The core features of chromosome 9 FTD/ALS
There was a relatively characteristic spectrum of core findings among individuals with FTD and/or ALS associated with the hexanucleotide repeat expansion in
C9ORF72, which we suggest should be termed c9FTD/ALS. The phenotype was typically behavioural variant FTD, ALS or a combination of both. Features of parkinsonism were found in nearly one half of patients with behavioural variant FTD or FTD-ALS but were not seen in patients with ALS in these cohorts. No patient had a primary dementia phenotype of primary progressive aphasia. The age of onset was in the 33–75 year range, and survival ranged widely, with most succumbing to the disease within 7 years from onset. Those with ALS by itself or in conjunction with behavioural variant FTD tended to have a shorter disease course. Most had one or more relatives with dementia and/or ALS. The neuroimaging findings of symmetric bilateral frontal
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parietal and/or temporal cortical changes were relatively consistent. All patients with autopsies and neuropathological examinations had TDP-43 pathology consistent with either type A or Type B, as well as frequent motor neuron pathology, frequent substantia nigra degeneration and ubiquitin-positive, TDP-43-negative cerebellar and hippocampal neuronal inclusions.
Demographic considerations
Among all subjects with dementia and/or ALS, and among only those who were examined by one of the co-authors, there was a slight male predominance (52–54%). Considering these three cohorts together, males with the primary diagnosis of behavioural variant FTD were over-represented (63%), and females with ALS
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FTD were slightly over-represented (18/30 or 60%), particularly in the ALS phenotype (61%), but whether these slight male–female differences reflect a sampling bias or have biological relevance in terms of sex factors impacting the topography of degeneration and hence the clinical phenotype will require further study.
Genetic/inheritance considerations
Among the three cohorts of subjects, the frequency of the mutation was highest in the Mayo Clinic Rochester FTD cohort (11.2% of all FTD
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ALS cases) and lowest in the Mayo Clinic Florida FTD cohort (3.6%), which likely reflects the strong focus of recruiting familial FTD kindreds in the Mayo Clinic Rochester centre and likely higher frequency of northern European ancestry in the Midwest (which is where most Mayo Clinic Rochester participants reside). Among those with a positive family history, the frequency of the expansion was highest in the Mayo Clinic Florida ALS cohort (at least 23%), but also reasonably high in the Mayo Clinic Rochester FTD cohort (19.1%). There are likely two factors that explain this. The newly identified
C9ORF72 mutation explains a high proportion of familial ALS based on the available data (
DeJesus-Hernandez et al., 2011;
Renton et al., 2011). Furthermore, other data suggest that a common founder from Scandinavia might underlie the disease (
Mok et al., 2012). More work is needed to determine if the mutation stems from a common founder, represents a ‘weak point’ in the genome which allows an increased frequency of expansions to randomly occur, or a combination of these and other factors.
As shown in , the frequency of the C9ORF72 mutation is highest in the FTD/ALS phenotype compared with the behavioural variant FTD and ALS phenotypes regardless of whether they are considered among all cases or familial cases only. Furthermore, no mutations in MAPT or PGRN were detected in the cases with sporadic or familial FTD/ALS and ALS. Also, at least in this Mayo Clinic series, the frequency of familial behavioural variant FTD being explained by the C9ORF72 mutation is higher than that explained by MAPT and PGRN despite the strong interest in recruiting familial FTD families over the past 20 years and contributions to the original identification of mutations in MAPT and PGRN causing familial FTD. These data therefore indicate that a sizable proportion of cases with familial FTD and/or ALS is explained by the C9ORF72 mutation. Importantly, however, an even greater proportion is not explained by mutations in these three or other known genes (>50% for each phenotype), indicating that other yet-to-be-identified genes are associated with FTD and/or ALS.
