Neurobiology of Aging 36 (2015) 1603.e5e1603.e9

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p62/SQSTM1 analysis in frontotemporal lobar degeneration Louise Miller a, Sara Rollinson a, Janis Bennion Callister a, Kate Young a, Jenny Harris b, Alex Gerhard b, David Neary b, Anna Richardson b, Julie Snowden b, David M.A. Mann b, Stuart M. Pickering-Brown a, * a b

Faculty of Medical and Human Sciences, Institute of Brain, Behaviour and Mental Health, University of Manchester, Manchester, UK Institute of Brain, Behaviour and Mental Health, Salford Royal Hospital NHS Foundation Trust, Salford, UK

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 August 2014 Received in revised form 7 August 2014 Accepted 11 August 2014 Available online 18 October 2014

Mutations in the gene p62/SQSTM1 have been reported as a relatively rare cause of frontotemporal lobar degeneration (FTLD). To establish whether this was the case for cases of FTLD from the United Kingdom, we sequenced the sequenced the entire open reading frame of this gene in a large cohort of patients. We identified 3 novel mutations in p62/SQSTM1 in 4 patients. One of these was a premature stop codon that removed the last 101 amino acids of the protein that presumably has a negative effect on protein function. Another mutation was also found in a case with a repeat expansion mutation in C9orf72 confirmed by Southern blot. These findings confirm a role of p62/SQSTM1 as a cause of FTLD. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: p62 SQSTM1 C9orf72 Frontotemporal lobar degeneration FTLD

1. Introduction Frontotemporal lobar degeneration (FTLD) is now recognized as the second most common form of early-onset dementia. The 3 most common clinical syndromes associated with FTLD are behavioral variant frontotemporal dementia, semantic dementia, and progressive nonfluent aphasia (Neary et al., 1998). Neuronal pathologic inclusions associated with FTLD are composed of the protein TDP43, tau, or occasionally FUS (Mackenzie et al., 2010). FTLD can also co-occur with amyotrophic lateral sclerosis (ALS) and sometimes parkinsonism. Up to 40% of patients with FTLD report a family history of similar disease, indicating that genetics plays a major defining role in the etiology of this disease. In recent years, there has been much progress in understanding genetic causes of FTLD. Until recently, the main known genetic risks for familial FTLD were associated with mutations in MAPT (Hutton et al., 1998; Spillantini et al., 1998) and progranulin (Baker et al., 2006; Cruts et al., 2006). However, it is now known that the most common genetic cause of FTLD is a hexanucleotide repeat expansion in a noncoding region of the gene, * Corresponding author at: Faculty of Medical and Human Sciences, Institute of Brain, Behaviour and Mental Health, University of Manchester, Manchester M13 9PT, UK. Tel.: þ44 (0) 161 275 1341; fax: þ44 (0) 161 275 3938. E-mail address: [email protected] (S.M. Pickering-Brown). 0197-4580/$ e see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.08.035

C9orf72 (DeJesus-Hernandez et al., 2011; Renton et al., 2011). This particular mutation similarly is a major cause of ALS demonstrating that these 2 conditions are part of a disease spectrum. This had long been considered to be so given that about 50% of cases of FTLD, and around 85% of cases of ALS, demonstrate TDP-43-positive pathologic inclusions in their brains. Likewise, ALS can be inherited and numerous genes causing autosomal dominant disease have been identified, including SOD1, TARDBP, and FUS in addition to C9orf72 (Conte et al., 2012). Recently, mutations in the gene p62/sequestosome1 (p62/ SQSTM1) have been claimed to be associated with ALS and FTLD (Chen et al., 2013; Hirano et al., 2013; Kwok et al., 2013; Le Ber et al., 2013; Rubino et al., 2012; Shimizu et al., 2013; Teyssou et al., 2013). p62 is a ubiquitin-binding protein involved in protein homeostasis and is also present in the TDP-43 pathologic neuronal inclusions in both FTLD and ALS. In addition, mutations in p62/SQSTM1 are major causes of Paget disease of bone (Michou et al., 2006). How certain mutations in p62/SQSTM1 can lead to neurodegeneration, whereas other lead to Paget disease in the absence of any apparent neuronal dysfunction, is currently unclear. Given the shared etiology between FTLD and ALS, we sequenced the whole coding open reading frame of p62 in a large cohort of FTLD patients from the North West of England to establish to what extent mutations in p62/sequestosome-1 can also be a cause of FTLD.

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Table 1 A list of the known variants detected in our cohort with their relative frequency in our patient population. Exon

RS number

Base/AA change

MAF in local controls

5 6 6 6 6 6 7 8 8

rs11548633 rs4797 rs4935 rs145001811 rs55793208 rs56092424 rs146164139 rs104893941 rs143511494

AAG: Lys to GAG: Glu AGG to AGA; silent Arg GAC to GAT; silent Asp CAC to CAT; silent His GAG: Glu to GAC: Asp TCC to TCT; silent Ser TCG to TCA; silent Ser CCG: Pro to CTG: Leu GGC: Gly to AGC: Ser

0.00027 0.477 0.49 0.00134 0.0174 0.01474 0.0068 0.00131 0.00131

Key: AA, amino acid; MAF, minor allele frequency.

