Clinical characteristics of patients with familial amyotrophic lateral sclerosis carrying the pathogenic GGGGCC hexanucleotide repeat expansion of C9ORF72

Introduction

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of adult life characterized by a progressive loss of upper and lower motorneurons at spinal and bulbar level. Its course is invariably fatal within 3–5 years from symptom onset, and there are currently no effective therapies (Kiernan et al., 2010). Approximately 5% of the cases are familial in nature, mostly inherited in an autosomal dominant manner (Chiò et al., 2008).

Recently, we described a large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72, a gene located on the short arm of chromosome 9 and responsible for ∼40% of familial ALS cases of European ancestry (Dejesus-Hernandez et al., 2011; Renton et al., 2011). Although this expansion accounted for an unprecedented large proportion of patients with familial ALS, little clinical data have been reported about the phenotypic characteristics of patients with ALS carrying this type of mutation.

The aim of the current article was to address this gap in our knowledge by describing the phenotype of ALS cases carrying the expansion by providing a detailed clinical description of affected cases from representative multi-generational kindreds, and by analysing the age of onset, gender ratio and survival in a large cohort of patients with familial ALS. As the underlying genetic lesion was a repeat expansion, we also examined effects of the parental gender transmitting the mutant allele on the manifestation of disease in their children and analysed variation of the age of onset in parent–child couples looking for evidence of anticipation.

Patients and methods

Results

We screened 120 unrelated familial ALS cases from mainland Italy and 21 unrelated familial ALS cases from Sardinia for C9ORF72 hexanucleotide repeat expansions using a repeat-primed polymerase chain reaction assay. Pathogenic repeat expansions were detected in 45 (37.5%) patients from mainland Italy and 12 (57.1%) patients of Sardinian ancestry. Nine (22.0%) of the 41 German index familial ALS cases were previously reported to carry the repeat expansion (Renton et al., 2011).

The clinical characteristics of patients with familial ALS carrying the C9ORF72 expansion with different ethnic origin (Italians versus Sardinians versus Germans) are reported in Table 1. No statistically significant differences were found in age at onset, gender, site of onset and frequency of FTD between these cohorts.

Nearly half of the patients with ALS carrying the pathogenic repeat expansion developed comorbid cognitive impairment during the course of their illness (44.2%, n = 30 of 68 patients). All of these cases presented with behavioural variant FTD. More specifically, all patients assessed with the Frontal Systems Behaviour Scale (Family Rating form) (Grace and Malloy, 2001) (n = 23) had impairment in the apathy and executive domains (median T-scores: apathy 79, range 66–91, executive dysfunction 83, range 70–94; normal value < 65), while the disinhibition domain was pathological in only three patients. Patients with ALS with C9ORF72 mutations and comorbid FTD also had pathological scores in tests for executive function, such as Stroop Colour-Word Interference Test, letter and category fluency test and Trail Making A and B. In contrast, tests for memory function, such as Rey Figure and Digit Span, were generally normal. Three index cases displayed prominent delusions and hallucinations, whereas one case presented with an acute-onset obsessive–compulsive disorder.

Discussion

We found that the GGGGCC hexanucleotide repeat expansion of the C9ORF72 gene is the most common mutation in our large cohort of patients with familial ALS of Italian, Sardinian and German ancestry. This confirmed our previous findings and demonstrated the importance of this gene in the pathogenesis of the familial form of ALS (Renton et al., 2011). Overall, the discovery of the repeat expansion means that the genetic defect has now been elucidated for 102 (57.6%) of our consecutive series of 177 Italian familial ALS index cases collected across the Italian mainland. Furthermore, the high rate of this mutation in the isolated population of Sardinia, together with the previously described prevalence of TARDBP mutations in the same population (Chiò et al., 2011a), means that the cause of nearly two-thirds of all familial ALS on this island is now explained.

The frequency of familial ALS carrying the C9ORF72 hexanucleotide repeat expansions in Italy and Germany is lower than that reported in the Finnish population (Renton et al., 2011) and similar to that of the American series (Dejesuz-Hernandez et al., 2011; Renton et al., 2011), supporting the notion of the possible north-European origin of the mutation.

