8. Ehlers B, Diringer H. Dextran sulphate 500 delays and prevents mouse scrapie by impairment of agent replication in spleen. J Gen Virol 1984;65:1325–1330. 9. Kimberlin RH, Walker CA. Suppression of scrapie infection in mice by heteropolyanion 23, dextran sulfate, and some other polyanions. Antimicrob Agents Chemother 1986;30:409 – 413. 10. Doh-ura K, Iwaki, Caughey B. Lysosomotropic agents and cysteine protease inhibitors inhibit scrapie-associated prion protein accumulation. J Virol 2000;74:4894 – 4897. 11. Supattapone S, Nguyen H-O, Cohen F, et al. Elimination of prions by branched polyamines and implications for therapeutics. Proc Natl Acad Sci USA 1999;96:14529 –14534. 12. Caughey B, Raymond G. Sulfated polyanion inhibition of scrapie-associated PrP accumulation in cultured cells. J Virol 1993;67:643– 650. 13. Korth C, May BC, Cohen F, Prusiner SB. Acridine and phenothiazine derivatives as phamacotherapeutics for prion disease. Proc Natl Acad Sci USA 2001;98:9836 –9841. 14. Love R. Old drugs to treat new variant Creutzfeldt-Jakob disease. Lancet 2001;358:563. 15. Caughey B, Race R. Potent inhibition of scrapie-associated PrP accumulation by Congo red. J Neurochem 1992;59:768 –771. 16. Caughey B, Ernst D, Race R. Congo red inhibition of scrapie agent replication. J Virol 1993;67:6270 – 6272. 17. Diringer H, Ehlers B. Chemoprophylaxis of scrapie in mice. J Gen Virol 1991;72:457– 460. 18. Caughey WS, Raymond LD, Horiuchi M, Caughey B. Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines. Proc Natl Acad Sci USA 1998;95: 12117–12122. Cree Leukoencephalopathy and CACH/VWM Disease Are Allelic at the EIF2B5 Locus Anne Fogli, PhD,1 Kondi Wong, MD,2 Eleonore Eymard-Pierre, PhD,1 Jack Wenger, BSc,2 John-Paul Bouffard, MD,2 Ehud Goldin, PhD,3 Deborah N. Black, MD,4 Odile Boespflug-Tanguy, MD, PhD,1 and Raphael Schiffmann, MD3 Cree leukoencephalopathy is a rapidly fatal infantile autosomal recessive leukodystrophy of unknown cause observed in the native North American Cree and Chippewayan indigenous population. We found in the brain of affected individuals the typical foamy cells with the oligodendroglial phenotype described in central hypomyelination syndrome/vanishing white matter, a syndrome related to mutations in the genes encoding the five subunits of the eucaryotic translation initiation factor eIF2B. In three patients of two Cree families, we found a homozygous missense mutation resulting in a histidine substitution at arginine 195 of -eIF2B. Ann Neurol 2002;52:506 –510 An infantile leukoencephalopathy among the native Cree and Chippewayan indigenous population in Northern Quebec and Manitoba was first described in 1988.1 The onset of Cree leukoencephalopathy (CLE) is between 3 and 9 months of age with death in 100% by 21 months of age. Hypotonia often is noted in early infancy followed by relatively sudden onset of seizures, spasticity, hyperventilation, vomiting, and diarrhea, often in the setting of a febrile illness. Onset is followed by developmental regression, lethargy, blindness, and cessation of head growth seen as flattening of the head circumference curve.1 Computerized tomography of From the 1Institut National de la Santé et de la Recherche Médicale UMR 384, Facultéde Médecine, Clermont-Ferrand, France; 2Department of Neuropathology at the Armed Forces Institute of Pathology, Washington, DC; 3Developmental and Metabolic Neurology Branch, National Institutes of Health, Bethesda, MD; and 4 Department of Psychiatry, Université de Montréal, Montréal, Quebec, Canada. Received Apr 24, 2002, and in revised form Jun 13. Accepted for publication Jun 14, 2002. Published online Aug 28, 2002, in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.10339 Address correspondence to Dr Boespflug-Tanguy, INSERM UMR 384, Faculté de Médecine, 28, place Henri Dunant BP 38, 63001 Clermont-Ferrand Cedex, France. E-mail: odile.boespflug@inserm. u-clermont1.fr 506 © 2002 Wiley-Liss, Inc. the head shows symmetrically hypodense white matter. A similar image was seen on T1-weighted head magnetic resonance imaging that showed symmetrical diffuse attenuation of hemispherical and often cerebellar white matter.2 T2-weighted magnetic resonance images showed hyperintense white matter that