2. Methods 2.1. Patients The study group comprised 465 consecutive patients, 42% men and 58% women, referred to the Cerebral Function Unit, University of Manchester, who were diagnosed with one of the clinical syndromes associated with FTLD (Neary et al., 1998; Rascovsky et al., 2011). All patients had undergone clinical and neuropsychological assessments within the Cerebral Function Unit, and in 1 case the diagnosis had been pathologically confirmed at postmortem. Patients’ mean age at onset of symptoms was 61 years (30e82 years). A positive family history of dementia in a first degree relative was recorded in 40% of cases. Controls, usually spouses of affected patients (n ¼ 525), were neurologically normal and had a mean age of 59 years (36e79 years) at time of sampling. 2.2. Immunohistochemistry This was performed as described previously (Mann et al., 2013). 2.3. p62/SQSTM1 sequencing All exons of p62/SQSTM1 were amplified as previously described (Le Ber et al., 2013). Sequence analysis was performed using an ABI3730.

Fig. 1. Southern blot confirming a repeat expansion in C9orf72 in case DA634. The figure represents ladder, negative control, lymphoblastoid cell line positive control; and case DA634.

2.4. Genotyping analysis Single-nucleotide polymorphisms were genotyped using the appropriate Taqman assay (ABI) and genotyped using ABI7900HTS. Southern blotting was performed as described in Mann et al. (2013). 3. Results By sequencing the entire open read frame of p62/SQSMT1 in our FTLD cohort identified a range of previously known SNPs (Table 1). In addition, we identified 3 novel mutations in 4 patients with FTLD (Table 2). These mutations were absent from dbSNP, 1000 Genomes

Project, and 525 local control samples, the latter genotyped using taqman. Two apparently unrelated patients shared the same R183C mutation. The patient with the G351A mutation, with a SIFT score of 0.01, also had a confirmed repeat expansion in C9orf72 with approximately 2500 repeats (Fig. 1). This case came to autopsy and had an unremarkable FTLD-TDP type B pathology. In addition, this case also exhibited dipeptide repeat (DPR) inclusions that were positive for all 5 DPR antibodies(Mann et al., 2013), which are normally associated with a C9orf72 expansion (Fig. 2). The other mutation (W321X) introduced a premature stop codon that removes the final 101 amino acids from the protein, including the

Table 2 Summary of clinical features of patients with novel variants in p62. Case

Sex

Age at onset (y)

Diagnosis

Family history

Genetic variation

Sift value

DA275 DA266 DA443

M M F

65 68

SD CBS bvFTD

R183C R183C W321X

DA634

F

52

bvFTD

AD brother Father Schizophrenia father and brother bvFTD mother and sister

0.03 0.03 Loss of last 101 AA 0.01

G351A

Key: AD, Alzheimer’s disease; bvFTD, behavioral variant frontotemporal dementia; CBS, corticobasal syndrome; SD, semantic dementia.

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Fig. 2. DP pathology in the cortex resembles type B (A), DPR (B) and p62 (C) NCI in CA4, DPR (D), and p62 (E) in dentate granule cells and DPR in cerebellar granule cells (F). All DPR staining is poly-Gly-Ala. Abbreviations: DPR, dipeptide repeat.

ubiquitin-binding region. None of the patients exhibited any signs of Paget disease nor had a family history of such. The 4 patients with novel mutations had atypical clinical presentations. Patient 1 (DA275) presented with difficulty recognizing faces and objects, against a background of depressed mood and personality change. He showed loss of sympathy and empathy, was routine bound, and he acquired a sweet tooth. The neuropsychological profile was of striking semantic impairment, more marked for visual (faces, objects) than verbal (words) material, together with problems in memory, executive functions, and praxis. Functional neuroimaging showed bitemporal hypoperfusion, more marked on the right, extending into the right parietal lobe. The primary clinical phenotype was of (right-predominant) semantic dementia, albeit not pure given the cooccurrence of memory, executive, and praxic difficulties. His brother exhibited a circumscribed amnestic syndrome reminiscent of Alzheimer’s disease. Patient 2 (DA266) presented with limb apraxia, more marked on the left, alien limb phenomena, mild-limb rigidity, and action myoclonus. Language, perceptual, spatial, and memory skills were relatively preserved, and he retained a warm affect. Imaging showed temporoparietal hypoperfusion, especially on the right. The profile was in keeping with corticobasal syndrome. With progression, his speech became palilalic, and he experienced visual hallucinations. Patient 3 (DA443) presented with personality and behavioral change, set against a background of long-standing mental health illness and previous diagnoses of endogenous depression, schizophrenia, and schizoaffective disorder. She was lacking in sympathy and empathy, verbally and sexually disinhibited, stereotyped in her behavior, and preoccupied by somatic complaints. She experienced visual hallucinations and delusions. Neurology was normal. Neuropsychology revealed problems in executive skills and

