Clinico-pathological features in amyotrophic lateral sclerosis with expansions in C9ORF72

Determination of the clinical phenotype of patients with ALS with the C9ORF72 hexanucleotide expansion

Clinical notes of patients found to carry the C9ORF72 expansion were reviewed in a systematic fashion to identify details of the disease phenotype including gender, age of onset, disease duration, disease variant, details of family history and the presence of any cognitive impairment. It should be noted that patients in this cohort only underwent formal cognitive evaluation when a clinical problem was identified and did not undergo routine serial neuropsychological evaluation during the disease course. Therefore, the recorded incidence of frontotemporal dysfunction in this cohort is likely to be an underestimate, as prospective neuropsychological studies show a higher prevalence of frontal lobe dysfunction in patients with ALS (Phukan et al., 2011).

Screening for the C9ORF72 hexanucleotide repeat sequence by repeat primed polymerase chain reaction

Genomic DNA (100 ng) was amplified using the primers and method described by Renton et al. (2011) with a minor adjustment to the primer ratio: Forward:Reverse:Anchor = 8:1:8. Detailed methodology is provided in the Supplementary Material. Fragments were analysed on an ABI3730 capillary analyser (Applied Biosystems, Life Technologies Corporation) using a 60-s injection time. Fragment data were analysed using Peak Scanner Software (Applied Biosystems, Life Technologies Corporation).

Neuropathological evaluation

The brain and spinal cord tissues were donated to the Sheffield Brain Tissue Bank for research, with the consent of the next of kin. The donation procedure and use of the tissue in this project were undertaken with research ethical committee approval. Tissue was available for detailed pathological evaluation from 19 of the 22 brain tissue bank cases with the hexanucleotide expansion of C9ORF72. These cases were compared with up to 96 (see below and Table 3) ALS cases, which were negative for the C9ORF72 expansion and three neurologically normal controls. For some cases, one cerebral hemisphere, half the midbrain and brainstem, a portion of the cerebellum and segments of the spinal cord at various levels were rapidly frozen in liquid nitrogen at autopsy and stored at −80°C. The remainder of the CNS was formalin-fixed. For the other cases, only a portion of the cerebellum was frozen, and the whole brain and spinal cord were formalin fixed. Selected blocks (including lumbar, thoracic and cervical spinal cord, medulla, midbrain, hippocampus, and frontal, temporal and motor neocortex) were processed to paraffin.

In addition to routine neurohistology with tinctorial preparations, immunohistochemistry was performed for p62/sequestosome 1, TAR DNA-binding protein 43 (TDP-43), FUS, OPTN, CD68 and C9ORF72 (Supplementary Table 1) where paraffin tissue was available. The latter antibody is commercially available (Santa Cruz Biotechnology Inc.) and was selected on the basis that it labelled a protein of the molecular weight of C9ORF72 on western blotting (data not shown). Immunohistochemistry was performed on all available cases with the hexanucleotide expansion as well as three cases with sporadic ALS without the expansion and three neurologically healthy controls.

To characterize the distribution of pathology in cases with ALS in the Sheffield Brain Tissue Bank, the extent of p62-positive pathology was assessed. In all regions assessed, a single 6-µm section was examined and the number of neuronal cytoplasmic inclusions in the region of interest assessed semi-quantitatively as 0–4 (low), 5–9 (intermediate) or 10 or more (high). The regions of interest assessed were the anterior horn of the spinal cord, at mid-cervical and lumbar levels; the hypoglossal, dorsal vagal and ambiguus nuclei of the medulla; the dentate granule cell layer and CA4 subregion of the hippocampus and the frontal and motor cortices. In the cortical regions, quantification of neuronal cytoplasmic inclusions was carried out in 10 fields (×25 objective).

In addition, the number of neuronal cytoplasmic inclusions in the CA4 subregion of the hippocampus was assessed using the same semi-quantitative scheme on sections that had been immunostained for TDP-43 and OPTN.

Clinical features of ALS cases with pathological expansions in C9ORF72

The clinical features of the cases with pathological expansions in C9ORF72 are summarized in Table 1. The mean age of onset was 57.3 years (range 27–74 years). Of these, 60% of cases had limb onset disease, 31% had bulbar onset disease; 6% had multi-focal disease onset, one patient presented with dementia and for one patient the time of onset was not known. The mean disease duration from symptom onset in these patients was 30.5 months (range 7–60 months); three patients are alive at the time of writing and for one patient survival information was not available.

