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UBQLN2/Ubiquilin 2 Mutation and Pathology in Familial Amyotrophic Lateral Sclerosis Kelly L

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UBQLN2/Ubiquilin 2 Mutation and Pathology in Familial Amyotrophic Lateral Sclerosis Kelly L Neurobiology of Aging 33 (2012) 2527.e3–2527.e10 www.elsevier.com/locate/neuaging UBQLN2/ubiquilin 2 mutation and pathology in familial amyotrophic lateral sclerosis Kelly L. Williamsa,b, Sadaf T. Warraicha,b, Shu Yanga, Jennifer A. Solskia, Ruvini Fernandoa, Guy A. Rouleauc, Garth A. Nicholsona,b,d, Ian P. Blaira,b,* a Northcott Neuroscience Laboratory, ANZAC Research Institute, Sydney, New South Wales, Australia b Sydney Medical School, University of Sydney, New South Wales, Australia c Department of Medicine, University of Montreal, Montreal, Canada d Molecular Medicine Laboratory, Concord Hospital, New South Wales, Australia Received 9 March 2012; received in revised form 18 May 2012; accepted 20 May 2012 Abstract Amyotrophic lateral sclerosis (ALS) shows clinical and pathological overlap with frontotemporal dementia that includes the presence of hallmark ubiquitinated inclusions in affected neurons. Mutations in UBQLN2, which encodes ubiquilin 2, were recently identified in X-linked juvenile and adult-onset ALS and ALS/dementia. As part of an established exome sequencing program to identify disease genes in familial ALS, we identified a novel missense UBQLN2 mutation (c.1460CϾT, p.T487I) in 2 apparently unrelated multigenerational ALS families with no evidence of frontotemporal dementia. This mutation segregated with the disease and was absent in 820 healthy controls and all public single nucleotide polymorphism databases. The UBQLN2 p.T487I mutation substitutes a highly conserved residue and is located immediately upstream of a PXX region where all previous mutations have been identified. Immunostaining of spinal cord from a patient with UBQLN2 p.T487I mutation showed colocalization of ubiquilin 2 with ubiquitin in all neuronal inclusions examined and frequent colocalization with TAR DNA-binding protein 43 (TDP-43) and fused in sarcoma protein (FUS). To examine ubiquilin 2 pathology in broader ALS, we showed that ubiquilin 2 pathology also extends to ALS with a FUS mutation. These data further support the importance of ubiquilin 2 in the pathogenesis of ALS. © 2012 Elsevier Inc. All rights reserved. Keywords: Amyotrophic lateral sclerosis; Exome sequencing; Mutation; UBQLN2; Ubiquilin 2 1. Introduction brains and spinal cords of familial and sporadic patients (Mackenzie et al., 2010). TAR DNA-binding protein 43 Amyotrophic lateral sclerosis (ALS) is a late-onset fatal (TDP-43) has been shown to be a principal component of disorder characterized by the rapidly progressive degenera- these inclusions (Neumann et al., 2006). tion of the upper and lower motor neurons. Frontotemporal Most ALS cases occur sporadically but approximately dementia (FTD) is observed in about 20% of ALS cases, 10% of cases show familial inheritance (FALS). To date, and as many as 50% of ALS cases show some frontotem- mutations in SOD1, TARDBP, and FUS account for approx- poral impairment (reviewed by Neumann et al., 2007). A pathological hallmark of ALS and ALS-FTD is the presence imately 25% of FALS (Kwiatkowski et al., 2009; Rosen et of ubiquitin positive neuronal cytoplasmic inclusions in the al., 1993; Sreedharan et al., 2008; Vance et al., 2009). Expansions of a hexanucleotide repeat in C9ORF72 cause ALS and FTD linked to chromosome 9p21 and may account for approximately a third of FALS with outbred European * Corresponding author at: Northcott Neuroscience Laboratory, ANZAC ancestry (DeJesus-Hernandez et al., 2011; Renton et al., Research Institute, Concord Hospital, NSW 2139, Australia. Tel.: ϩ61 2 9767 9118; fax: ϩ61 2 9767 9101. 2011). Rare mutations in OPTN, VCP, SQSTM1 (encoding E-mail address: [email protected] (I.P. Blair). p62), and FIG4 are also believed to account for a small 0197-4580/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2012.05.008 2527.e4 K.L. Williams et al. / Neurobiology of Aging 33 (2012) 2527.e3–2527.e10 percentage of FALS cases (Chow et al., 2009; Fecto et al., expansion was performed using the method described by 2011; Johnson et al., 2010; Maruyama et al., 2010). Rare Renton et al. (2011). mutations in TARDBP, FUS, and VCP have also been iden- Validation and screening of the UBQLN2 p.T487I mu- tified in FALS patients with FTD. tation was performed by polymerase chain reaction (PCR) Deng et al. (2011) recently identified a missense muta- amplification of a 487 base pair amplicon encompassing the tion in the ubiquilin 2 gene (UBQLN2) in a large X-linked mutation. PCR products were sequenced using Big-Dye dominant ALS-dementia family. Subsequent mutation anal- terminator sequencing and an ABI 3730XL DNA analyzer ysis in a cohort of 188 families without male-to-male