RESEARCH ARTICLE A novel hypomorphic splice variant in EIF2B5 gene is associated with mild ovarioleukodystrophy Agustı Rodrıguez-Palmero1,2 , Agatha Schlu € ter1,3 , Edgard Verdura1,3 , Montserrat Ruiz1,3 , Juan Jose Martınez , Isabelle Gourlaouen , Chandran Ka4,5,6, Ricardo Lobato7, 1,3 4,5 Carlos Casasnovas1,3,8 , Gerald Le Gac4,5,6,9 , Ste phane Fourcade1,3 & Aurora Pujol1,3,10 1 Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, 08908, Spain 2 Pediatrics Department, University Hospital Germans Trias i Pujol, Badalona, 08916, Spain 3 Center for Biomedical Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain 4 INSERM U1078, Brest, France 5 Laboratory of Excellence GR-Ex, Paris, France 6 Laboratoire de G enetique Moleculaire et Histocompatibilit ^pital Morvan, Brest, France e, CHRU de Brest, Ho 7 Neurology Department, Hospital Universitario Infanta Sofıa, San Sebasti an de los Reyes, 28703, Spain 8 Neuromuscular Unit, Neurology Department, Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat, 08908, Spain 9 Universit e Bretagne Loire, Universite de Bretagne Occidentale, IBSAM, Brest, France 10 Catalan Institution of Research and Advanced Studies (ICREA), Barcelona, Spain Correspondence Abstract Aurora Pujol, Neurometabolic Diseases Laboratory, IDIBELL, Hospital Duran i Reynals, Objective: To identify the genetic cause in an adult ovarioleukodystrophy Gran Via 199, 08908 L’Hospitalet de patient resistant to diagnosis. Methods: We applied whole-exome sequencing Llobregat, Barcelona, Spain. Tel: +34 (WES) to a vanishing white matter disease patient associated with premature 932607137; Fax: +34 932607414; E-mail: ovarian failure at 26 years of age. We functionally tested an intronic variant by apujol@idibell.cat RT-PCR on patient’s peripheral blood mononuclear cells (PBMC) and by mini- Funding Information gene splicing assay. Results: WES analysis identified two novel variants in the This study was supported by grants from the EIF2B5 gene: c.725A > G (p.Tyr242Cys) and an intronic noncanonical muta- Hesperia Foundation, the Asociacio n Espan ~ola tion (c.1156 + 13G>A). This intronic mutation resulted into generation of vari- contra las Leucodistrofias (ALE-ELA Espan ~a), the ous isoforms both in patient’s PBMC and in the minigene splicing assay, Autonomous Government of Catalonia [SGR showing that ~20% residual wild-type isoform is still expressed by the intronic- 2014SGR1430, 2017SGR1206], the PERIS mutated allele alone, concordant with an hypomorphic effect of this variant. Program [SLT002/16/00174] from the Conclusion: We report two novel variants in EIF2B5, one of them a noncanon- Autonomous Government of Catalonia and the Center for Biomedical Research on Rare Diseases ical intronic splice variant, located at a +13 intronic position. This position is (CIBERER) [ACCI19-759] to A.P. This study has mutated only in 0.05% of ClinVar intronic mutations described so far. Further- been funded by the Marato  de TV3 [345/C/2014] more, we illustrate how minigene splicing assay may be advantageous when val- as well as by Instituto de Salud Carlos III through idating splice-altering variants, in this case highlighting the coexistence of wild- the projects [FIS PI14/00581] to C.C. (Co-funded type and mutated forms, probably explaining this patient’s milder, late-onset by European Regional Development Fund. ERDF, phenotype. a way to build Europe), [Miguel Servet Program CPII16/00016 (Co-funded by European Social Fund. ESF investing in your future)] to S.F, [Sara Borrell program, CD19/00221 (Co-funded by European Social Fund. ESF investing in your future)] to E.V., and the Center for Biomedical Research on Rare Diseases (CIBERER) to M.R. CIBERER ER20P2AC759/2020, substituting the current CIBERER grant Received: 5 June 2020; Accepted: 27 June 2020 Annals of Clinical and Translational Neurology 2020; 7(9): 1574–1579 doi: 10.1002/acn3.51131 1574 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. A. Rodrıguez-Palmero et al. EIF2B5 Ovarioleukodsytophy USA) with 100-bp paired-end read sequences generated Introduction on a HiSeq2000 (Illumina, Inc. USA) in the Centro Vanishing white matter disease (VWMD; OMIM Nacional de Analisis Gen omico in Barcelona (CNAG). #603896) is a leukodystrophy caused by recessive muta- Sequence processing was carried out with BWA aligner, tions in any of the five genes encoding subunits of trans- the Genome Analysis Toolkit (GATK), SAMtools, and lation initiation factor EIF2B. Manifestations usually start Picard Tools as previously described.6 between late infancy and early childhood, and mainly More information regarding methods are detailed in consist of pyramidal and cerebellar signs with mental the Data S1. decline and episodes of acute deterioration following stressors. However, 15% of cases are adult-onset forms associated with a more benign course and fewer decom- Results pensations.1 Infrequently, VWMD can appear associated Clinical findings with premature ovarian failure (POF), a clinical syndrome that has been called ovarioleukodystrophy.2 Although A 26-year-old woman was referred to the neurology EIF2B1-5 mutations are the main cause of leukodystrophy department because of a leukoencephalopathy detected with POF, other genes have been described.3 during the study of amenorrhea with hyperprolactinemia More than 120 mutations have been reported in >250 (108 ng/mL). She was born from nonconsanguineous par- patients with an EIF2B-related disorder, mostly in EIF2B5 ents and she had one asymptomatic sister. Physical exam and EIF2B2.4 No mutational hotspots have been found, showed right hand clumsiness, dystonic left foot postures, although some recurrent mutations seem to occur in and generalized hyperreflexia in absence of cognitive, paired cytosine/guanine (CpG) dinucleotides. The vast behavior or psychiatric symptoms. MRI evidenced T2 majority of pathogenic mutations are missense, whereas periventricular and pontine white matter hyperintensities truncating mutations (frameshifts, nonsense, splice site with malacic areas in the frontal and atrial horns. Corti- mutations) are rare and have been reported only in com- cal-subcortical, spinal, and corpus callosum atrophy were pound-heterozygous state,5 indicating that total loss of also reported (Fig. 1A). Magnetic resonance spectroscopy function may be incompatible with life. was unremarkable. In the next 5 years, she developed Here, we report a patient with an adult-onset ovari- unsteady gait, more evident pyramidal signs and lower oleukodystrophy carrying two novel disease-causing vari- limb paresthesia and cramps. Her cognitive level ants in EIF2B5 identified by WES, one of which is an remained normal and she did not develop seizures. MRI intronic mutation leading to activation of a cryptic splice performed four years after the initial one did not show donor site. The impact of this rare, noncanonical splice significant changes. Routine hematology and clinical variant on EIF2B5 pre-mRNA processing was evaluated chemistry tests, as well as thyroid and adrenal function, both in cDNA from patient’s PBMC, and using minigene were normal. Nerve conduction tests and electromyogra- splicing reporter assays. These experiments revealed resid- phy were normal, whereas visual-evoked potentials ual wild-type splicing, probably accounting for the mild showed bilateral latency enlargement of the P100 wave late-onset clinical phenotype in this patient. and somatosensory-evoked potentials exhibited bilaterally prolonged latencies with normal amplitudes. Methods WES analysis Participant and ethics Whole-exome sequencing (WES) revealed two suspicious Blood was processed by centrifugation within 2 h of col- novel variants in EIF2B5. The first one is a missense vari- lection using a gradient of Histopaque to separate plasma, ant (NM_003907:c.725A > G; p.Tyr242Cys) referenced in erythrocytes, and PBMC. Plasma and PBMC were stored dbSNP database as rs750767613, and has a frequency of at 80°C. The use of all samples was approved by the 3,98.10e-6 in gnomAD database, with 0 homozygotes. Clinical Research Ethics Committee of the Bellvitge This ultrarare missense mutation alters a highly conserved University Hospital (PR076/14). Informed written consent residue, and is predicted to be damaging by SIFT and was obtained from all patients and control individuals. PolyPhen-2. The second variant is intronic (NM_003907: c.1156 + 13G>A) and is predicted by different algorithms to increase the use of a cryptic donor splice site located at Exome sequencing and variant calling positions + 11/+12 (NNSplice, MaxEntScan, FSplice). In-solution exome capture was performed using the Seq- Conversely, Human Splicing Finder and SpliceView did Cap EZ Human Exome Kit v3.0 (Roche Nimblegen, not predict a significant impact on splicing (Table S1). ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 1575 EIF2B5 Ovarioleukodsytophy A. Rodrıguez-Palmero et al. Figure 1. Clinical and genetic features. (A) Periventricular T2 white matter hyperintensities with small areas of cystic degeneration, and corpus callosum and mild cerebellar atrophy. (B) Family tree and cosegregation analysis of EIF2B5 variants. (C) PCR amplified products of cDNA from II-1 and a healthy individual (CTL) resolved on a 4% agarose gel. L: Ladder (GeneRuler 50bp DNA ladder). (D) Partial sequence chromatograms of cDNA from CTL and II-1. Nucleotide and amino acid sequences of normal and mutated alleles are written above of each chromatogram. (E) RT- PCR from transfected HEK 293T17 and sequencing profiles of the products with retention of intron 7. Arrows indicate the relative positions of forward and reverse primers; the different constructs and sizes obtained are mentioned in the figure. L: Ladder; No RT: No Reverse Transcriptase; pSP: pSplicePOLR2G vector alone, pSP-EIF2B5: WT EIF2B5 cloned into pSP and pSP-EIF2B5-mut: intronic variant of EIF2B5 cloned into pSP. (F) Capillary electrophoresis of fluorescent RT-PCR products from wild-type and derived mutant minigenes, for the short and large constructs. 1576 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association A. Rodrıguez-Palmero et al. EIF2B5 Ovarioleukodsytophy This variant is absent from gnomAD database. Cosegrega- donor cryptic splicing site. Two splicing reporter vec- tion analysis revealed that the patient inherited these vari- tors were constructed in the background of the pSplice- ants in trans (Fig. 1B). Despite their rare frequency, POLR2G plasmid13: a small construction containing cosegregation, conservation of affected amino acids, in sil- EIF2B5’s exon 7 and flanking intronic sequences ico predictions for missense and splicing variants, and the (143 bp of intron 6202 bp of intron 7), and a large patient’s highly concordant phenotype, these variants construction encompassing EIF2B5’s exons 7 and 8 and were classified as variants of unknown significance (VUS) the whole intron 7 sequence. Both vectors, with either according to the ACMG/AMP (American College of Med- the wild-type or the mutated sequence, were transfected ical Genetics and Genomics) guidelines for variant evalua- into HEK293T/17, U-251MG, and COS-7 cell lines. tion.7,8 Results obtained from the smaller pSplicePOLR2G- EIF2B5 construct in HEK293T cells show that the 10 intronic bp insertion was mainly, but not only, In silico analysis of previously reported observed with the mutated c.1156 + 13G>A allele splicing variants (Fig. 1E). Similar results were observed in U-251MG So far, 11998 single-nucleotide variants probably affecting and COS7 cells (Fig. S2A–C). Semi-quantitative fluores- splicing and considered as pathogenic or likely pathogenic cent RT-PCR revealed a normal/abnormal-splicing ratio are listed in the ClinVar database. Splice-site mutations are of 0.9 for the wild-type allele and 0.2 for the mutated most commonly detected at the G(+1), T(+2), G(+5), A allele (547 bp peak area/557 bp peak area) (Fig. 1F). ( 2), G( 1) canonical sequences of introns, as shown in Similar results were obtained from the larger pSplice- Figure S1: +1 (4273 hits), 1 (2484), 2 (2181), +2 POLR2G-EIF2B5 construct. These results indicate that: (1429), and +5 (551). Frequencies then decrease dramati- (1) a cryptic donor splicing site is active in EIF2B5 cally as distance from the acceptor/donor splice site intron 7 and is moderately used by the splicing increases. Only two variants have been reported in position machinery in different cell types; and (2) this cryptic +13: the A > G transition in intron 10 of the MAPT gene, splicing site is significantly more active in the associated with familial frontotemporal dementia with c.1156 + 13G>A EIF2B5 pre-mRNA where it overcomes parkinsonism,9 and the C > T transition in FGB gene’s use of natural donor’s splicing site. intron 6, associated with congenital afibrinogenemia.10 In conclusion, we have identified a new intronic variant in the EIF2B5 gene which activates acryptic 5’ donor splice site of intron 7, probably leading to synthesis of a Functional assays truncated protein (if not degraded by nonsense-mediated Given c.1156 + 13G>A variant’s absence from databases mRNA decay). This splicing variant is hypomorphic, as it and the discordant results of splicing predictors, we leads to a