included the subcortical fibers, basal ganglia, and thalamus. Previous gross neuropathological examination showed that white matter was grayish white with translucent zones and subcortical cavitation. Microscopic examination showed diffuse white matter vacuolation in some cases and astrogliosis with presence of oligodendrocytes and cells described as lipid-laden macrophages.1,2 Parents of affected children are normal, and because there is a strong history of consanguinity in this population, CLE is considered autosomal recessive. We microscopically investigated three brains of CLE patients and found the typical foamy oligodendrocytes we earlier reported in childhood ataxia with diffuse central hypomyelination syndrome (CACH)3,4 also called myelinopathia centralis diffusa5 or vanishing white matter disease (VWM).6 Mutations in each of the five subunits of the translation initiation factor 2B (eIF2B) have been reported recently in this syndrome.7,8 We first investigated the EIF2B5 gene, which encodes the ε subunit, and is localized in the 3q27 region,9 a region involved in CACH/VWM including in a severe variant we recently reported.10,11 Subjects and Methods Light Microscopy Paraffin blocks of tissue were histologically sectioned to a 9m thickness and stained with standard hematoxylin and eosin, periodic acid–Schiff (PAS)/Luxol fast blue, Bielschowsky, and modified Alcian blue (8GX)/PAS. The modifications to the standard AB8GX/PAS staining procedure consisted of placing the histological sections in 3% acetic acid at pH 2.5 for 5 minutes before the Alcian blue 8GX reagent in 3% acetic acid solution for 30 minutes. After a 5-minute wash, the sections were placed in periodic acid solution for 10 minutes followed by Schiff’s reagent (Colmann’s Feulgen reagent) for 20 minutes. The sodium metabisulfite rinse was purposefully omitted. The slides were washed in warm tap water until the water was pink, and the slides were left to stand in the warm pink tap water for 5 minutes. (The rational being that the pink dye neutralizes chlorine; otherwise, chlorine bleaches the Schiff’s reagent resulting in loss of coloration.) The sections then were counterstained with Richard Allen Scientific (Kalamazoo, MI) hematoxylin I counterstain. EIF2B5 Analysis SUBJECTS. We analyzed two CLE affected families (894 and 866; Fig 1). Patients II1 and II2 of Family 894 had mild motor developmental delay during early infancy followed by acute neurological deterioration during a febrile illness at ages 7 months and 5 months, respectively. Both patients died within 10 days of onset of acute neurological symptoms. The patient of Family 866 presented at age 4 months with developmental delay followed by progressive deteriora- Fig 1. EIF2B5 mutations in Cree leukoencephalopathy (CLE) families. A G584A mutation was found in an homozygous state in the three CLE affected individuals (Family 866-II1, Family 894-II1 and II2) and in an heterozygous state in both parents (Family 866-I1 and I2, Family 894-I1 and I2) and in one unaffected individual (Family 894-II4). Two individuals were homozygous for the wild-type G nucleotide (Family 894-II3 and Family 894-II5). Fogli et al: CLE and EIF2B 507 tion to a decerebrate, nonresponsive state by age 13 months and death 1 month later. The appropriate institutional review boards approved the study, and written informed consent was obtained from all participating subjects or from their legal guardian. DNA AND SEQUENCE ANALYSES. Genomic DNA was extracted from blood lymphocytes using the Nucleon kit (Amersham, Buckinghamshire, UK). We used 10 sets of oligonucleotide primers to amplify by polymerase chain reaction and sequence the 16 coding regions of the EIF2B5 gene including 39 to 173 nucleotides of the intronic regions (primer sequences and conditions are available upon request). Results “Foamy” Oligodendrogial Cells Are Observed in Cree Leukoencephalopathy Brains Cerebral cortical and cerebellar white matter was rarefied, with profound myelin loss, but with relative sparing of the axons. The cerebellar cortex was atrophic with thinning of the foliae and loss of cells in the granular layer. Marked neuronal loss also was observed in the hippocampus. Numerous cells with round to oval