Analiza proteinskih karakteristika i patogenosti varijanti optineurina u amiotrofičnoj lateralnoj sklerozi i glaukomu primjenom bioinformatike

Amyotrophic lateral sclerosis (ALS) is a severe, rapidly progressive neurodegenerative disease that affects upper and lower motor neurons. ALS is characterized by high genetic heterogeneity – so far 17 confirmed or “gold standard” genes and 100 potential risk factors - have been linked to this disease. One of the confirmed genes is OPTN, which encodes for optineurin, a multifunctional, ubiquitin binding adaptor protein proposed to act in various biological processes. OPTN mutations have also been found in glaucoma, another neurodegenerative disease, encompassing a group of eye conditions in which the optic nerve is damaged. The aim of this work was to examine optineurin protein features and to compare OPTN variants present in ALS and glaucoma with naturally occurring variants through different in silico programs. We also examined if the mutations were more frequent in the evolutionary conserved regions of optineurin. The OPTN variants and subsequent protein changes were compiled by expert literature curation and taken from UniProt, OMIM, ClinVar and gnomAD databases. In silico species alignment analysis of optineurin domain conservation was done using ClustalOmega. Our results showed that OPTN variants in both ALS and glaucoma predominantly affect coiled-coil regions of optineurin, but this is comparable to their length. However, ALS variants were enriched in the zinc finger (ZF) domain. They were curiously not enriched in one of the main functional domains of optineurin, the ubiquitin-binding region of ABIN and NEMO (UBAN), but clustered around it. Pathogenicity of OPTN mutations patient mutations was predicted using Polyphen-2, SIFT and PROVEAN programs. Although prediction of pathogenicity differed between the programs for variants present in both ALS and glaucoma, they were consistent in assigning K59N, R83C, Q314L, M447R, E478G, K557T, D564H, L568S, H571Q as damaging protein changes present in ALS, and in assigning E50K as damaging in glaucoma. Eight different species representing major vertebrate genera were taken for in silico species alignment analysis of the optineurin conservation. This analysis showed that 89% of predicted pathogenic variants reported in ALS patients mapped to the conserved regions of OPTN, suggesting that they are more likely to be pathogenic. The highest degree of the conservation, both total and partial, was observed in the UBAN and ZF regions of optineurin. The optineurin E50K glaucoma-linked variant mapped to conserved regions as well. Although variant E322K is located on the conserved position of the optineurin protein, it is predicted to be benign according to Polyphen-2, SIFT and PROVEAN analysis. This in silico research of OPTN variants represents the basis for the further in vivo and in vitro investigations and could possibly help conceive future experimental directions relevant for uncovering optineurin function in neurodegenerative diseases.Amiotrofična lateralna skleroza (ALS) je teška, brzo progresivna neurodegenerativna bolest koja zahvaća gornje i donje motoričke neurone. ALS karakterizira visoka genetska heterogenost s do sada 17 potvrđenih ili ''gena zlatnog standarda'' te >100 potencijalnih faktora rizika povezanih s ovom bolešću. OPTN gen kodira za optineurin, višenamjenski, adaptorski protein koji veže ubikvitin te za koji se pretpostavlja da djeluje u raznim biološkim procesima. Mutacije OPTN gena također su pronađene u još jednoj skupini neurodegenerativnih bolesti – glaukomu – u kojima dolazi do oštećenja i gubitka neurona vidnog živca. Cilj ovog rada bio je usporediti značajke varijanti proteina optineurina i OPTN gena prisutnih u ALS-u i glaukomu s prirodnim varijantama putem različitih in silico programa. Također smo ispitali jesu li mutacije pronađene u pacijentima bile češće u evolucijski očuvanim regijama optineurina. OPTN varijante navedene su stručnim pregledom literature i preuzete iz baza podataka UniProt, OMIM, ClinVar i gnomAD. In silico analiza sličnosti vrsta i očuvanje domene optineurina među vrstama analizirana je pomoću programa ClustalOmega. Naši rezultati pokazuju da OPTN varijante i kod ALS-a i glaukoma pretežno zahvaćaju zavijene zavojnice (Coiled-coil) optineurina, ali to je usporedivo s njihovom duljinom. Međutim, ALS varijante obogaćene su u domeni cinkova prsta (engl. Zinc Finger, ZF). Suprotno očekivanjima, varijante nisu bile obogaćene u ubikvitin-vezujućoj regiji ABIN i NEMO proteina (engl. ubiquitin-binding region of ABIN and NEMO, UBAN), koja predstavlja glavnu funkcionalnu domenu optineurina, ali su bile grupirani oko nje. Patogenost OPTN mutacija je predviđena pomoću programa Polyphen-2, SIFT i PROVEAN. Iako se predviđanje patogenosti djelomično razlikovalo među programima i za varijante u ALS-u i glaukomu, programi su bili dosljedni u označavanju K59N, R83C, Q314L, M447R, E478G, K557T, D564H, L568S, H571Q kao štetne promjene proteina u ALS-u te E50K kao štetne u glaukomu. Osam različitih vrsta koje predstavljaju glavne rodove kralježnjaka uzeto je za in silico analizu sličnosti između vrsta te za ispitivanje očuvanja optineurina i utjecaja konzerviranosti na pojavu mutacija. Analiza sličnosti između vrsta pokazala je da je 89% predviđeno patogenih varijanti optineurina prisutnih u bolesnika s ALS-om mapirano na očuvane regije, što sugerira njihovu patogenost. Najviši stupanj očuvanosti (potpuni i djelomični) uočen je u UBAN i ZF regijama optineurina. Optineurin E50K varijanta, pronađena kod pacijenata s glaukomom, također je smještena u očuvanim regijama. Iako se varijanta E322K nalazi na očuvanom položaju proteina optineurina, prema Polyphen-2, SIFT i PROVEAN analizi je benigna. Zaključno, ovo in silico istraživanje OPTN varijanti predstavlja temelj za daljnja in vivo i in vitro istraživanja i isto bi moglo pomoći u osmišljavanju budućih eksperimenata relevantnih za razumijevanje funkcije optineurina u neurodegenerativnim bolestima