More than 80% of our probands were familial, of which more than half had both the behavioural variant FTD and ALS phenotypes represented in the kindreds, a third had only dementia or behavioural variant FTD, and the remaining minority had only ALS represented in the families. Therefore, the repeat expansion should be considered in any kindred with familial FTD and/or ALS. The other 16% of our probands did not meet predefined criteria for a positive family history and hence were considered sporadic.
The observation that the GGGGCC hexanucleotide repeat expansion in
C9ORF72 may be associated with FTD, FTD/ALS and ALS in the same pedigree supports recommendations included in recently proposed criteria for the diagnosis of familial ALS (
Byrne et al., 2011). In the Mayo Clinic Florida ALS cohort we classified eight patients who carried the
C9ORF72 repeat mutation and had no known family history of ALS in a first- or second-degree relative, as sporadic ALS. However, available information suggests that five of these eight patients might be reasonably classified as having a familial disorder given a family history of dementia in a first-degree relative in four and in a second-degree relative in one patient. The latter classification is in keeping with criteria we applied in designating patients with FTD and FTD/ALS as familial or sporadic. This leaves a small number of patients with FTD or ALS in whom family history suggests sporadic disease, but evaluation of parental DNA will be important in future investigation to determine whether these pedigrees represent non-penetrance or spontaneous expansion of the GGGGCC hexanucleotide repeat from a non-pathogenic parental form. Adoption of stringent criteria for the diagnosis of familial ALS along the lines of those recently proposed by
Byrne et al. (2011) seems especially important in giving increasing recognition that expanded phenotypes such as extrapyramidal disease may be associated with genes linked to FTD and ALS.
Whether the sporadic cases represent incomplete penetrance or de novo mutations is being investigated. Two subjects appeared to be obligate carriers but died while neurologically asymptomatic—one at age 34 from an accident, and another at age 72 from a myocardial infarction. The younger subject might have exhibited features had he survived decades more, and the 72-year old may have done likewise if he had survived a few more years. However, considering that 72 was the maximal age of onset of any clinically examined subject in this series, and the subject with the Alzheimer's disease phenotype whose onset was at the age of 74 and autopsy revealed FTLD-TDP-43 pathology (the repeat expansion has not been confirmed in this case but is presumed), this asymptomatic case who died at age 72 suggests that incomplete penetrance might occur even among aged individuals.
A curious observation is the apparent earlier age of onset (>10 years) in the youngest generation compared with the previous generation in 11 (26% of all probands) of our families with c9FTD/ALS (
Supplementary Fig. 1). Considering that the mechanism of this genetic defect involves a hexanucleotide repeat expansion, and the concept of anticipation (i.e. an earlier age of onset in successive generations occurs with increasing repeat length) is well established in many of the nucleotide repeat disorders (
Lindblad and Schalling, 1999), it is tempting to hypothesize that a similar mechanism may be at play in these families. However, this will be challenging to study. First, the repeat expansion is unstable and variable across cells, and variability in single subjects has already been seen (
DeJesus-Hernandez et al., 2011). Furthermore, the current methodology for quantifying repeat length is imperfect, particularly when several hundreds of repeats are present. Investigating this issue further is clearly worthy to support or refute an anticipation mechanism associated with this hexanucleotide repeat. Repeat length may also impact the topography of neurodegeneration and hence the clinical phenotype expressed.
