ALS phenotypes with mutations in CHMP2B (charged multivesicular body protein 2B)

Approximately 10% of ALS cases are familial, linked at present to nine distinct loci. In 5 to 10% of motor neuron disease (MND) cases, a frontotemporal type dementia (FTD) is present.¹ Here we show that CHMP2B (charged multivesicular body protein 2B), a gene that was recently shown to be linked to FTD,² was altered in two unrelated patients with ALS-spectrum disorders. One mutation is predicted to alter a conserved functional domain. Pathology from this case strengthens the hypothesis of a common molecular pathology between ALS and FTD.³ The second case showed a point substitution that has been previously described as a low-frequency variation in the CHMP2B gene.⁴

Case reports.

Methods.

Results.

Mutation analysis of CHMP2B identified previously undescribed heterozygous mutations. A single nucleotide change, A161G, was identified in Patient 2 predicting an isoleucine to valine substitution (I29V) (figure E-1A). A different single nucleotide change, A694C, in exon 6 (RefSeq NM_014043) was identified in Patient 1 predicting a glutamine to histidine substitution (Q206H) (figure E-1B). Q206H and I29V were not identified in 640 control samples (120 CEPH individuals, 100 Danish individuals, 420 UK white individuals), in the public SNP databases, or any of the other 170 ALS samples screened as part of this study, nor in 400 FTD samples previously published.²

Neuropathology in Patient 1 showed no upper motor neuron pathology in the motor cortex (figure, A, or in multiple levels of the corticospinal tracts based on conventional stains and immunocytochemistry for CD68. Lower motor neurons (LMNs) in the ventral horn of the spinal cord and hypoglossal nuclei were depleted. Surviving LMNs showed classic ubiquitylated inclusion bodies, negative for tau and α-synuclein, characteristic of ALS/MND, but there were no Bunina bodies. The case was assigned a pathologic diagnosis of PMA. After the discovery of Q206H genetic change in CHMP2B, the histology was reviewed with additional stains including p62/sequestosome1, a marker of ubiquitylated inclusions in a variety of neurodegenerative disorders.⁵ Antibody to p62/sequestosome1 labeled LMN inclusions intensely (figure, B and C) and revealed a previously unrecognized pathology in the motor cortex comprising both neuritic profiles and coiled body type inclusions (figure, D and E). These coiled bodies were localized to oligodendroglia by double-labeling immunocytochemistry to carbonic anhydrase II (figure, F through H) but were negative for ubiquitin, glial fibrillary acidic protein, tau, and α-synuclein. They appear to represent a novel pathology, indicate that there was motor cortex involvement at a pathologic but not clinical level, and provide a potential candidate pathology that may characterize CHMP2B-related familial FTD. These lesions were also present at much lower densities in the premotor cortex (Brodmann area 6) and in a prefrontal block (Brodmann area 9) and were not identified in the neocortex of a series of 25 sporadic ALS cases.

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Figure. Photomicrographs of motor system regions from Patient 1. (A) Motor cortex showing intact Betz cells. (B) Low-power view of spinal anterior horn showing numerous motor neuron compact inclusions typical of ALS. These lesions were immunoreactive both to ubiquitin and p62/sequestosome 1 but negative for tau, neurofilament, and α-synuclein. (C) Compact inclusion in a spinal cord motor neuron. (D) Motor cortex showing numerous cell profiles immunoreactive for p62/sequestosome1. (E) At high power, these cortical inclusion bodies show coiled body morphology. They were not demonstrated by conventional ubiquitin immunocytochemistry or to antibodies to tau, neurofilament, and α-synuclein. Double-labeling immunocytochemistry was performed using either SMI32 (neurons), glial fibrillary acidic protein (astrocytes), CD68 (microglia), or carbonic anhydrase II (oligodendroglia) and p62/sequestosome 1 to identify the cell type involved by coiled body inclusion formation. (F) Coiled bodies immunostained for p62/sequestosome 1. (G) As in F under fluorescence illumination showing cell profiles stained by carbonic anhydrase II. (H) Merged images of parts F and G showing colocalization of coiled bodies to oligodendroglia. (Cresyl fast violet [A], immunoperoxidase [3,3′-diamino-benizidine chromogen] forp62/sequestosome 1 [B, C, D, E, F, H]; immunofluorescence [Texas Red for carbonic anhydrase II] [G, H]; magnification: ×2 [B,D]; ×40 [A, C, E, F, G, H]).

