A novel optineurin truncation mutation identified in a consanguineous Palestinian family with Amyotrophic Lateral Sclerosis confirms loss of function as a disease mechanism
Creators: name: Gotkine, Marc
name: Majo, Martina De
name: Hao, Wong Chun
name: Topp, Simon
name: Moein  Kanaan
name: Michaelson-Cohen, Rachel
name: Litman, Silvina Epsztejn
name: Eigess, Rachel
name: Nishimura, Agnes L.
name: Smith, Bradley N.
name: Shaw, Christopher E.
Publisher: name: F1000Research
Year: 2016

description: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease (incidence of ~2 per 100,000 per year) with an average survival of three years from diagnosis. A clear genetic component is apparent in approximately 10% of cases, and mutations in four major genes: SOD1, TDP43, FUS and C9ORF72 account for approximately 50% of familial and 15% of sporadic cases. The genetic factors involved in the majority of ALS cases remain to be elucidated. Optineurin (OPTN) mutations have been described in open angle glaucoma—and  more recently, in ALS—with recessive mutations found in  consanguineous Japanese, Turkish and Moroccan Jewish kindreds; dominant Caucasian cases with variable penetrance have also been reported. Here we describe, for the first time, the genetic and functional characterization of a novel recessive mutation in OPTN found in a consanguineous Palestinian family with an aggressive, adult form of ALS. Furthermore, this study is the first to characterize endogenous OPTN from primary fibroblast lines cultured from carriers of OPTN mutations.<br><br>OPTN is a 577 amino acid protein involved in a variety of cellular pathways, including autophagy, which seems to be the most important in ALS pathogenesis. OPTN acts as autophagy receptor and it is involved in the clearance of protein aggregates through the autophagy-lysosome pathway in an ubiquitin dependent and ubiquitin independent manner. Within the same pathway OPTN is phosphorylated on Ser177 by tank binding kinase 1 (TBK1), a protein linked to ALS.<br><br>A novel homozygous truncation mutation in OPTN (S174X) was identified in this family, via exome sequencing from genomic DNA of the affected family member; this results in removal of both the Ubiquitin binding in ABIN and NEMO (UBAN) domain and the TBK1 phosphorylation site, as well as other important protein interaction sites. Notably, the truncated protein retains the TBK1 binding region of OPTN. The unaffected mother was confirmed to be a heterozygous carrier, and sequencing of three unaffected siblings of the proband showed two of them to be heterozygous carriers and one to harbor a homozygous S174X change. <br><br>Fibroblast primary lines were obtained from the unaffected mother and three siblings. Mutation status was confirmed by Sanger sequencing. Interestingly, Western blot analysis of fibroblast lysates, probing for endogenous OPTN, revealed OPTN expression for the wild type and heterozygous carriers but no expression of the truncated S174X OPTN allele. These observations support the hypothesis that early truncation results in an unstable protein; this was also confirmed by immunocytochemistry. However, transcript analysis revealed that full length and truncated OPTN message was produced from both the heterozygous and homozygous carriers. Further investigation with quantitative PCR will identify the level of OPTN mRNA transcription, and whether the mutation promotes nonsense mediated decay in homozygous and heterozygous individuals.<br><br>The absence of the mutated copy of OPTN suggests that the pathogenic mechanism could be associated with a loss of function, which could result in a knock on effect abolishing interaction with TBK1. Further research will aim to characterise the mechanism behind the pathogenicity of this truncation mutation in OPTN and how this affects its interaction with TBK1. <br>