Clinical considerations
As noted above, the core phenotypes associated with this repeat expansion were behavioural variant FTD, ALS or combined FTD/ALS, with one-third of total cases and nearly half with behavioural variant FTD or FTD/ALS also having some degree of parkinsonism, which was typically an akinetic-rigid syndrome without rest tremor. When parkinsonism was present, it tended to evolve over the initial 2 years of the disease course. When both behavioural variant FTD and ALS occurred in the same individual, the features almost always evolved within 2 years. Even among subjects with the primary behavioural variant FTD syndrome, almost 40% in this series had some degree of upper and/or lower motor neuron dysfunction but not of sufficient breadth to warrant the FTD/ALS diagnosis. A comprehensive neurological examination in any patient with dementia will permit identification of these clinical findings. Almost 20% of the subjects with a primary diagnosis of ALS had some degree of behavioural variant FTD features but either did not fully meet criteria for the FTD/ALS diagnosis or could not be formally evaluated owing to motor impairment. Features of behavioural variant FTD may be subtle and not characteristic of fully developed dementia in ALS resulting in potential misattribution of signs of cognitive dysfunction to situational factors or depression. Weakness and fatigue may limit the practical assessment of FTD in subjects with ALS, and eliciting the historical and clinical features of behavioural variant FTD may not be routinely performed in clinics focused on standard elements of ALS patient care. Thus, the 20% frequency of behavioural variant FTD features in this cohort of subjects with ALS may be an underestimate. Yet the overlapping features of behavioural variant FTD and ALS in many subjects with the repeat expansion in
C9ORF72 underscores several key points: (i) this genetic alteration can impact cognition, behaviour and motor functioning via degeneration in cerebral and/or spinal cord neurons; (ii) clinicians will often identify ALS features in subjects with behavioural variant FTD and behavioural variant FTD features in subjects with ALS if detailed questioning, clinical examinations and electromyograms are performed (
Lomen-Hoerth et al., 2002,
2004); and (iii) assessments over time permit the identification of more widespread findings as the disease process evolves.
Neuropsychiatric morbidity was frequent in the subjects in whom this information was collected. The caregivers of subjects endorsed the presence of changes in appetite and eating behaviour in all who were queried, and almost half endorsed the presence of delusions or hallucinations. These findings are consistent with other recent publications on the neuropsychiatric features in FTD/ALS and/or those with TDP-43 positive pathology (
Lillo et al., 2010;
Burrell et al., 2011;
Piguet et al., 2011).
Among the clinically examined subjects, most with ALS features fulfilled El Escorial criteria for definite, probable or possible ALS, and the site of onset was approximately equal across the bulbar, upper and lower spinal cord segments. The rare variants included atypical primary lateral sclerosis (no DNA available), monomelic ALS (mutation identified), and progressive muscular atrophy (mutation identified). Although most had ALS-like findings, neuropathological studies also showed patients with motor neuron degeneration consistent with progressive muscular atrophy. Hence, this genetic alteration should be considered in any ALS variant, particularly with a positive family history of FTD and/or ALS.
Importantly, none of our cases with FTD/ALS with early and profound aphasia, nor any of our non-fluent/agrammatic and semantic subtypes of primary progressive aphasia, were found to have a hexanucleotide repeat expansion. This observation not only has diagnostic relevance, but also suggests the repeat expansion on C9ORF72 affects cerebral networks in a more bilateral, patchy or diffuse manner as opposed to a focal/asymmetric manner which is what the primary progressive aphasia syndromes reflect. If this repeat expansion is also found to be rare or non-existent in other focal/asymmetric cortical degeneration syndromes such as the corticobasal syndrome, associative agnosia/prosopagnosia, posterior cortical atrophy, etc., this concept of clinical features reflecting more symmetric, patchy or relatively diffuse cerebral neurodegeneration will need to be explained based on the mechanism of how the repeat expansion causes cerebral dysfunction.
Neuropsychological considerations
Consistent with the phenotypic presentation of FTD/ALS, the predominant cognitive profile in subjects with the C9ORF72 hexanucleotide repeat expansion is associated with slowed processing speed, complex attention/executive dysfunction and impairment in rapid word retrieval. This pattern of dysfunction implicates frontal lobe structures and subcortical pathways involving dorsolateral prefrontal cortex and anterior cingulate. In contrast, naming, episodic memory, word reading and gross visuoperceptual/spatial skills are relatively spared, suggesting infrequent or minimal involvement of temporal and parietal lobe structures.
