The role of UBQLN2 in disease: a protocol for a systematic review and meta- analysis. Bethany Waddington1,2, Dr Jenna Gregory 2,3, Dr George Gorrie4 and Dr Thimo Kurz1,2. 1Molecular, Cell and Systems Biology, University of Glasgow, Davidson Building, Glasgow, G12 8QQ, UK 2 Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Chancellor’s Building, Edinburgh, EH16 4SB, UK. 3 Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor’s Building, Edinburgh, EH16 4SB, UK. 4 Department of Neurology, Institute of Neurological Sciences, Queen Elizabeth University Hospital, Glasgow, UK. *Correspondence to [email protected] Keywords UBQLN2, disease, systematic review, meta-analysis 1. INTRODUCTION There are 5 Ubiquilin proteins within the family in humans, named UBQLN 1-4 and UBQLNL. Whilst UBQLN3 and UBQLNL are restricted to the testis, the remaining members are widely expressed (Marı´n, 2014), with UBQLN2 most prominently expressed in the brain and nervous system, spleen, heart, liver and pancreas, amongst other tissues. (Zhang and Saunders, 2011). UBQLN2 is composed of 3 key domains: The Ubiquitin Associated Domain (UBA), the central domain (STI Like), and the Ubiquitin Like Domain (UBL) (Walters et al., 2004). Within the central domain lies the unique PxxP motif, a region which contains nearly all the disease-causing UBQLN2 mutations, but with unknown function in the activity of UBQLN2 (Deng et al., 2011). UBQLN2 has been found to contribute to numerous neurodegenerative diseases and accumulations (Deng et al., 2011; Hjerpe et al., 2016; Williams et al., 2012; Zeng et al., 2015). Of recent interest is the role of UBQLN2 mutations in the development of Amyotrophic Lateral Sclerosis (Deng et al., 2011). Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of motor neurones. A major characteristic of ALS and other forms of neurodegenerative diseases is the presence of protein aggregates in the affected areas (Al-Chalabi et al., 2012; Blokhuis et al., 2013). However, the nature and content of these aggregates can vary between individuals, creating problems discerning between causal and coincidental pathology. Nonetheless, this inability to efficiently clear mutated, damaged or aggregated proteins points to a dysfunction in the homeostatic proteolytic systems within the motor neurones. Protein degradation is crucial in post-mitotic cells, such as neurones; without proliferation the toxic proteins or aggregations will not be diluted out (Hara et al., 2006; Komatsu et al., 2006). Therefore, in order to ensure efficient progression through the system, numerous proteins and complexes are formed and utilised. Of interest in this case is Ubquillin-2 (UBQLN2), a protein known to cause X-linked juvenile- and adult-onset ALS (Deng et al., 2011). Of note is the presence of UBQLN2 positive aggregations in non-mutant-UBQLN2 patients; ALS patients with no genetic mutation in UBQLN2 still present with aggregations positive for UBQLN2 (Deng et al., 2011; Williams et al., 2012). This reaffirms the belief that UQBLN2 activity is central to the cellular homeostasis and protein degradation within motor neurones. The complexity of the formation and clearance of these aggregates is yet to be understood, although much evidence points to an impairment in protein degradation pathways as human spinal cord autopsy of UBQLN2 patients found skein-like inclusions containing UBQLN2, Ubiquitin, p62, TDP-43, Fus and OPTN (Deng et al., 2011; Williams et al., 2012). Together, this data provides the justification that further research is needed into the mechanistic actions of UBQLN2, and its impact in the development of ALS. As no other systematic review and meta-analysis has been carried out investigating UBQLN2, this review aims to collate the existing literature regarding UBLQN2 in all diseases. If appropriate, quantitative analysis will be carried out using studies of the same pathway. Gathering a broad range of data allows the possibility of grouping, and potentially analysing if appropriate, data sets based on the disease implicated, model used, or pathways involved. In using this primarily wide spanning, qualitative approach, techniques, methodologies and results could be extrapolated from the better- established research, to provide suggestions and tools for moving forward in the elusive fields of neurodegenerative disease and ALS. 2. APPROACH A systematic review will be performed to assess the contribution of UBQLN2 mutations to the development, progression and severity of disease in both preclinical models and clinical cases. The search strategy will include clinical cases and human post-mortem, animal models and cellular models providing the model and mutation is clearly stated. Individual meta-analyses will be consequently performed for the outcomes measured and further sub-group analysis carried out based on their suggested cause of phenotype, if appropriate. 