Discussion.

We report two patients with ALS-spectrum disorders and mutations in CHMP2B. Both have a possible family history. Both cases were negative for other known ALS mutations. Both were screened for SOD1 and angiogenin, Patient 1 was screened for VAPB, and Patient 2 for SOD2, SOD3, VEGF-A1, and dynactin. They were phenotypically dissimilar: Patient 1 showed PMA during life (El Escorial category suspected ALS) confirmed by conventional neuropathologic methods. Patient 2 showed features of ALS-dementia presenting with frontal lobe features before developing ALS (El Escorial category ALS + syndrome). Q206H is highly conserved from humans to Drosophila, whereas I29V is positioned within two conserved regions, the snf-7 and coiled coil domains (figure E-2). In silico prediction of structure with the I29V mutation suggests that stability would be significantly reduced in the mutant protein. The Q206H change likely represents a pathogenic mutation because it was not found in this study in 450 controls or in 400 samples from FTD patients,² or in 141 patients with frontotemporal lobar degeneration (FTLD).⁴ The I29V change has been suggested to be a nonpathogenic variation on the basis that it was present in one of 141 probands with FTLD, and at a frequency of 0.5% in 200 control chromosomes.⁴ Whether I29V represents a benign variation or a pathogenic mutation of variable penetrance remains to be elucidated. In this context, it is of interest that at least 16 of the 128 disease-associated mutations in the SOD1 gene in patients with ALS are associated with reduced disease penetrance.⁷ The finding that a gene implicated in FTD is also present in patients with ALS supports the hypothesis that pathogenic mechanisms in ALS underlie a diverse phenotypic spectrum that spans from pure LMN disease (PMA; Patient 1) through to FTD with no clinical motor system neurology.³

Mutation within the 3′ acceptor splice site of exon 6 and a point mutation in exon 5 of CHMPB2 were previously identified in FTD.² Those patients were not recognized to have features of ALS. CHMP2B is a component of the endosomal sorting complex (ESCRT) involved in sorting cargoes to form multivesicular bodies. Dysfunction of the ESCRT impairs the ability to internalize membrane bound cargoes and leads to dysmorphic endosomes.⁸ Overexpression of mutant CHMP2B causes the formation of aberrant endosomes in cell culture.² The changes in CHMP2B reported here support endosomal dysfunction as a potential mechanism of motor system degeneration. This mechanism is also postulated in Alsin-related ALS2⁹ and in vesicle-associated membrane protein (VAMP)/synaptobrevin-associated membrane protein B gene (VAPB)–related ALS8.¹⁰

The upper motor neuron pathology of Patient 1 is unusual and apparently novel. Inclusions in oligodendroglia are a feature of the cortical pathology of ALS-dementia and FTD where they are immunoreactive either for ubiquitin or tau epitopes. The combination of p62 immunoreactivity, in the absence of tau, α-synuclein, and ubiquitin, is unusual and suggests the possible utility of p62 as a potential molecular pathologic signature of CHMP2B gene–related neurodegeneration.

Appendix

Frontotemporal Dementia Research in Jutland Association (FReJA) Consortium: Peter Johannsen, MD, Anders Gade, PhD, Jørgen Nielsen, MD, and Tove Thusgaard, RN, Memory Disorders Research Unit, Copenhagen University Hospital, Copenhagen, Denmark; Susanne Gydesen, MD, Department of Psychiatry, Central Hospital, Holbaek, Denmark; Ida Holm, MD, Department of Pathology Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark; Elisabet Englund, MD, Department of Pathology, Lund University Hospital, Lund, Sweden; Jerry Brown, MD, Department of Neurology, Addenbrooke's Hospital, Cambridge, UK; Martin Rossor, MD, Dementia Research Group, Institute of Neurology, UK; John Collinge, MD, MRC Prion Unit, and Elizabeth Fisher, PhD, Department of Neurodegenerative Disease, Institute of Neurology, University College London, London, UK.

MRC Proteomics in ALS Study: Maria Spillantini, PhD, University of Cambridge, Cambridge, UK; Robert Layfield, University of Nottingham, Nottingham, UK; Paul G. Ince, MD, and Pamela J. Shaw, MD, University of Sheffield, Sheffield, UK.