nuclei and abundant foamy-vacuolated cytoplasm were scattered throughout the cerebral and cerebellar deep white matter on hematoxylin and eosin staining (Fig 2A and B). The foamy cells had light blue cytoplasm and a bright red Golgi region on modified PAS/Alcian blue 8GX staining (see Fig 2C). The foamy cells were strongly immunoreactive to myelin oligodendrocyte glycoprotein antibody (see Fig 2D), as were the foamy oligodendroglial cells seen in CACH/VWM (see Fig 2F). The abundance of abnormal foamy oligodendroglial cells was in direct proportion to the amount of white matter left in the brain. There were no parenchymal macrophages identifiable with the CD68 marker (see Fig 2E). Glial fibrillary acidic protein staining showed atypical astrogliosis with blunted processes (see Fig 2G) and many nests of atypical gliofibrillar cells scattered throughout the white matter (see Fig 2H and I). The Gene Causing Cree Leukoencephalopathy Syndrome Is EIF2B5 Similarities in pathological features between CLE and CACH/VWM led us to look for possible mutations in the EIF2B genes. Because a severe variant of CACH is linked to chromosome 3q27, we first investigated the EIF2B5 gene encoding the ε-subunit of the eIF2B factor. We found a homozygous G584A missense mutation in exon 4 of EIF2B5 in all three affected individuals resulting in a histidine substitution at arginine 195 of ε-eIF2B (see Fig 1). Unaffected parents in both families were heterozygous for the mutation as well as one unaffected sibling, whereas two nonaffected children were homozygous for the wild-type allele (see Fig 1). 508 Annals of Neurology Vol 52 No 4 October 2002 This homozygous G584A mutation was not present in a control group of 110 individuals including 50 nonaffected adults from the Cree population. In this Cree control group, five individuals have this mutation in a heterozygous state yielding a mutated allele frequency of 1 of 20. Discussion We recently noticed similarities and differences between CLE and a severe variant of CACH/VWM. Investigating microscopically the brain of three CLE patients, we found neuropathological hallmarks of CACH/VWM: cavitating orthochromatic leukodystrophy with rarity of myelin breakdown and relative sparing of axons.12 In all three Cree brains examined, we observed abnormal cells identical to the foamy oligodendroglial cells that we described in CACH/VWM.4 Atypical astrogliosis with blunted processes and many nests of atypical gliofibrillar cells scattered throughout the white matter were observed in CLE and absent in CACH/VWM. Subsequently, using molecular analysis, we demonstrated that CLE is allelic to CACH/VWM at the 3q27 locus: all affected Cree infants analyzed showed a founder homozygous missense mutation R195H in the ε-eIF2B. The eucaryotic translation initiation factor 2B (eIF2B) is composed of five different subunits (␣, ␤, ␥, ␦, and ε) and converts the protein synthesis initiation factor 2 (eIF2) from an inactive guanine diphosphate (GDP)-bound form to an active eIF2–guanine tri-phosphate (GTP) complex. In CACH/VWM patients, mutations has been found in each of these subunits without correlation between severity of the phenotype and the mutations detected in the eIF2B genes; both childhood and juvenile forms were observed in siblings.7 In contrast, all CLE patients express a very severe phenotype. Homozygous R195H mutation that affects the guanine exchange factor domain of the ε-eIF2B has never been reported in CACH/VWM7,8 to our knowledge, or in our large patient population, including in a recently reported severe form of CACH/VWM. We recently identified a heterozygous R195H ε-eIF2B mutation in only one CACH/VWM family from Highlands in Scotland (A. Fogli, D. Rodriguez, E. Bertini, R. Shiffmann, D. Boespflug-Tanguy, unpublished results). The northern Quebec Cree first encountered Europeans in the early 1700s; these were Scottish fur traders from the Hudson Bay Trading Company.14 Probands from the two Cree families in this report can trace their paternal ancestors to three English Hudson Bay Company employees around 1770 (J. Pachano, personal communication). A founder effect arising from a common ancestor may explain the presence of the ε-eIF2B mutation in this population and