The study examined characteristics of 32 patients with mutations in the C9ORF72 gene, identified from a large cohort of 398 patients with clinical syndromes of FTLD. Demographic characteristics of gender, age at onset of symptoms and duration of illness at referral did not distinguish patients with C9ORF72 gene mutations from those without, indicating that the mutation bearers were representative of the larger group with respect to those demographic variables. Not surprisingly, however, a positive family history of dementia and/or MND was significantly more common in patients with C9ORF72 mutations.
There was, as anticipated, a significant association with MND: the presence of MND more than doubled the likelihood of a patient having the C9ORF72 mutation. Nevertheless, there was no absolute concordance. First, there were patients with the C9ORF72 mutation who had no signs of MND (to our knowledge at any time during the disease course), nor family history of MND. This strongly suggests that MND is not an inevitable manifestation of mutations in the C9ORF72 gene. Secondly, and crucially, there were patients in the study cohort with familial FTD–MND, who did not have the C9ORF72 mutation. This important finding indicates that hexanucleotide repeat expansion in C9ORF72 is not the cause of all cases of FTD–MND. Another genetic mutation or mutations must be responsible.
A striking and unanticipated finding was the strong association of C9ORF72
gene mutations with psychotic symptoms: delusions, hallucinations, paranoid ideation and disordered thinking. More than a third of patients presented with florid psychosis and were initially classified by their psychiatrist using conventional psychiatric diagnostic labels: delusional psychosis, mono-delusional psychosis, somatoform psychosis, paranoid schizophrenia. Other patients exhibited paranoid and delusional thinking as part of their behavioural disorder. In two-thirds of patients behaviour was bizarre and illogical. None of these patients had a history of psychiatric illness. The high prevalence of psychotic symptomatology is important because of its rarity in FTD in general (Bathgate et al., 2001
; Mendez et al., 2008a
). In their study of 86 patients with FTD Mendez et al. (2008b)
reported delusions in only 2.3% and hallucinations in 0%, figures significantly lower than for Alzheimer's disease. Although psychotic symptoms have been described in association with MAPT
mutations (Sumi et al., 1992
; Poorkaj et al., 1998
), this appears to be unusual. In line with previous findings, in the present cohort, <4% of patients without C9ORF72
mutations presented with psychosis or exhibited psychotic symptoms at the time of their referral. The presence of psychosis increased the odds of having the C9ORF72
gene mutation by 15-fold. It is noteworthy, moreover, that where psychosis was recorded in patients without the C9ORF72
mutation this was in the context either of a prodromal depressive illness or else a long-standing psychiatric disorder, and bore little resemblance to the florid, yet often circumscribed, delusional disorder found in people with the C9ORF72
Interestingly, and in contrast to the paucity of psychosis in FTD in general, there have been several case reports of patients with FTD–MND or a family history of MND who present with psychotic symptoms (Nitrini and Rosenberg, 1998
; Larner, 2008
; Lillo et al., 2011
; Loy et al., 2011
). Moreover, in a series of 43 patients with behavioural FTD and 18 with FTD–MND (Lillo et al., 2011
), delusions were found to be significantly more common in the FTD–MND group. The prediction would be that those patients had mutations in the C9ORF72
gene. This prediction remains to be substantiated. Psychosis has previously been reported in individual patients with chromosome 9-linked FTD–MND (Pearson et al., 2011
) but there has thus far, to our knowledge, been no systematic study of its incidence. It is, however, pertinent that only one of the patients with FTD–MND without C9ORF72
mutations in the present series exhibited psychotic symptoms, this being in the context of a longstanding bipolar affective disorder. This finding is important because it suggests that it is the C9ORF72
mutation that is crucial to the development of psychotic symptoms and not FTD–MND per se
Aside from patients with FTD–MND, the only other subgroup of FTD patients that has been associated with a relatively high prevalence of psychotic symptoms are rare cases with FUS pathology (Seelaar et al., 2010
; Urwin et al., 2010
; Snowden et al., 2011b
). Importantly, FUS pathology and mutations in the FUS
gene have been associated with MND. Psychosis has not typically been reported in association with mutations in either the MAPT
(Hutton et al., 1998
) or PGRN
(Baker et al., 2006
) genes, which are not linked to MND.