Approximately one-third of our subjects did not demonstrate impairment on cognitive screening measures. This finding may reflect the known poor sensitivity of screening measures to executive dysfunction or the presence of cases with behavioural variant FTD that have yet to develop cognitive deficits. It also bears noting that neuropsychological tests in common practice may not capture the entire spectrum of cognitive impairment possible in behavioural variant FTD or FTD/ALS (
Torralva et al., 2009). Thus, the need for broad-based and more sensitive measures of cognitive assessment will become paramount when evaluating subjects in the presymptomatic and early symptomatic stages of the disease.
Neuroimaging considerations
The syndrome of behavioural variant FTD is typically associated with mild to marked frontal and/or temporal lobe abnormalities, particularly in the non-dominant cerebral hemisphere (usually right), on structural and functional neuroimaging studies (
Miller et al., 1991;
Rosen et al., 2002;
Foster, 2003;
Whitwell et al., 2004,
2005,
2009;
Whitwell and Jack, 2007;
Rohrer, 2011). On MRI, the degree of cerebral cortical atrophy in patients with
C9ORF72 mutation tended to be rather mild, and was most apparent in the dorsolateral prefrontal and insular cortex. The temporal lobes were normal to only mildly affected. Some degree of parietal cortical atrophy was evident in some cases. And, the pattern of atrophy was never in a strikingly focal or asymmetric manner in any of our cases. Signal changes on T
2-weighted and fluid attenuation inversion recovery images in the cortex or subcortical white matter were also minimal or non-existent.
The findings on SPECT and PET were similar to those on MRI, with the dorsolateral prefrontal cortex, and anterior
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medial cingulate cortex involved. This topography of atrophy, hypoperfusion and hypometabolism correlates well with other features such as the prominent apathy and inertia as part of the behavioural variant FTD phenotype. The neuroimaging findings are also correlated with poor performance on neuropsychological measures that assess psychomotor speed, word fluency and sustained attention. Similar to the MRI findings in some subjects, the degree of hypoperfusion on SPECT or hypometabolism on PET was quite mild in some despite the presence of an obvious behavioural variant FTD syndrome. Also, a few cases had atrophy on MRI or hypometabolism on PET that was apparent in the biparietal/precuneus regions.
Therefore, abnormalities on neuroimaging studies that maximally involve the dorsolateral prefrontal cortex and anteromedial cingulate cortex were the most consistent findings. Parietal and/or temporal cortex abnormalities were present in some. Findings were mild in many, particularly early in the course, and relatively symmetric cerebral cortical involvement was the rule.
Neuropathological considerations
All cases had TDP-43 pathology, and more than half also had evidence of motor neuron degeneration and substantia nigra degeneration. Overt clinical evidence of MND or parkinsonism was not always associated with motor neuron or substantia nigra degeneration, respectively. Cortical neuronal loss, gliosis and spongiosis was most prominent in frontal lobe, with less in parietal and temporal lobes. Hippocampal sclerosis, which is common in FTLD-TDP (
Josephs and Dickson, 2007) was detected in less than half the cases with c9FTD/ALS, possibly owing to the greater frequency of FTLD-MND in this series, where it has been previously shown that hippocampal sclerosis is less frequent (
Josephs and Dickson, 2007). In an unselected autopsy series of c9FTD/ALS, MND was less common, while hippocampal sclerosis was more common than in this series (
Murray et al., 2011). The paucity of prominent amnestic syndromes in the present series may be accounted for partly by the low frequency of hippocampal sclerosis.