Clinical characteristics of patients with the C9ORF72 expansion were compared with the remainder of the screened cohort (Table 2). The C9ORF72 patients had a significantly lower age at onset than non-C9ORF72 cases (mean age of onset 57.3 years, SD 8.9 years compared with mean 60.1 years, SD 12.3 years; P = 0.03, d.f. = 494, t = 2.20), but not significantly different to the overall familial ALS cohort (P = 0.43, d.f. = 107, t = 0.78). Likewise, patients with the C9ORF72 expansion had a significantly shorter duration of disease than non-C9ORF72 cases [mean (SD) duration of disease 30.5 (13.3 months) compared with mean (SD) 36.3 (28.5 months); P = 0.01, d.f. = 397, t = 2.44] but not significantly different to the overall familial ALS cohort (P = 0.32, d.f. = 100, t = 1.00). The subgroup of patients with sporadic ALS with the C9ORF72 expansion differed from patients with familial ALS with the expansion, in that they had a significantly higher age of onset [mean 59.5 (SD 7.0 years) in sporadic ALS, 54.3 (SD 10.4 years) in familial ALS, P = 0.03, d.f. = 59, t = 2.21], but no difference in age of onset to the screened cohort overall. There was no difference between patients with sporadic ALS and patients with familial ALS with the expansion, with respect to duration of disease [mean (SD) 28.6 (12.0 months) in sporadic ALS, and 33.3 (14.8 months) in familial ALS, P = 0.21, d.f. = 56, t = 1.26].

In our cohort of patients with the C9ORF72 expansion, 5/27 (19%) familial ALS cases and 5/35 (14%) sporadic ALS cases had evidence on clinical and neuropsychological testing of FTD. An additional 12 cases had a family history of dementia in first- or second-degree relatives, six of whom had familial ALS and six of whom had sporadic ALS. Overall 22/62 (35%) of patients with the expansion had either a personal diagnosis of dementia or a family history of dementia in first- or second-degree relatives. In this cohort, routine neuropsychological assessment was not performed in the absence of a clinically apparent cognitive problem, so subclinical cognitive dysfunction was not evaluated.

Several of the patients carrying the hexanucleotide expansion in C9ORF72 were either diagnosed with or had a family history of other non-dementia neurological or neuromuscular disease, particularly neurodegenerative disease: four patients had a family history of Parkinson's disease and one patient had a comorbid diagnosis of Parkinson's disease that was confirmed at post-mortem; thus 4/62 (6.5%) patients had either a diagnosis of Parkinson's disease or a family history of Parkinson's disease. Two patients had evidence of demyelinating disease and one further patient had a family history of multiple sclerosis; thus 3/62 (5%) patients had either a diagnosis or a family history of demyelinating disease. Other noteworthy findings included a patient with a family history of ALS, Charcot–Marie–Tooth disease and dementia; one patient with early onset cataracts which also occurred in his mother; one patient with a family history of Huntington's disease and one patient with a brother who died with a diagnosis of muscular dystrophy.

The patients with C9ORF72 expansion had limb onset disease in 37/62 (60%) cases and bulbar onset disease in 19/62 (31% cases) (Table 2). These proportions are similar to those for the cohort without the C9ORF72 expansion (56 and 25%, respectively). There was no significant difference in gender ratio (P = 0.09, d.f. = 1, χ² = 2.85) or site of onset (P = 0.74, d.f. = 1, χ² = 0.10) between the two groups although the patients with familial ALS with the expansion had a female preponderance (ratio males:females = 1:1.7), which was not present in the patients with sporadic ALS with the expansion (males:females = 1.1:1) or the screened cohort overall (males:females = 1.3:1).

Patterns of inheritance

Hexanucleotide expansions of C9ORF72 account for a large proportion (43%) of familial ALS cases in our cohort from the North of England and many of these families exhibit clear autosomal dominant inheritance. The high incidence of the C9ORF72 repeat expansions in apparently sporadic cases is an important observation to explain. It may represent a genuinely inherited pathogenic genotype with variable penetrance or it may represent a high rate of new mutation consistent with the suggestion that the C9ORF72 gene may be inherently unstable (Renton et al., 2011). That incomplete penetrance can occur is demonstrated by a family within our cohort where the expansion is present in the index case and his maternal uncle who are both patients with ALS, and in the mother of the index case who is disease free into old age (Fig. 3). It is noteworthy that for a proportion of the apparently sporadic cases there is a family history of neurological disease; it seems likely that some of these cases represent unrecognized or undisclosed familial disease. In particular, some of the patients with relatives who developed dementia may in fact have familial ALS/FTD. It is interesting that, with regard to age of onset and gender ratio but not disease length, the patients with sporadic ALS with the C9ORF72 expansion are distinct from the patients with familial ALS with the expansion. The earlier age of onset of familial ALS cases with the mutation may suggest the occurrence of genetic anticipation, although this could not be confirmed in this study as the precise length of the pathological repeat sequences has not yet been determined.