Patients with C9ORF72 hexanucleotide repeat expansions display phenotypic differences compared with patients with mutations of other genes or with unknown mutations. The most notable difference is a significantly higher frequency of cognitive impairment both within the index patients and within the extended kindreds (Fig. 2). Mutations in other genes, particularly TARDBP and valosin containing protein, have been occasionally associated with FTD syndromes (Borroni et al., 2010; Chiò et al., 2010; Johnson et al., 2010). However, the high frequency of cognitive impairment associated with the GGGGCC pathogenic repeat expansion supports the hypothesis that the overlap between ALS and FTD phenotypes is largely driven by the chromosome 9p21 genetic lesion (Renton et al., 2011). In this way, our genetic findings confirm what was already known about the neuropathology of ALS and FTD: these clinically disparate syndromes are characterized by the mislocalization and cytoplasmic accumulation of TDP-43 leading this group of disorders to be reclassified as TDP43-proteinopathies (Geser et al., 2009). Indeed, the neuropathological features of chromosome 9p-linked ALS/FTD include a TDP43 proteinopathy, with the peculiar involvement of hippocampal pyramidal neurons (Boxer et al., 2011).

In our cohort, FTD in C9ORF72 mutated cases almost invariably presents with behavioural symptoms, and there was an absence of cases presenting with semantic dementia or progressive non-fluent aphasia. Moreover, we identified psychotic-like symptoms, such as delusions and hallucinations both in three index cases with ALS–FTD and in several relatives within the extended kindreds (see, for example, Pedigree ITALS#7). These findings are consistent with previous observations of families linked to chromosome 9.21, where behavioural symptoms were reported to be prominent (Morita et al., 2006; Boxer et al., 2011; Pearson et al., 2011). The neuroanatomical substrate underlying these psychotic symptoms is not clear, though it is noteworthy that pathological involvement of the hippocampus is prominent in cases carrying the C9ORF72 repeat expansion, a CNS region that is thought to have a role in the production of psychotic-like symptoms (Goto and Grace, 2008).

Studies on family aggregations of neurodegenerative disorders are relatively scarce in ALS, but seem to confirm the co-occurrence of ALS and dementia in the same families. In an article based on a retrospective analysis of a series of 197 Irish patients with ALS and 235 general neurology controls, there was a significantly higher risk of Parkinson's disease and dementia in first-degree relatives of patients with ALS compared with controls (Fallis and Hardiman, 2009). Similarly, a case control study on 635 patients with ALS and 1616 controls in the Netherlands found a mild increase of dementia among siblings and parents of patients with sporadic ALS (Huisman et al., 2011). However, in both papers no distinction between the different types of dementia (in particular, Alzheimer's disease versus FTD) was made, making it difficult to interpret their findings in light of our new understanding of the genetics of chromosome 9p21-linked ALS/FTD.

The reason why C9ORF72 hexanucleotide repeat expansions manifest as disparate ALS and FTD clinical syndromes is unknown. It could be related to variation in the size of the GGGGCC expansion, to the effect of unknown regulatory genes, or to environmental causes. However, an affected child was more likely to manifest symptoms of cognitive dysfunction if the affected parent had a FTD or ALS–FTD phenotype (see, for example, Pedigrees ITALS#4, ITALS#7, ITALS#8 and ITALS#9). This strongly suggests that phenotype is being influenced by a heritable factor, rather than by a pure stochastic event.

Familial patients with ALS with C9ORF72 gene repeat expansions had nearly double the frequency of bulbar-onset disease compared with those with mutations of other genes. This finding is in line with the observation that bulbar onset is more frequent in Scandinavian and north-European populations (Logroscino et al., 2010), which have been reported to have the highest frequency of C9ORF72 mutations (Laaksovirta et al., 2010; Mok et al., 2011).

