Characterisation of novel gene mutations causing familiar amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a complex fatal degenerative disease selectively affecting motor neurones. The cause of the disease is still uncharacterised for the sporadic cases (SALS), whereas several disease-linked genes, such as SOD1 and VAPB, have been identified in families with ALS (FALS). However, since the pathological features are indistinguishable between FALS and SALS, it is generally believed that a common ALS-causing mechanism is responsible for both forms of disease. To gain a better understanding of ALS pathogenesis, which could also provide cues for therapeutic treatment, studies on the characterisation of FALS-linked mutations have been carried out. In this study, we screened for novel FALS-linked mutations and found three TDP-43 mutations in five unrelated FALS families, that caused marked changes at evolutional conserved amino acids and were absent in previously screened control populations. In addition, functional characterisation of novel ALS linked mutations in DAO (R199W) and VAPB (T46I) were undertaken whereby both mutations were shown to disrupt the physiological properties of wild type proteins. Whereas the T46I mutation in VAPB led to a sub-cellular redistribution and shift in solubility of VAPB protein, and an impairment in the unfolded protein response (UPR) and ubiquitin-proteasome system, R199W significantly abolished DAO enzyme activity. Cell culture-based functional studies showed that the expression of both mutant proteins triggered pathological features including ubiquitin-positive aggregates and cell death. Furthermore, an association study was conducted to investigate the cause of a dramatic VAPB down-regulation seen in SALS. An intronic SNP close to exon 5 of VAPB, rs6100067, was found to be significantly linked to the expression levels of VAPB in SALS. As the down-regulation of VAPB profoundly affects the activation of IRE1/XBP1, the most characterised UPR pathway, this SNP may present as a risk factor for ALS development. From these results, we propose an ALS pathogenesis mechanism, whereby VAPB plays a central role in sustaining multiple cellular events, such as UPR. Disruption or deprivation of VAPB causes impairments in these cellular functions and leads to motor neurone death.