Of note, we have now identified a pathogenic change in 42/63 (67%) of our familial cohort. In two cases, an expansion in C9ORF72 was found to coexist with another identified putative mutation linked to ALS. That the expansion was absent from most of our patients with a previously identified mutation is supportive of the independent pathogenesis of those changes. An expansion was found in one case (Patient 37) who had a p.Ala321Val change in TARDBP. The pathogenesis of mutations in the C-terminal region of TARDBP is well established (Sreedharan et al., 2008; Kirby et al., 2010) and it is notable that the remainder of patients in our cohort with described changes in TARDBP did not carry the C9ORF72 expansion. It is interesting that the age of onset in this patient was relatively young (37 years), perhaps consistent with a synergistic effect of both genetic changes on the disease pathogenesis. However, the disease duration in this patient was relatively long at 58 months. Since mutations in TARDBP and expansions of C9ORF72 are both associated with neuronal cytoplasmic TDP-43 inclusions, it is conceivable that they may be affecting the same pathway in a non-additive manner. Unfortunately, no post-mortem material was available from this case.

An unreported familial ALS case with a p.Glu322Lys OPTN substitution also showed a C9ORF72 expansion. While this substitution has been reported as a polymorphism in sub-Saharan African samples (Liu et al., 2008), it was absent from 375 neurologically normal Caucasian controls from the same geographical region. This patient had bulbar onset ALS, with onset of disease aged 50 years and a disease course of 29 months.

Dementia

An association between ALS and FTD is well established (Phukan et al., 2007). However, a previous population study reported that the incidence of dementia (5%) in relatives of patients with ALS was only slightly higher than in controls (Huisman et al., 2011) and only in first-degree relatives. ALS is thought to be the common end-point of various disease mechanisms (Ferraiuolo et al., 2011) and it is possible that only some of these mechanisms result in an associated dementia. Thus, disease heterogeneity may have meant that a stronger association in some patients within this population study was masked by a lack of association in other patients. In our cohort of patients with an expansion in C9ORF72, 35% either had a diagnosis of dementia or a family history of dementia. We propose that the subtype of ALS caused by hexanucleotide repeat expansions of C9ORF72 shows a striking association with dementia clinically. This proposal is strongly supported by our finding of extra-motor neuropathology in all C9ORF72 expansion cases examined at autopsy, in combination with a relative paucity of extra-motor pathology in cases found to have normal repeat lengths of C9ORF72. This hypothesis would be supported further by routine cognitive testing of ALS cases during life, with subsequent correlation with post-mortem neuropathology. The TDP-43 proteinopathies ALS, ALS with FTLD and pure FTLD-TDP have often been seen as a continuum of disease at both the clinical (Lillo and Hodges, 2009) and pathological levels (Mackenzie and Feldman, 2005; Geser et al., 2009). This has raised the question of what governs where on this spectrum a patient with a TDP-43 proteinopathy will manifest disease pathologically, and how they will present clinically. Our data suggest that repeat expansions of C9ORF72 have a strong influence on the clinical phenotype and the spectrum of pathology. The precise mechanism(s) by which the repeat expansion has this effect needs to be elucidated, and it will clearly be of interest to compare the distribution of pathology in brains of FTLD-TDP cases with and without the repeat expansion.

Other neurological disease

Several of the patients carrying the hexanucleotide expansion in C9ORF72 were either diagnosed with or had a family history of other neurological, particularly neurodegenerative disease. This is illustrated by the case with both ALS and multiple sclerosis confirmed at autopsy. Previous work has suggested that the clinical spectrum of ALS may be wider than initially recognized. In particular, mutations in the VCP gene appear to cause a clinical spectrum including inclusion body myositis, FTD and Paget's disease (Johnson et al., 2010). ALS has also been reported to coexist with glaucoma in a patient with a mutation in the OPTN gene, although the authors went on to suggest that this may have been a coincidence (Maruyama et al., 2010). Analysis of our cohort suggests that expansion of C9ORF72 may also produce a broad clinical phenotype although we have not yet demonstrated segregation of the expansion with non-ALS neurological disease.

