 dilution, (sc-2380, Santa Cruz Biotechnology, Santa Cruz, USA) for 2h at room temperature, the protein bands were detected using the ECL+Plus kit (Amersham) according to the manufacturer's instructions.
eIF2B GEF Activity Measurement
The GEF activity of eIF2B was measured as previously described (Fogli et al., 2004b) using one million lymphoblastoid cells. Each assay was performed in triplicate.
Identification of an EIF2B5 Splice Site Mutation at the Genomic Level
Sequencing analysis for the patient G569-3 allowed us to identify two mutations at the heterozygous state in the EIF2B5 gene: the already described and fairly common c.338G>A mutation (p.Arg113His), on the maternal allele and a novel mutation affecting the acceptor splice site of exon 6: c.766-1G>A, on the paternal allele (Figure 2A). Using the splice prediction software SpliceView™, this novel splice site mutation is predicted to delete the entire exon 6.
Consequences of the EIF2B5 Splice Site Mutation at mRNA and Protein Levels
In order to analyse the effects of the mutation, we extracted total RNAs from lymphoblastoid cell lines from each subject. PCR amplification of the EIF2B5 cDNA from exons 2 to 7 generated a 612 bp product for the proband G569-3 and her father (G569-1), in addition to the 690 bp product which was also found in the mother (G569-2) cDNA (Figure 2B). Sequencing of the abnormal smaller product showed an EIF2B5 cDNA variant deleted from nucleotides 766 to 843 (r.766-843del) corresponding to the skipping of exon 6 (78bp), which should result in an eIF2Bɛ protein lacking 26 amino acids (p.256_281del) (Figure 2C). Sequencing of the 690 bp PCR product amplified in the proband G569-3 and her mother RNA confirmed the presence of the c.338G>A mutation in the heterozygous state.
Western blot analysis of protein extracts from patient G569-3 lymphoblastoid cells showed only one band at 80 kDa corresponding to the normal molecular weight of eIF2Bɛ (Figure 2D). Western analysis of the father's lymphocytes, expressing the mRNA r.766_843del variant at the heterozygous state, also detected exclusively the normal 80 kDa band similarly to the mother's and control lymphocytes. Therefore in these cell lines, the splice site mutation is expressed at the RNA level whereas the predicted deleted protein is not detected by Western Blot analysis.
Functional Consequences of the EIF2B5 Mutations: Measurement of the eIF2B GEF Activity
The eIF2B GEF activity was measured in lymphoblastoid cells from the affected patient G569-3 and her parents. These activities were compared to those of two wild-type individuals. A significant decrease in the GEF activity was observed in cells from the affected mutated patient G569-3 (40 ± 2%) compared to cells from normal controls. Cells from both parents G569-1 and G569-2 exhibited normal GEF activities, respectively 98.6 ± 4.5% and 104.4 ± 9.3%versus 100.3 ± 11.6% for controls. These results demonstrate that a reduction in eIF2B GEF activity correlates with the disease and is observed only when both alleles of the same EIF2B gene are mutated.
EIF2B5 mutations are found in 63% of CACH/VWM patients, the majority being missense mutations (Fogli & Boespflug-Tanguy, 2006). Frameshifts and premature terminations causing major alteration at the protein level are always found in the compound heterozygous state with “milder” mutation such as the p.Arg113His mutation (Fogli & Boespflug-Tanguy, 2006; Pronk et al., 2006; Scali et al., 2006). Here, we describe disease associated with a novel mutation at the acceptor splice site of EIF2B5 exon 6, underscoring the importance of screening for mutations in EIF2B non-coding regions for the molecular diagnosis of eIF2B-related disorders. The c.766-1G>A mutation identified on the acceptor splice site of the exon 6 of the EIF2B5 gene has severe consequences: (1) the two sisters who inherited the mutation from the father have a late infantile form of CACH/VWM despite the presence of the “mild” c.338G>A mutation (p.Arg113His) on the maternal allele, (2) the resulting eIF2B GEF activity measured in lymphoblastoid cells of one of the affected sisters is low (40% of the normal value), compared to the 65.8 ± 1.8% value usually found for patients homozygous for the p.Arg113His mutation (Fogli et al., 2004b). We demonstrated that both mutations are expressed at the mRNA level with the identification of an aberrantly spliced transcript resulting from exon 6 skipping (r.766_843del). However, the predicted p.256_281del eIF2Bɛ protein could not be detected by Western blot analysis. Similarly, for the father heterozygote for the splice site mutation, the r.766_843del transcript has been identified but not the p.256_281del eIF2Bɛ protein. The absence of the predicted abnormal protein could result from its degradation by the ubiquitin-proteasome system due to maturation or folding defects and/or to its low incorporation into the eIF2B complex. The normal eIF2B enzymatic activity observed in the father heterozygote cells suggests that the level of eIF2Bɛ subunits produced by the normal allele and incorporated into the final eIF2B pentameric complex is adjusted in order to compensate the defect of the mutated allele. Identically, the normal eIF2B GEF activity observed in cells carrying only one heterozygous EIF2B missense mutation (Fogli et al., 2004b) could be explained by the preferential incorporation of the normal subunit in the eIF2B complex. When both EIF2B alleles carry missense mutations, the mutated subunits are incorporated into the eIF2B complex, causing instability of the complex and reduced GEF activity (Richardson et al., 2004; Li et al., 2004) in keeping with the observation that the defect in GEF activity correlates with the type of EIF2B mutation and to the age of disease onset (Fogli et al., 2004b). The clinical phenotype and the degree of eIF2B GEF activity detected in the compound heterozygous patients carrying the splice site mutation and the p.Arg113His mutation are more severe than those of patients homozygous for the p.Arg113His mutation. Interestingly, they are similar to those of compound heterozygous patients carrying the p.Arg113His mutation and a mutation in the second allele leading to a truncated eIF2Bɛ subunit affecting the catalytic domain (Fogli & Boespflug-Tanguy, 2006; Scali et al., 2006; Pronk et al., 2006). As it has been shown by Li et al., 2004, a protein in which the catalytic domain of the eIF2Bɛ subunit is absent impairs eIF2B complex formation suggesting its importance in eIF2B complex stability. In this study, we show that deletion of 26 amino-acids in the non-catalytic region which mediates interactions with the other eIF2B subunits, is also important for eIF2B complex stability and activity despite the presence of the catalytic center. These observations confirm the importance of the N-terminal region of the eIF2Bɛ subunit in the eIF2B complex formation.
Together, these results demonstrate the severe deleterious consequences of an acceptor splice site mutation in the compound heterozygous state with the “mild” p.Arg113His mutation. However, when the same mutation is associated with a normal allele there is normal eIF2B complex formation and activity. Further functional analysis to confirm this hypothesis would be of great interest for gene therapy strategies.
We gratefully acknowledge the participation of the patients and their family. We thank F. Gauthier and P. Combes for technical help in sequencing and western blot analysis and F. Cambi for review of the manuscript. This work was supported by grant for the European Leukodystrophy Association (ELA) Research Foundation.