The TDP-43 pathology was variable, with the majority being type A. Type A (Mackenzie type 1) is characterized by many cortical neuronal cytoplasmic inclusions and dystrophic neurites often with intranuclear inclusions and widespread involvement of subcortical areas, while type B (Mackenzie type 3) have many neuronal cytoplasmic inclusions, but few dystrophic neurites and more limited subcortical pathology (
Josephs et al., 2009). These findings contrast to what was expected from previously suggested pathological correlates of c9FTD/ALS (
Boxer et al., 2011;
Mackenzie et al., 2011), which was suggested to be associated with type B (Mackenzie type 3) TDP-43 pathology. The explanation for the heterogeneity in TDP-43 pathology remains to be determined. It is of note that none of the cases had type C (Mackenzie type 2) pathology, which is associated with prominent cortical neuritic pathology with few cortical neuronal inclusions and clinically by the semantic subtype of primary progressive aphasia, was also not a clinical syndrome found in any patient in this series. The present findings contrast with detection of at least a few cases with type C in an unselected autopsy series of cases with the
C9ORF72 mutation (
Murray et al., 2011).
It has been suggested that ubiquitin-positive (P62-positive) inclusions in cerebellar granule neurons are characteristic of c9FTD/ALS (
Al-Sarraj et al., 2011;
Boxer et al., 2011;
Murray et al., 2011), and we were able to confirm this except for three cases in which such inclusions could not be detected. It has also been suggested that in the hippocampus, cases with c9FTD/ALS have neuronal inclusions that are disproportionately positive for ubiquitin compared with TDP-43 (
Al-Sarraj et al., 2011). We were able to confirm this finding. These are promising pathological features that might one day prove to lead to a specific and sensitive biomarker for c9FTD/ALS when their protein composition is eventually determined.
Comparison of ante-mortem and post-mortem features and findings in subjects with mutations in C9ORF72, MAPT and PGRN
Comparing and contrasting the clinical, neuropsychological, neuroimaging and neuropathological data between
C9ORF72, MAPT and
PGRN features will provide insights for clinicians to better target genetic testing (particularly for those with the behavioural variant FTD phenotype), and also enlighten our understanding of neurodegeneration associated with different mutations. A summary of the known features associated with these three mutations is shown in . Future analyses will likely include comparisons such as those shown in
Supplementary Figs 5 and
6.
| Table 6Key features of c9FTD/ALS due to the GGGGCC hexanucleotide repeat expansion in C9ORF72 compared with FTLD due to mutations in the genes encoding MAPT and PGRN |
Implications for research and future clinical practice
These findings suggest that genetic testing for the hexanucleotide repeat expansion in
C9ORF72 should be considered in anyone with the FTD and/or ALS phenotype and a positive family history of dementia, parkinsonism or ALS. The presence of multiple affected relatives, absence of primary progressive aphasia in any affected relatives, symmetric and relatively mild frontal
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parietal and/or temporal atrophy on MRI, and FTLD-TDP pathology in any relative should particularly raise suspicion of c9FTD/ALS. Clinical genetic testing will surely be developed in the near future. Considering that the frequency of the hexanucleotide repeat expansion in
C9ORF72 already exceeds the frequency of mutations in the other known genes associated with familial FTD and/or ALS, it is highly likely that a significant proportion of individuals with familial FTD and/or ALS without a mutation in the other known genes will be found to harbour the hexanucleotide repeat expansion in
C9ORF72.
1 Kevin B. Boylan,2 Neill R. Graff-Radford,2 Mariely DeJesus-Hernandez,3 David S. Knopman,1 Otto Pedraza,4 Prashanthi Vemuri,5 David Jones,5 Val Lowe,5 Melissa E. Murray,6 Dennis W. Dickson,6 Keith A. Josephs,1 Beth K. Rush,4 Mary M. Machulda,7 Julie A. Fields,7 Tanis J. Ferman,4 Matthew Baker,3 Nicola J. Rutherford,3 Jennifer Adamson,3 Zbigniew K. Wszolek,2 Anahita Adeli,1 Rodolfo Savica,1 Brendon Boot,1 Karen M. Kuntz,1 Ralitza Gavrilova,1 Andrew Reeves,1 Jennifer Whitwell,5 Kejal Kantarci,5 Clifford R. Jack, Jr,5 Joseph E. Parisi,8 John A. Lucas,4 Ronald C. Petersen,1 and Rosa Rademakers3
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