On average, patients carrying the C9ORF72 expansion manifested disease in their fifth decade of life. A similar pattern has been observed for patients with SOD1 and TARDBP mutations. The biological substrate underlying this late-adult onset is a matter of speculation at present. Normal cellular ageing, environmental exposures, other genetic variants or a combination of these factors may play a role in determining clinical onset. However, we have observed in our cohort that patients with ALS manifesting cognitive impairment had a significantly higher age at onset. One possible explanation for this observation is that the clinical phenotype is, at least in part, driven by the age at onset. A strong influence of the age at onset of ALS phenotype has been already found in sporadic ALS, with a prevalence of bulbar onset in older patients and a prominent upper motor neuron (pyramidal) phenotype in younger patients (Chiò et al., 2011b).

In our series, there was a trend towards an earlier age at onset in younger generations. This 7-year difference in age of onset between parents and children may represent anticipation, a phenomenon known to occur in repeat expansion diseases, such as Huntington's disease and myotonic dystrophy, and may reflect increase in the expansion size with each meiosis. Care is required in interpreting these data, because of the relatively small number of the cohort, and because the individual kindreds were not sufficiently detailed to detect an effect beyond two generations (e.g. grandparent and great-grandparent generation). Accurate measurements of the repeat expansions size by Southern blot analysis will be required to confirm if there truly is repeat expansion instability from generation to generation: such analysis could not be performed in the current cohort due to lack of sufficient quantities of DNA required for such genomic analysis.

The likelihood of detecting the C9ORF72 repeat expansion mutation was not related to the strength of family history of ALS. In fact, in several pedigrees the relative affected by ALS was more distant than a first- or second-degree relative (see, for example, Pedigree ITALS#6). In fact, careful examination of most pedigrees showed that a first- or second-degree relative was more likely to be affected by FTD, strongly supporting the motion of the ALS–FTD continuum of disease. It also highlights the importance (and difficulties) of obtaining a detailed family history of all neurodegenerative disease from patients with ALS and argues against current efforts to limit the definition of what is familial ALS (Byrne et al., 2011).

The current European Federation of Neurological Society (EFNS) guidelines clinical DNA analysis for gene mutations should only be performed in cases with a known family history of ALS, and in sporadic ALS cases with the characteristic phenotype of the recessive D90A mutation (EFNS Task Force, 2011). The complexity of the phenotype of pedigrees related to C9ORF72 mutations, which include cases with pure ALS, ALS–FTD and pure FTD, challenge the current notion of familial ALS, expanding its boundaries to families with a single patients with ALS phenotype and relatives with pure FTD, as exemplified by pedigree ITALS#9 of our series.

In conclusion, C9ORF72 hexanucleotide repeat expansions represent the most frequent genetic mutation among Italian familial ALS. Together with mutation of SOD1, TARDBP and FUS, mutations of C9ORF72 account for ∼60% of familial ALS in Italy. A large number of ALS cases with C9ORF72 mutations present a comorbid behavioural variant of FTD, sometimes associated with hallucinations and delusions. The identification of specific phenotypes related to the different ALS genes is still in its beginnings, but early indications are already emerging that can aid the clinical decision-making. In particular, patients with ALS carrying C9ORF72 repeats are characterized by a high incidence of bulbar-onset disease and comorbidity with FTD, and their pedigrees typically display a high frequency of cases with pure FTD.

Funding

Federazione Italiana Giuoco Calcio (Grant 2010 #1 to M.S. and A.C.); Fondazione Vialli e Mauro per la Sclerosi Laterale Amiotrofica onlus (to A.C.); Ministero della Salute (Ricerca Sanitaria Finalizzata, 2007) (to A.C. and G.R.); European Community's Health Seventh Framework Programme (FP7/2007-2013) under grant agreement 259867 (to A.C.); The Intramural Research Programmes of the National Institutes of Health (NIH); National Institute on Aging (Z01-AG000949-02); and National Institute on Neurological Disorders and Stroke (NINDS); The Packard Centre for ALS Research at Hopkins (to B.J.T.); the ALS Association (to B.J.T. and A.C.); Microsoft Research (to B.J.T.); the Deutsche Forschungsgemeinschaft (SFB 581 and TP B1 to C.D. and M.S.).