Updated project metadata. Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease leading to motor neuron loss. It has a strong genetic contribution, but most ALS-associated gene mutations remain poorly characterized. Therefore, we performed comparative interactome analyses of recently discovered ALS-associated proteins which highlighted many novel binding partners, and both unique and shared interactors. This suggests that different ALS-associated proteins can act through distinct and/or similar cellular pathways. The analysis identified C21ORF2 as a strongly connected protein. The role of C21ORF2 mutations in ALS is largely unknown. Our data show C21ORF2 expression in affected neurons and that the prominent C21ORF2-V58L variant induces apoptosis in mouse neurons and movement defects in zebrafish embryos. iPSC-derived motor neurons from C21ORF2-V58L-ALS patients, but not isogenic controls, show increased apoptosis, and changes in DNA damage response (DDR), mitochondria and electrophysiological properties. In addition, C21ORF2-V58L induced post-transcriptional downregulation of NEK1, an ALS-associated protein implicated in apoptosis and DDR. In all, our data define the pathogenic effects of C21ORF2-V58L and implicate reduced NEK1 levels, due to impaired proteosomal degradation, downstream of this ALS mutation.