Is psychosis the only feature that distinguishes the behaviour of patients who have C9ORF72
gene mutations from those who do not? Patients exhibited a range of behaviours associated with FTD: apathy, disinhibition, loss of sympathy and empathy, repetitive behaviours, dietary change and loss of insight. At first glance, therefore, they appear indistinguishable from other patients with FTD. Nevertheless, close examination suggests some distinctive features. First, abnormal behaviours are, to some extent, influenced by patients’ delusional beliefs: for example, repeated telephone calls to the police by a patient who believes himself to be persecuted. Secondly, repetitive behaviours are typically complex. Simple, low-level motor mannerisms such as hand rubbing, tapping, humming, or grunting, which are found in many patients with FTD (Bathgate et al., 2001
; Mateen and Josephs, 2009
) were rare. This is particularly noteworthy in view of the patients’ general apathy: simple motor stereotypies have been reported most frequently in apathetic patients with FTD (Snowden et al., 2001
). Moreover, repeated cleaning and hand washing observed in some patients has characteristics of obsessive–compulsive disorder, a feature that is rare in FTD in general (Bathgate et al., 2001
). In the dietary domain, an altered preference for sweet foods was notable for its rarity. Gluttony or increased appetite, a feature that has been linked particularly to TDP-43 pathology (Piguet et al., 2011
), did not differ in frequency in patients with and without the C9ORF72
mutation, suggesting that it is the sweet food preference in particular, rather than dietary change in general that is a relevant discriminator. There are therefore behavioural pointers that, taken together, might predict the presence of C9ORF72
mutations: psychotic symptoms, complex repetitive behaviours linked to a mono-delusion or with an obsessional–compulsive quality, absence of sweet food preference. The additional presence of MND in the patient or family member would greatly strengthen the prediction.
Notwithstanding these clinically distinct characteristics, absolute accuracy of prediction is likely to prove difficult by virtue of the phenotypic variability in patients with C9ORF72
mutations. Not only do some patients have MND and others not, the cognitive/behavioural phenotype also varies. As found in the Finnish cohort reported by Renton et al. (2011)
, patients may present with progressive non-fluent aphasia or semantic dementia, as well as with behavioural FTD. Patient 30 with progressive non-fluent aphasia, showed no psychotic symptoms, none of the behavioural features of FTD, and no signs of MND. There would, therefore be no a priori
basis on which to predict the presence of a C9ORF72
gene mutation. In general, however, the prevailing cognitive profile of patients with C9ORF72
mutations was similar, characterized by executive impairment, combined with a dynamic form of aphasia, with additional semantic deficits present in some patients. No patient showed evidence of speech or limb apraxia.
The limitation of any retrospective study is the possibility that symptoms were present but not recorded. The likelihood is reduced, in this study, by the fact that the clinical history uses a structured proforma, which addresses individual behavioural and cognitive symptoms systematically. Moreover, the history was taken for the most part by the same consultant neurologist (D.N.) or neurologist trained by him, providing uniformity of data collection. Similarly, the neuropsychological examination was uniform in all patients and carried out or supervised by the same neuropsychologist (J.S.). Salient features of patients’ clinical presentation, such as psychosis, were recorded on a database prior to the results of genetic screening becoming available, so the recording procedure was carried out in blind fashion. Most importantly, any potential biases arising from ‘missing data’ cannot explain statistical differences in people with and without mutations in the C9ORF72 gene, since these patients were drawn from a single cohort and examined by the same people in the same way.
Findings from the present study not only highlight features likely to be of predictive value in identifying patients with mutations in the C9ORF72
gene but also inform the debate about the nature of the relationship between FTD and MND. Some authors have referred to a ‘continuum’ between FTD and MND (Talbot et al., 1995
; Murphy et al., 2007a
), implying that they are different manifestations of the same disease process. Others propose that FTD–MND should be regarded as an aetiological entity distinct from FTD (Bak et al., 2001
; Mitsuyama et al., 2009
; Bak, 2011
). The present findings point to truth in both views. Mutations in the C9ORF72
gene are associated with a spectrum—or continuum—of clinical manifestations, with isolated MND at one end, isolated FTD or progressive aphasia at the other and a combination of behavioural/cognitive and motor neuron symptomatology in the middle. Yet, the form of FTD associated with C9ORF72
mutations appears qualitatively distinct from other forms of FTD, suggesting that it represents a distinct clinical phenotypic variant linked to a distinct aetiology.