trans- (Applied Biosystems, Foster City CA, USA). Primer se- mission identified 4 additional UBQLN2 missense muta- quences and amplification conditions are available on re- tions. Two of these segregated with the disease in quest. multigenerational ALS-FTD families and 2 were found in Single nucleotide polymorphism (SNP) genotyping of single patients each from small families with classic ALS. controls was performed using a custom TaqMan SNP geno- Ubiquilin 2 pathology was demonstrated in affected tissues typing assay according to the manufacturer’s instructions of cases with UBQLN2 mutations. Ubiquilin 2 was also (Life Technologies, Carlsbad, CA, USA) and analyzed us- shown to be a component of spinal cord neuronal inclusions ing a Viia 7 real time PCR system (Life Technologies). SNP in a subset of ALS patients that included sporadic ALS, genotyping for haplotype analysis was performed by PCR FALS, and ALS-dementia cases (Deng et al., 2011; Fecto and Big-Dye terminator sequencing as described above. and Siddique, 2011). Microsatellite marker genotypes were determined by PCR As part of an established exome sequencing program to and analysis using an ABI 3730XL DNA analyzer (Applied identify disease genes in an Australian familial ALS cohort Biosystems). without dementia, we identified a novel UBQLN2 mutation Conservation of ubiquilin 2 orthologs was examined by that segregated in 2 apparently unrelated ALS pedigrees and aligning sequences from a variety of species (Entrez protein demonstrated pathological features of ubiquilin 2 in patient database, ncbi.nlm.nih.gov) using ClustalW2, Version neuronal tissues and nonneuronal cells. 2.0.12 (www.ebi.ac.uk/Tools/msa/clustalw2/). 2.3. Cell culture 2. Methods Fibroblasts were cultured from skin biopsies taken from 2.1. Participants the inner forearm of individual IV: 18 (Fig. 1A) and 2 neurologically normal age-matched controls. Fibroblasts were One hundred FALS patients, 6 related “married-in” con- seeded at a density of 20,000 cells per well on 12-mm diameter trols, and 190 unrelated controls were recruited through thickness number 1.5 glass coverslips in Dulbecco’s Modified neurogenetic clinics at Concord Hospital, Sydney, as well as Eagle’s Medium (Sigma-Aldrich, St Louis, MO, USA) con- at the Molecular Medicine Laboratory, Concord Hospital. taining 2 mM L-glutamine, 100 U/mL penicillin, 100 ␮g/mL An additional 770 healthy controls were obtained from the streptomycin, and 10% (vol/vol) heat-inactivated fetal bovine Australian MND DNA Bank. Most families were of Euro- serum (Sigma-Aldrich, St Louis, MO, USA). Cells were main- pean descent. All patients were diagnosed with definite or tained in a humidified 37 °C incubator with 5% CO2. probable ALS according to El Escorial criteria (Brooks et al., 2000). Patients and family members provided informed 2.4. Immunohistochemistry and immunofluorescence written consent regulated by the human research ethics Immunohistochemistry and immunofluorescence were committee of the Sydney South West Area Health Service. performed on spinal cord tissue from 2 ALS patients using All families had previously been screened for mutations in a mouse monoclonal anti-ubiquilin 2 antibody (Novus Bi- known ALS genes including TARDBP, SOD1, FUS, OPTN, ologicals, St Louis, MO, USA), rabbit polyclonal anti- VCP, ANG, FIG4, DCTN1, and CHMP2B. TDP-43 antibody (Proteintech, Chicago, IL, USA), rabbit 2.2. Genetic analysis polyclonal anti-FUS antibody (Abcam, Cambridge, UK) and rabbit polyclonal antiubiquitin antibody (Dako, Genomic DNA was extracted from peripheral blood using Glostrup, Germany). Spinal cord sections (5-␮m) were standard protocols. The genomic DNA of family FALS5 in- deparaffinized with xylene, and rehydrated with a descend- dividual IV: 10 was derived from formalin-fixed, paraffin- ing series of diluted ethanol and water. Antigens were re- embedded kidney tissue using an FFPE Repli-G kit (Qiagen trieved by heating sections in 10 mM citrate buffer (pH 6.0). Valencia, CA, USA). The exomes of 106 individuals (ALS Endogenous peroxidase activity was blocked for immuno- patients, obligate carriers, and related “married-in” controls) histochemical studies using 3% hydrogen peroxide in meth- from 65 ALS families were captured using TruSeq exome anol. Nonspecific background was blocked with either 1.5% Enrichment kit and sequenced with HiSeq2000 sequencing goat serum (Vector Laboratories, Burlingame, CA, USA) system (Illumina, San Diego, CA, USA). for immunohistochemistry, or 1% bovine serum albumin Analysis of the C9ORF72 hexanucleotide repeat and (Sigma-Aldrich, St Louis, MO, USA) for immunofluores- K.L. Williams et al. / Neurobiology of Aging 33 (2012) 2527.e3–2527.e10 2527.e5 Fig. 1. A novel UBQLN2 mutation in familial amyotrophic lateral sclerosis (ALS). Pedigree of family FALS5 (A) and FALS14 (B) showing segregation of the UBQLN2
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