residual 20% of WT splicing, and should be evaluated the functional significance of this variant considered pathogenic after applying the ACMG crite- in vitro. Reverse-transcription PCR encompassing exons ria.7,8 In consequence, as VUS missense variant 7-8 of the EIF2B5 mRNA produced a single 193 bp p.Tyr242Cys is in trans with the pathogenic intronic vari- product from the control samples, whereas two bands ant, this missense is reclassified as Likely Pathogenic7,8 (193 and 203 bp) were observed in the patient’s sam- and our case is solved. ple. Sanger sequencing showed that the longest product included the first 10 nucleotides of intron 7. This 10- Discussion bp insertion creates two adjacent premature termination codons (TGA and TAA), leading to a truncated protein Splicing mutations represent approximately one-third of (Fig. 1C and D). To confirm the association between disease-causing mutations, most of them occurring in the c.1156 + 13G>A EIF2B5 variant and partial reten- conserved consensus splice sites.14 However, the wide- tion of intron 7, we performed a minigene splicing spread use of high throughput technologies has increased assay. This technique consists of the construction of an the detection of variants in cryptic splice sites, which rep- expression vector containing a minimal gene fragment resent around 10% of total splicing mutations according encompassing the variant sequence of interest along to ClinVar data. In these cases, functional analysis is with flanking intronic sequences, and then is transfected mandatory to confirm variant’s pathogenicity since, as in into cultured cells in order to evaluate splicing pat- the case here reported, splicing predictors may provide terns.11,12 This strategy allowed us to study the monoal- discordant results. Our case also underscores the impor- lelic effect of this EIF2B5 variant compared to a wild- tance of thorough clinical evaluation guiding the bioinfor- type situation, and to evaluate more precisely the influ- matic analysis, to uncover variants in intronic regions. As ence of the G > A change on the use of the identified incomplete coverage of intron-exon boundaries in WES ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 1577 EIF2B5 Ovarioleukodsytophy A. Rodrıguez-Palmero et al. studies may hamper detection of intronic variants, WGS References may still be required to unravel some cases. 1. Hamilton EMC, van der Lei HDW, Vermeulen G, et al. To the best of our knowledge, only four splicing vari- Natural history of vanishing white matter. Ann Neurol ants have been reported in EIF2B genes so far,15–18 of 2018;84:274–288. which only one is noncanonical, although it was not 2. Schiffmann R, Tedeschi G, Philip Kinkel R, et al. functionally validated.15 Thus, our work is the first func- Leukodystrophy in patients with ovarian dysgenesis. Ann tional validation report of a novel, noncanonical splicing Neurol 1997;41:654–661. mutation in EIF2B5 associated with ovarioleukodystrophy. 3. Dallabona C, Diodato D, Kevelam SH, et al. Novel In this case, the c.1156 + 13G>A hypomorphic mutation (ovario) leukodystrophy related to AARS2 mutations. generates a novel splice site 10 bp beyond, that competes Neurology 2014;82:2063–2071. with the wild-type splicing donor site, therefore generat- 4. Maletkovic J, Schiffmann R, Gorospe RJ, et al. Genetic and ing two coexisting populations of transcripts, the clinical heterogeneity in eIF2B-related disorder. J Child mutated, and the WT form. Our work has benefited from Neurol 2008;23:205–215. the capacity of the minigene assay to quantify the mRNA 5. Bugiani M, Boor I, Powers JM, et al. Leukoencephalopathy production in an allele-specific manner. Indeed, minigene with vanishing white matter: a review. J Neuropathol Exp splicing assays have several advantages, such as: (1) cir- Neurol 2010;69:987–996. cumventing use of patient’s RNA if sample is not avail- 6. Pant DC, Boespflug-tanguy O, Pujol A, et al. Loss of the able; (2) analysis and quantification of the splicing sphingolipid desaturase DEGS1 causes hypomyelinating outcome of mutant alleles without interference of the leukodystrophy graphical abstract find the latest version. J wild-type allele; (3) high reproducibility of results; and Clin Invest 2019;129:1240–1256. (4) testing of variants located in any human disease gene, 7. Richards S, Aziz N, Bale S, et al. Standards and guidelines independently of gene