Neuropathological investigation carried out in five patients with C9ORF72
mutations revealed some unexpected findings. The prediction had been that patients would consistently show FTLD-TDP pathology and this would likely conform to type B (Mackenzie et al., 2011
). Three patients did indeed exhibit type B pathology, in keeping with prediction. However, in Patient 10 the pathology was more akin to type A, suggesting that the underlying pathological characteristics are not entirely uniform. A recent study (Murray et al., 2011
) of 15 cases of FTLD with C9ORF72
mutations (eight with FTLD
MND and seven with FTLD) showed similar heterogeneity, with cases of FTLD-MND being more likely to have type B histology and those with FTLD alone tending to be type A, though there was no absolute concordance.
More surprisingly, in the present study, in one mutation bearer (Patient 24) TDP-43 based histology was absent, and a corticobasal degeneration pathology (not associated with parkinsonism or apraxia) was seen. It is not possible, at present, to know whether the chromosome 9 hexanucleotide expansion in this patient was silent, or whether it in some way promoted the corticobasal degeneration pathology. The lack of TDP-43 staining in this case was unlikely to be due to post-mortem interval or agonal state since normal physiological nuclear staining was present and TDP-43 pathology was adequately immunostained in other patients who died in hypoxic states with longer post-mortem delays. Interestingly, Patient 24 had a strong family history of Huntington's disease. The patient did not himself carry the huntingtin mutation. Nevertheless, since Huntington's disease is a trinucleotide repeat expansion disorder it raises the question whether there is an increased susceptibility within the family to have an altered replication of repeat expansions. In any event, the absence of TDP-43 pathology inevitably raises questions regarding the specificity of the C9ORF72 mutation for FTLD.
Pathogenic repeat expansions can cause disease through haploinsufficiency, in which expression or splicing of the gene in question is disrupted. The expansion can also generate abnormal amounts of toxic RNA species, which interfere with normal cellular activity by sequestering normal RNA and proteins involved in transcription regulation (Wojciechowska and Krzyzosiak, 2011
). The large size of the expansion and its non-coding localization within C9ORF
72 gene may favour the latter (Renton et al., 2011
). Presently, however, the function of C9ORF72 protein is unknown, though in normal individuals at least three alternatively spliced transcripts are expressed (DeJesus-Hernandez et al., 2011
). The absence of C9ORF72 protein within the TDP-43 pathological lesions that characterize cases with the mutation suggests that the C9ORF
72 expansion does not induce a ‘toxic gain-of-function’ by the protein per se
through some process of aggregation and inclusion body formation akin to TDP-43 and FUS proteins in cases of MND bearing mutations in transactive response DNA-binding protein
) and FUS
genes (Kabashi et al., 2008
; Kwiatkowski et al., 2009
; Vance et al., 2009
). The presence of C9ORF72
RNA intranuclear inclusions in mutation carriers suggest that gain of RNA toxicity is a likely mechanism in these cases (DeJesus-Hernandez et al., 2011
However, a putative loss-of-function effect (irrespective of the actual mechanism whereby this might be induced) might be anticipated to lead to loss of protein within brain tissue. Nonetheless, present immunostaining with antibodies against C9ORF72 did not detect any obvious loss of immunoreactivity or change in pattern of staining in mutation bearers compared with cases with FTLD associated with MAPT or GRN mutations, or with FTLD-tau, FTLD-TDP, FTLD-FUS, MND or Alzheimer pathology. However, given that careful characterization of the present commercial antibodies available to us for this study has not been performed, (we were unable to obtain recombinant C9ORF72 protein ourselves in order to pre-adsorb the antibody prior to immunostaining), the specificity of the histological findings reported here cannot be guaranteed. Nonetheless, the observations that many of those particular cells and neuroanatomical pathways that are affected by TDP-43 pathological changes in FTD–MND (i.e. hippocampus, inferior olives, motor neurons) are those that appear normally rich in C9ORF72 protein would argue that the findings presented are credible and suggest a functional dependency between C9ORF72 and TDP-43, though the nature of this remains to be elucidated. Present immunohistochemical staining may represent patterns of normal physiological activity, and this would be consistent with the variable preservation or accessibility of the antigen following death even with antigen retrieval, post-mortem delay or differential fixation effects.
