Exome sequencing reveals VCP mutations as a cause of familial ALS

Abstract

Using exome sequencing, we identified a p.R191Q amino acid change in the valosin-containing protein (VCP) gene in an Italian family with autosomal dominantly inherited amyotrophic lateral sclerosis (ALS). Mutations in VCP have previously been identified in families with Inclusion Body Myopathy, Paget disease, and Frontotemporal Dementia (IBMPFD). Screening of VCP in a cohort of 210 familial ALS cases and 78 autopsy-proven ALS cases identified four additional mutations including a p.R155H mutation in a pathologically proven case of ALS. VCP protein is essential for maturation of ubiquitin-containing autophagosomes, and mutant VCP toxicity is partially mediated through its effect on TDP-43 protein, a major constituent of ubiquitin inclusions that neuropathologically characterize ALS. Our data broaden the phenotype of IBMPFD to include motor neuron degeneration, suggest that VCP mutations may account for ∼1%-2% of familial ALS, and provide evidence directly implicating defects in the ubiquitination/protein degradation pathway in motor neuron degeneration.

Figure 1

Pedigrees of patients with VCP mutations. Mutant alleles are indicated by mt, whereas wild-type alleles are indicated by wt. Probands are indicated by arrows. Sex of the pedigree members is obscured to protect privacy. Pedigrees of the familial samples from the NINDS repository at Coriell have been constructed based on available family history information from the Coriell website (www.coriell.org).

Figure 2

Filters applied to variants and indels detected by exome sequencing in affected individuals of the ITALS#1 pedigree. Regions with LOD score greater than zero were based on linkage analysis of ITALS#1 pedigree (see also Figure S1). Variants that were found in exome data of 200 Neurologically normal control subjects were filtered. Prediction of effect of variant on protein function was based on SIFT analysis (www.sift.jcvi.org).

Figure 3

Distribution of VCP mutations detected in familial ALS patients. An ideogram of chromosome 9 is shown at the top. The 17 exons of VCP are numbered. Mutations detected in familial ALS cases are indicated in red, whereas mutations previously described to cause IBMPFD are shown in black. Corresponding chromatograms showing mutant and wild-type alleles are as indicated, and conservation of amino acid residue across species is highlighted at the bottom (generated using the ClustalW2 online tool, www.ebi.ac.uk/clustalw/). See also Figure S2 for location of mutations on VCP protein structure.

Figure 4

Histopathological images from autopsy tissue, family UP731, individual III:1. (A) Haematoxylin-eosin (H&E) stained low power view (100x) of a lumbar spinal cord section demonstrates extensive motor neuron loss in the anterior horn, with a few remaining motor neurons (arrow). (B) Motor neurons from the hypoglossal nucleus in the medulla with abundant round eosinophilic cytoplasmic inclusions consistent with Bunina bodies (arrows), H&E, 200x. (C) (200x) and (D) (400x). TDP-43 immunohistochemistry on the hypoglossal nucleus shows TDP-43 positive punctate cytoplasmic inclusions with concomitant loss of nuclear stain in two motor neurons (arrows). Normal nuclear staining is preserved in the surrounding normal cells. Of note, additional tissue was not available from this autopsy. The current images were generated by removing the cover slip from selected old slides, followed by de-staining and processing for TDP-43 immunohistochemistry. See also Figure S3 for histopathological images of a muscle biopsy from patient III:4 of the ITALS#1 family.