Exome sequencing links corticospinal motor neuron disease to common neurodegenerative disorders

Abstract

Hereditary spastic paraplegias (HSPs) are neurodegenerative motor neuron diseases characterized by progressive age-dependent loss of corticospinal motor tract function. Although the genetic basis is partly understood, only a fraction of cases can receive a genetic diagnosis, and a global view of HSP is lacking. By using whole-exome sequencing in combination with network analysis, we identified 18 previously unknown putative HSP genes and validated nearly all of these genes functionally or genetically. The pathways highlighted by these mutations link HSP to cellular transport, nucleotide metabolism, and synapse and axon development. Network analysis revealed a host of further candidate genes, of which three were mutated in our cohort. Our analysis links HSP to other neurodegenerative disorders and can facilitate gene discovery and mechanistic understanding of disease.

Fig. 1. Functional validation of private HSP genes in zebrafish

(A) Quantification of 24-hours-post-fertilization (hpf) embryos mortality (black) and curly-tail (gray) phenotypes for noninjected (NI), scrambled, and morphants (MO) at stated nanogram concentrations. Overt phenotypes were observed for all MOs except MO^pgap1. (B) Average touch-response distance (in arbitrary units, A.U.) in 72-hpf larvae, showing blunted response for all MOs. (C) Immediate touch-response trajectory of example larvae, each shaded uniquely. Mars2 MO was too severe to be tested, whereas others showed reduced response. (D to F) Spontaneous locomotion at 6 days post fertilization. (D) Average percent of time spent moving over a 30-min window showed a reduction for all for at least one dose. (E) Average active period duration, showing reduction for all. (F) Representative kymographs recording fish position (black dot) over 30-min recording. MOs showed either short distance traveled (MO^arl6ip1) or reduced movements per recording (MO^pgap1 and MO^usp8). *P < 0.01 (t test). N > 2 experiments with n > 20 animals per experiment. Error bars indicate standard error.

Fig. 2. Hereditary spastic paraplegia interactome

(A) HSP seeds + candidate network (edge-weighted force-directed layout), demonstrating many of the genes known to be mutated in HSP (seeds, blue) and new HSP candidates (red), along with others (circles) constituting the network. (B and C) Comparison of statistical strength of HSP subnetworks with 10,000 permutations of randomly selected proteins. Dots denote the value of the metric on the true set (i.e., seeds or seeds + candidates). Box and whisker plots denote matched null distributions (i.e., 10,000 permutations). (B) Seed (known mutated in HSP) versus random proteins drawn with the same degree distribution. (C) Seed plus candidate HSP versus a matching set of proteins. (Left) Within group edge count (i.e., number of edges between members of the query set). (Middle) Interaction neighborhood overlap (i.e., Jaccard similarity). (Right) Network random walk similarity.

Fig. 3. Genes from HSP networks found mutated in HSP

(A) HSP candidate genes predicted from the HSPome found mutated in the HSP cohort. BICD2, MAP, and REEP2 were subsequently found mutated in HSP families 1370 (B), 1226 (D), and 1967 (F), respectively. (C) Homozygosity plot from family 1370. Red bars, regions of homozygosity; arrow, homozygous block containing BICD2. (E) Linkage plot of family 1226; arrow, MAG locus. (G) Homozygosity plot; arrow, REEP2 locus. (H to J) Zoom in from HSPome for specific interaction identifying candidates CCDC64 (a paralog of BIC2D), MAG, and REEP2 (yellow) with previously published (blue) and newly identified (red) genes mutated in HSP. Blue lines denote manually curated interactions.

Fig. 4. Functional link between HSP genes and genes of other neurodegenerative conditions

(A) Density distribution representing random walk distances of OMIM-derived neurodegeneration gene networks along with 10,000 permutations of randomly selected protein pools compared with the HSP seeds plus candidates pool. The top 5%ile distance is shaded. Only for Parkinson’s, Alzheimer’s, and ALS do the HSP seeds plus candidates fall within this 5%, whereas epilepsy and autism spectrum disorder show no statistical overlap. (B) Bipartite network showing the top links between the set of HSP and ALS proteins. Clear circles, HSP seeds; yellow circles, HSP candidates; boxes, ALS genes (VCP and ALS2 are implicated as causative of both HSP and ALS); line thickness, diffusion similarity between the two proteins.