The finding of C9ORF72 protein within Pick bodies of the granule cells of the dentate gyrus is interesting. However, this observation might simply reflect the passive sequestration and recruitment of C9ORF72 protein into these tau containing lesions, given that such cells normally appear to be unusually rich in C9ORF72 protein, and that Pick bodies within the relatively C9ORF72 impoverished pyramidal cells of the hippocampal pyramidal cell layer, and the cerebral cortex do not apparently display such immunoreactivity. Nonetheless, a specific interaction between C9ORF72 protein and tau is possible because the TDP-43 positive inclusions in cells of the dentate gyrus in cases with FTLD-TDP apparently did not contain C9ORF72 protein as might be expected if the argument that C9ORF72 protein is being merely sequestered into Pick bodies is correct.
The pathological findings of p62-positive, TDP-43 negative neuronal cytoplasmic inclusions within granule cells of the cerebellum in the four C9ORF72
mutation bearers with TDP-43 pathology are broadly consistent with previous reports (Pikkarainen et al., 2008
; King et al., 2011
; Murray et al., 2011
). These particular cases would support the suggestion by Boxer et al. (2011)
that the presence of ubiquitin/p62-positive, TDP-43 negative neuronal cytoplasmic inclusions within the cerebellar granule cells might potentially be used as a pathological surrogate to indicate linkage to chromosome 9p, and the presence of hexanucleotide repeat expansion in C9ORF72
, even in the absence of formal linkage studies or genetic analysis. However, three other cases of FTLD showed similar ubiquitin/p62-positive, TDP-43 negative neuronal cytoplasmic inclusions within cerebellar granule cells, in the absence of hexanucleotide repeat expansion in C9ORF72.
Moreover, no such inclusions were present in the cerebellum in the fifth patient with C9ORF72
mutation with corticobasal degeneration histology. Hence, although it is likely that most patients/families with FTLD and/or MND possessing repeat expansions in C9ORF72
will display a ubiquitin/p62-positive, TDP-43 negative cerebellar pathology, this does not seem to be a universal finding, and the presence of this cerebellar pathology per se
does not appear to be predictive of the presence of this particular mutation, or even linkage to chromosome 9.
The variability in clinical presentation of patients with C9ORF72 mutations is intriguing. Currently, it is unclear why some patients show signs of MND and others not, why some patients present with psychosis and others not, and why patients occasionally present with a highly circumscribed language disorder with neither behavioural nor psychotic features. The imaging and pathology data available in this study do not resolve these issues. The imaging findings reveal no obvious differences in terms of relative preponderance of frontal or temporal atrophy and hemispheric asymmetries in patients with and without psychosis. Predictably, patients with progressive aphasia all showed predominant left-sided atrophy, but asymmetries were present in other patients as well. No obvious pathological difference was apparent between the four patients with psychosis and the one patient without. There is a need for more sophisticated prospective neuroimaging studies of patients screened for the C9ORF72 mutation and pathological studies in larger cohorts of patients to address directly the question of the anatomical correlates of phenotypic variation. It is, of course, possible that phenotypic differences reflect differences in relative time course of symptom development rather than absolute differences. Patients who present with MND are likely to have an attenuated disease course, so it is plausible that they too have a susceptibility to the development of psychosis, but that this does not have time to become manifest. These issues require systematic investigation.
In conclusion, mutations in the C9ORF72 gene are responsible for some but not all cases of FTD–MND. The clinical presentation of patients who have the mutation is variable, the earliest symptoms being physical, behavioural or linguistic. The pathological characteristics are also variable. There are, nevertheless, clinical features that set these patients apart from other patients with FTD. The powerful association between C9ORF72 mutations and psychosis suggests that mutations in the C9ORF72 gene may have a crucial role not only in FTD–MND but also in the development of late onset psychosis.