expression. This approach allowed for the interpretation of sequence variants: a joint us to observe that the cryptic splice site activated by consensus recommendation of the American College of c.1156 + 13G>A is moderately functional in the wild-type Medical Genetics and Genomics and the Association for allele, and also that the mutated allele expresses a residual Molecular Pathology. Genet Med 2015;17:405–424. amount of wild-type mRNA. Competition between the 8. Amendola LM, Jarvik GP, Leo MC, et al. Performance of wild-type and the cryptic splice donor site could deter- ACMG-AMP variant-interpretation guidelines among nine mine the expression of some degree of functional EIF2B5 laboratories in the clinical sequencing exploratory research protein and consequently, the mild phenotype exhibited consortium. Am J Hum Genet 2016;98:1067–1076. by the patient. Our finding of a partial mis-splicing is 9. Hutton M, Lendon CL, Rizzu P, et al. Association of important in view of potential therapies since modulation missense and 5’-splice-site mutations in tau with the of splice processes represents a therapeutic approach for inherited dementia FTDP-17. Nature 1998;393:702–705. some genetic diseases.19 10. Spena S, Duga S, Asselta R, et al. Congenital Even if ovarioleukodystrophy seems to be an infrequent afibrinogenemia: First identification of splicing mutations cause of pure premature ovarian failure (POF),20 identifi- in the fibrinogen Bb-chain gene causing activation of cation of hyperprolactinemia or amenorrhea should cryptic splice sites. Blood 2002;100:4478–4484. prompt both an exhaustive neurological examination and, 11. Cooper TA. Use of minigene systems to dissect alternative possibly, the performance of a cranial MRI. If clinical and splicing elements. Methods 2005;37:331–340. neuroimaging studies are suggestive, WES or even WGS 12. Gaildrat P, Killian A, Martins A, et al. Use of splicing are recommended to detect intronic mutations in EIF2B reporter minigene assay to evaluate the effect on splicing genes that could be responsible for mild phenotypes. of unclassified genetic variants. Methods Mol Biol 2010;653:249–257. 13. Callebaut I, Joubrel R, Pissard S, et al. Comprehensive Acknowledgments functional annotation of 18 missense mutations found in suspected hemochromatosis type 4 patients. Hum Mol We thank CERCA Program / Generalitat de Catalunya for Genet 2014;23:4479–4490. institutional support. We thank Peio Aristu, Cristina 14. Sibley CR, Blazquez L, Ule J. Lessons from non-canonical Guilera, and Laia Grau for technical assistance. splicing. Nat Rev Genet 2016;17:407–421. 15. Fogli A, Schiffmann R, Bertini E, et al. The effect of Conflict of Interest genotype on the natural history of eIF2B-related leukodystrophies. Neurology 2004;11:1509–1517. The authors declare that this article was conducted in the 16. Horzinski L, Gonthier C, Rodriguez D, et al. Exon absence of any commercial or financial relationships that deletion in the non-catalytic domain of eIF2Be due to a could be construed as a potential conflict of interest. 1578 ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association A. Rodrıguez-Palmero et al. EIF2B5 Ovarioleukodsytophy splice site mutation leads to infantile forms of CACH/ panel of patients with premature ovarian failure. BMC VWM with severe decrease of eIF2B GEF activity. Ann Womens Health 2004;4:8. Hum Genet 2008;72(Pt 3):410–415. 17. Liu R, Van Der Lei HDW, Wang X, et al. Severity of vanishing white matter disease does not correlate with Supporting Information deficits in eIF2B activity or the integrity of eIF2B Additional supporting information may be found online complexes. Hum Mutat 2011;32:1036–1045. in the Supporting Information section at the end of the 18. Pronk JC, Van Kollenburg B, Scheper GC, Van Der Knaap article. MS. Vanishing white matter disease: a review with focus on its genetics. Ment Retard Dev Disabil Res Rev Data S1. In silico splicing predictors for variant 2006;12:123–128. c.1156+13G>A. 19. Spitali P, Aartsma-Rus A. Splice modulating therapies for Figure S1. Single-nucleotide splicing variants annotated human disease. Cell 2012;148:1085–1088. in ClinVar. 20. Fogli A, Gauthier-Barichard F, Schiffmann R, et al. Figure S2. Mini-gene splicing analysis of EIF2B5 Screening for known mutations in EIF2B genes in a large c.1156 + 13 G> A variant. ª 2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 1579