A previous population study on the Island of Guam described coexistence of Parkinson's disease and ALS both within individuals and within families (Yanagihara et al., 1983). However, the results of other population studies investigating the coexistence of more than one neurodegenerative disease have been inconsistent. A recent population-based study suggested that the rate of Parkinson's disease among relatives of patients with ALS was not significantly different from controls (Huisman et al., 2011). However, it is noteworthy that among our patients with the hexanucleotide repeat sequence, the frequency of Parkinson's disease in relatives (6.5%) is much higher than in either the disease or control population from the Huisman study (0.9%). One of our patients with the C9ORF72 expansion was diagnosed with both Parkinson's disease and ALS. His Parkinson's disease developed at a relatively early age of 38 years but was otherwise clinically typical, showing a good response to L-DOPA therapy; he had a deep brain stimulator inserted aged 47 years. Post-mortem he had neuropathological features of both Parkinson's disease and ALS including cell loss from the substantia nigra and 6/6 Braak grade α-syncluclein pathology with substantial staining both in the substantia nigra and in neocortical regions.

The coexistence of multiple sclerosis and ALS has been previously reported (Hader et al., 2008; Ismail et al., manuscript under review) and an increased incidence of multiple sclerosis in the offspring of patients with ALS has also been reported (Hemminki et al., 2009). The association of patients with ALS with the C9ORF72 expansion and multiple sclerosis in our cohort is consistent with these reports and raises the possibility that the association between ALS and multiple sclerosis may be related to this genotype. The diagnosis of Huntington's disease in the father of a patient carrying the C9ORF72 expansion is especially interesting given that Huntington's disease is also related to an aberrant repeat sequence. Perhaps a common underlying mechanism has led to the de novo occurrence of nucleotide repeat sequences in both of these individuals.

Neuropathological features

Qualitatively, the neuropathological features associated with hexanucleotide repeat expansion of C9ORF72 are those of classical ALS with ubiquitylated, TDP-43 and OPTN positive neuronal and glial cytoplasmic inclusions in upper and lower motor neurons as well as glia. This is seen in combination with Bunina bodies and degeneration of the pyramidal tracts. What is striking is the association of pyramidal cell pathology with the expansion in the hippocampus and frontal neocortex, while very little such pathology is seen in ALS cases without the repeat expansion. This differentiation is less marked in the context of the granule cells of the hippocampal dentate gyrus, a structure that is generally believed to be one of the most sensitive extra-motor structures to TDP-43 proteinopathy (Mackenzie and Feldman, 2003; Takeda et al., 2009). The extra-motor pathology seen is best characterized as type B according to the scheme of Mackenzie et al. (2011).

The subdivision of ALS cases into two subgroups on the basis of neuropathological involvement of extra-motor (principally hippocampal and frontotemporal neocortex) has been described elsewhere (Nishihira et al., 2008). Data presented in this report indicate that repeat expansions in C9ORF72 represent a major molecular basis for this pathological dichotomy.

It appears from our data derived from a large pathological cohort of ALS autopsies that the finding of extra-motor neocortical pathology, in particular ubiquitylated neuronal cytoplasmic inclusions in the CA4 subfield of the hippocampus is a relatively reliable indicator of the presence of a hexanucleotide repeat expansion in C9ORF72. Given that these changes can be detected at autopsy with relative ease, this neuropathological feature may be used to guide genetic investigation for the repeat expansion and thereby inform genetic counselling and further research. Interestingly, along with the characteristic hyaline conglomerate inclusions found in some patients with SOD1 mutations (Ince et al., 1998), this report adds another important element to neuropathological predictors of genotype in ALS. It is also noteworthy that in the hippocampus, the majority of the neuronal cytoplasmic inclusion pathology that is evident on p62 immunohistochemistry is not apparent on TDP-43 immunohistochemistry. In fact, for the CA4 subregion (where pathology is most specific for the repeat expansion), OPTN is a more sensitive marker than TDP-43, although neither is as sensitive as p62. This raises the significant issue of what protein forms the ubiquitylated lesion in the majority of these neurons if it is not TDP-43.

Immunohistochemistry to C9ORF72 protein reveals multiple minute, puncta of labelling throughout the neuropil of grey matter structures having the appearance of synaptic labelling in neuronal processes. In this hippocampus, this is most marked in the regions that show pathology that is specific for the hexanucleotide repeat expansion. There is additionally labelling of glial cells, many of which appear to be oligodendrocytes, and around axons, corresponding to some regions of myelin. These constitute initial observations, and the precise nature of C9ORF72 expression requires more detailed characterization, including finer localization studies, in order to begin to understand the function of the C9ORF72 protein in the CNS.

We thank all the ALS/MND patients from the Sheffield and Newcastle Care and Research Centres for Motor Neurone Disorders and their family members who contributed so generously to research by the donation of biosamples.