3. OBJECTIVES The overarching aim of this project is to elucidate the role of UBQLN2 mutations to the development of disease in pre-clinical models and clinical cases and provide a summary of the current data in the field. This summary will comprise of the methodology, including the mutations assessed and how these models or mutations were studied or created (Overexpression models for instance); and interventions in the treatment of disease caused by mutations in UBQLN2, including drug interventions, siRNA, or lentiviral rescue for instance. This overarching qualitative summary will be used to generate tools such as a frequency distribution model summarising the models and mutations used, and their phenotype. A more quantitative analysis will be performed retrospectively using standardised mean differences if appropriate. Specifically, we will gather insight into the contribution of UBQLN2 mutations to the development and severity of disease across all 3 platforms of research (Human, Animal and Cellular), where human denotes all clinical and post-mortem research, animal as whole animal or post-mortem/cellular subgroups and cellular as immortalised cell lines, including hiPSC work. In human cases, the primary outcome measure will be survival, with duration, age of onset, cognition, locomotion, behavioural, biochemical and anatomical as secondary objectives, where appropriate. Where animal models are concerned, again the primary objective will be survival, followed by duration, age of onset, cognition, locomotion, behavioural, biochemical and anatomical as secondary outcomes. Dependant on the experiments carried out, the primary outcome from the cellular models will be survival, followed by biochemical or electrophysiological as the secondary outcomes, but the cell line used must be clearly stated. The secondary aims of the pre-clinical models will be pooled together, whilst the primary outcome of mortality kept separate. From the results gathered a final set of outcomes will be assessed, to see whether any common suggestions of binding partners, modifiers or indicators of stress cause or contribute to the phenotype observed. 4. METHODS 4.1 Search Methods Sources: databases: 1. PubMed, 2. MEDLINE and 3. EMBASE. Search Date: There will be no publication date or language restrictions. 4.2 Search strategy Search terms: PubMed: (“UBQLN2” or “UBQLN” or “Ubiquilin-2” or “Ubuqillin 2” or “Ubquilin2” or “Ubiquilin” or “PLIC2” or “PLIC-2” or “DSK2” or “DSK-2” or “ALS15”) NOT (“UBQLN1” or “UBQLN3” or “UBQLN4” or “Ubiquilin- 1” or “Ubiquilin-3” or “Ubiquilin-4”or “Ubiquilin 1” or “Ubiquilin 3” or “Ubiquilin 4”). MEDLINE: (UBQLN2 or UBQLN or Ubiquilin-2 or Ubiquilin 2 or Ubiquilin2 or Ubiquilin or PLIC2 or PLIC-2 or DSK2 or DSK-2 or ALS15) not (UBQLN1 or UBQLN3 or UBQLN4 or Ubiquilin-1 or Ubiquilin-3 or Ubiquilin-4 or Ubiquilin 1 or Ubiquilin 3 or Ubiquilin 4). EMBASE: (UBQLN2 or UBQLN or Ubiquilin-2 or Ubiquilin 2 or Ubiquilin2 or Ubiquilin or PLIC2 or PLIC-2 or DSK2 or DSK-2 or ALS15) not (UBQLN1 or UBQLN3 or UBQLN4 or Ubiquilin-1 or Ubiquilin-3 or Ubiquilin-4 or Ubiquilin 1 or Ubiquilin 3 or Ubiquilin 4). 4.3 Screening The Systematic Review Facility online screening tool (app.syrf.org.uk) will be used by two independent authors in order to screen the title and abstract of each paper identified from our predetermined criteria, with regards to the inclusion and exclusion criteria. Duplicates will be removed during screening, but reviews kept to check for additional studies. A third, independent reviewer will assess 10% of the selected studies to assess inter-observer variability and bias and concordance will be set at <0.66. A quality score will be awarded, and the data extracted as outlined below. 4.4 Eligibility 4.4.1 Inclusion Criteria All models or cases of disease where mutations in UBQLN2 or UBQLN were the disease-causing mutation, or where levels of wild-type UBQLN2 or UBQLN were manipulated. Types of models included are: Immortal cell, e/iPS derived cell lines, primary cell cultures, whole animal models, post- mortem tissue (animal and human), clinical cases; Endogenous and exogenous gene expression; all known disease-causing patient mutations will be accepted in clinical cases or as Knock-In mutations, alongside UBQLN2 and UBQLN amplification or silencing in animal and cellular models. 4.4.2 Exclusion Criteria No data No controls or inappropriate control Case reports Letters and comments Reviews Any mutations/knock-ins or knock-outs of UBQLN 1,3 or 4 specifically. 4.5 Quality Score Checklist CAMRADES