perhaps also in Scotland. The indigenous North American population of Fig 2. Foamy oligodendroglial cells in the white matter of Cree leukoencephalopathy (CLE) brains. Light microscopy of CLE white matter brain sections (A, B, hematoxylin and eosin stain) shows numerous cells having small, eccentrically placed, round to oval nuclei (A, arrows) with fine chromatin and abundant foamy cytoplasm. When the nucleus is out of the plane of section, only a membrane-bound agglomeration of foamy cytoplasm is visible (A, arrowheads). Cytoplasmic vacuolations (B, arrows) occasionally are identified in the foamy cells that range in size from 12m to over 35m in diameter (B, arrowhead). Modified periodic acid– Schiff/Alcian blue 8GX staining (C) shows light blue–stained cytoplasm with a bright red Golgi region (C, arrow). The foamy cells react strongly to antibodies against myelin oligodendrocyte glycoprotein (MOG; D) but are nonreactive to CD68 (E) indicating an oligodendroglial rather than a monocytic phenotype. Compare the MOG immunoreactivity of foamy oligodendroglial cells in CLE (D, arrowheads) with those in central hypomyelination syndrome/vanishing white matter (F, arrowhead). Glial fibrillary acidic protein immunostaining (G–I) highlights atypical astrogliosis with coarse, blunted glial processes (G, arrowheads) and unusual gliofibrillary structures (H, I; arrowheads). A to D and F are ⫻400 original magnification; E and G to I are ⫻200 original magnification. northern Quebec may have evolved an adaptation to an extremely cold environment, rendering them particularly susceptible to dysregulation of protective mechanisms that respond to temperature elevation. Regulation of eIF2B represents a major protective mechanism of cells in response to heat stress.15 This mutation and other EIF2B5 mutations may increase the binding affinity of GTP/GDP preventing the cell from sensing a decrease in nucleotide availability during a traumatic event, thereby causing the protein to stay constitutively active. As a consequence during stress (eg, fever), eIF2B may be unable to decrease protein synthesis causing accumulation of denatured proteins. However, a reduction of eIF2B activity cannot be ruled out currently. A long presymptomatic phase, despite the presence Fogli et al: CLE and EIF2B 509 of severe white matter abnormalities on magnetic resonance imaging, has been observed in CACH/VWM, suggesting a pre-existing dysfunction in myelinogenesis.3 The basal ganglia and thalamic abnormalities described in CLE2 have not been observed in CACH/ VWM but are classically observed in other severe leukoencephalopathies with vacuolation of the white matter such as Alexander’s disease that is caused by astrocytic dysfunction caused by glial fibrillary acidic protein mutations (OMIM 137780).16 CLE may represent the most severe observed form of eIF2B-related disorders, possibly because of an exaggerated response to heat stress induced by a common infectious illness. In this population with a very high rate of consanguinity, the ability to detect heterozygous carriers and affected fetuses provides the possibility for genetic counseling and prevention of this very devastating disorder. This work was supported by grants from the European Leukodystrophy Association (ELA, G. Alba, president), the Institut National de la Santé et de la Recherche Médicale (INSERM projet PROGRES), the Jean Pierre and Nancy Boespflug myopathic research foundation, and the National Institute of Neurological Disorders and Stroke (protocol 97-N-0170). We gratefully acknowledge the participation of the patients’ families and the support of the Eeyou Awaash Foundation (A. Bearskin, President; W. Neacappo, Vice-President). We also thank L. Dauche, P. Combes, F. Gauthier, and G. Giraud for technical help in processing blood samples and in sequencing, and D. G. Schoenberg, MS, for editorial assistance. References 1. Black DN, Booth F, Watters GV, et al. Leukoencephalopathy among native Indian infants in northern Quebec and Manitoba. Ann Neurol 1988;24:490 – 496. 2. Alorainy IA, Patenaude YG, O’Gorman AM, et al. Cree leukoencephalopathy: neuroimaging findings. Radiology 1999; 213:400 – 406. 3. 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