Exome sequencing to identify de novo mutations in sporadic ALS trios

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease whose causes are still poorly understood. To identify additional genetic risk factors, we assessed the role of de novo mutations in ALS by sequencing the exomes of 47 ALS patients and both of their unaffected parents (n = 141 exomes). We found that amino acid-altering de novo mutations were enriched in genes encoding chromatin regulators, including the neuronal chromatin remodeling complex (nBAF) component SS18L1 (also known as CREST). CREST mutations inhibited activity-dependent neurite outgrowth in primary neurons, and CREST associated with the ALS protein FUS. These findings expand our understanding of the ALS genetic landscape and provide a resource for future studies into the pathogenic mechanisms contributing to sporadic ALS.

Figure 1

The SS18L1/CREST de novo mutation (Q388stop) identified in an ALS trio inhibits activity-dependent dendritic outgrowth. a) We sequenced the exomes of 47 ALS patients and both unaffected parents (n = 141 exomes) to identify de novo mutations. b) We identified a de novo mutation in the neuronal chromatin remodeling complex subunit SS18L1/CREST, which introduces a premature termination codon, deleting the CBP-binding motif contained within the last nine amino acids. h=human; m=mouse. c) SS18L1/CREST is expressed in motor neurons of the adult spinal cord and localizes to the nucleus (arrow). Scale bar 10 μm. d) Functional validation of the CREST de novo mutation in primary neurons. Primary cortical neurons were isolated from E18.5 mouse embryos, transfected with Vector-IRES-GFP, CREST-IRES-GFP or CREST _{AA 1–393}-IRES-GFP (The 1–393 truncation of mouse CREST corresponds to 1–388 of human CREST, which we identified in the ALS trio as Q388stop). Neurons were cultured for 5 days and stimulated overnight with 30mM KCl where indicated. Control vector and CREST overexpression do not affect dendrite outgrowth in response to KCl depolarization. CREST _{AA 1–393} significantly reduces total dendrite length in response to KCl depolarization. An example of the dendrite outline tracing used to quantify dendritic length and number of branch points is shown. Scale bar 10 μm. e) The average values are from three independent experiments, each with three coverslips per condition with 15–20 GFP+ neurons scored per coverslip. f) # branch points per cell is affected in a similar fashion as total dendrite length. Error bars, S.E. *P<0.02, **P<0.002, ***P<0.0005, Student’s t-test.

Figure 2

Identification of an additional SS18L1/CREST variant in FALS case and interaction with FUS. a) Novel SS18L1 missense variant in familial ALS. Genotypes of available DNA samples for the indicated SS18L1 variant are shown (‘wt’ denotes wild type, ‘I123M’ denotes mutant). The variant c.T369G (p.I123M) was identified in affected individual III:2 (the index case) and was absent in unaffected individuals II:3 and III:1. The genotype of sample II:2 (*) was inferred from the genotypes of spouse and progeny. The question mark (?) for individual I:2 indicates that no historical clinical notes were available. b) Motor neurons transfected with GFP or WT CREST respond to KCl stimulation by increasing the total dendritic length. Transfection of CREST_1–393 or CREST_I123M inhibits stimulation-induced dendrite outgrowth. The average values are from two independent experiments, each with four coverslips per condition with 30–35 GFP+ neurons scored per coverslip. c) # of branch points in motor neurons is affected in a similar fashion as total dendrite length. Error bars, S.E. *P<0.02, **P<0.002, ***P<0.0005, Student’s t-test. d,e) FUS and SS18L1/CREST can physically associate in mouse cortical neurons. d) As a positive control, nBAF complex core subunit Brg, was co-immunoprecipitated by SS18L1/CREST. The SS18L1/CREST antibody also co-immunoprecipitated FUS. e) Antibodies against several other nBAF subunits co-immunoprecipitate FUS. * FUS band is visible.