memory. Imaging was uninformative. The profile was in keeping with behavioral variant FTD, presenting with psychosis. Her father and brother had been diagnosed with “schizophrenia.” Patient 4 (DA 634) presented with problems in memory, loss of motivation, behavioral disinhibition, and delusions, set against a background of long-standing depressive illness. She remained physically well. The profile was in keeping with behavioral variant FTD, with delusions as an atypical feature. Genetic screening revealed expansions in the C9orf72 gene. She progressed slowly, dying 18 years after symptom onset. Pathologic examination revealed FTLD-TDP pathology, subtype B (Mackenzie et al., 2011). 4. Discussion Mutations in p62/SQSTM1 have been suggested to be a cause of FTLD and ALS in various populations (Fecto et al., 2011; Hirano et al., 2013; Le Ber et al., 2013; Rubino et al., 2012; Shimizu et al., 2013). Here, we undertook whole-gene sequencing to establish whether such mutations are also a cause of disease in our FTLD cohort from the North West of England. We detected 3 mutations in 4/465 patients confirming that p62/SQSTM1 mutations are, albeit, a rare (0.85%) cause of FTLD. This prevalance is consistent with that reported by van der Zee et al. (2014) who observed an overall frequency of 0.9% in a pan-European FTLD cohort. Although other family members were not available to demonstrate segregation, the absence of these 3 variants in our controls, and public databases argues in favor of them being pathogenic. The co-occurrence of G351A mutation with a repeat expansion mutation in C9orf72 is interesting. There are 3 possible explanations for this. First, that the G351A mutation is a benign polymorphism unrelated to disease. This is unlikely as it resides in the KIR domain of the gene, an area where many other confirmed mutations have been localized (Rea et al., 2014).

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Second, as it is known that the repeat expansion occurs in a small number of apparently healthy people (Beck et al., 2013), it is possible that the expansion is unrelated to disease in this patient, with the G351A mutation being causative. However, given that this case exhibited DPR pathology it indicates that the C9orf72 expansion was functionally active in this patient. Last, it is possible that the patient co-inherited both a p62/SQSTM1 mutation and a repeat expansion in C9orf72, both of which are pathogenic. However, if this was the case it might have been anticipated that the patient would have suffered a more severe form of disease, and this appears not to be the case. Hence, the status of G351A remains uncertain. The W321X mutation introduces a premature stop codon that removes the final 101 amino acids from the protein, including the ubiquitin-binding region. This presumably has a harmful effect on protein function and raises the possibility that a loss of function mechanism mediates disease. This would potentially lead to a reduction in protein turnover and/or degradation, which is consistent with the pathology observed in these patients. However, specific tests will be required to confirm that the other missense mutations result in loss of function effects. Furthermore, the original mutations reported in p62/SQSTM1 were found to cause Paget disease of bone in the absence of any obviously neurodegenerative changes. It is notable that none of the 4 patients with mutations in p62/SQSTM1, identified here, apparently showed any evidence of Paget disease of bone, thereby ruling out the possibility that the reported mutations might have been responsible for causing this condition, and that the presence of FTLD in such patients was simply coincidental and unrelated to the mutation. The reason as to why some mutations cause FTLD and ALS whereas others lead to Paget disease remains to be discovered. The 4 patients were heterogenous with respect to clinical phenotype. In 2 patients the predominant picture was of behavioral variant FTD, in another semantic dementia, and in another corticobasal syndrome. There were atypical features in each: (1) cooccurrence of apraxia in the patient with semantic disorder and a family history more suggestive of Alzheimer’s disease than FTLD; and (2) psychotic features and a history of psychiatric disorder in others. SQSTM1, or p62, is involved in numerous cellular process including the ubiquitin/proteasome pathway, growth control, receptor function, stress responses, DNA excision-repair, and cell signaling via protein kinases (Schwartz and Ciechanover, 1999). Furthermore, it is found within the pathologic inclusions of many neurodegenerative diseases, including FTLD and ALS. It is therefore a likely candidate gene for these diseases (Mackenzie et al., 2011), and the genetic variations reported here are in turn likely to be pathogenic. In conclusion, in the present study, we have identified a small number of mutations in p62/SQSTM1 in a cohort of FTLD from the North West of England which we believe are pathogenic, thereby supporting previous studies suggesting a causative role for this gene in the pathogenesis of disease. Disclosure statement The authors have no conflicts of interest to disclose. Acknowledgements This work was supported by a grant (G0701441) to Stuart M.Pickering-Brown from the Medical Research Council United Kingdom.

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