Frequency of the C9ORF72 hexanucleotide repeat expansion in ALS and FTD in diverse populations: a cross-sectional study

Introduction

Amyotrophic lateral sclerosis (ALS, Online Mendelian Inheritance in Man (OMIM) 105400) is a fatal neurodegenerative disease characterized by rapidly progressive paralysis and death from respiratory failure, typically within three years of symptom onset. Approximately five percent of the disease is inherited following a clear Mendelian pattern, whereas the vast majority of cases are classified as sporadic, as they appear to occur randomly in the community.¹ Considerable progress has been made in understanding the genetic underpinnings of familial ALS.² In contrast, the etiology of sporadic or idiopathic ALS is far less well understood. Mutations in the known familial ALS genes, SOD1, FUS, and TDP-43, occur only rarely in sporadic cases (each accounting for less than 1·0%)^3–5, and genome-wide association studies have identified a limited number of risk loci that have proven difficult to replicate.⁶

Frontotemporal dementia (FTD, OMIM 600274) is a degenerative disorder of the frontal and anterior temporal lobes, and is a common form of dementia affecting individuals below the age of 65 years. The syndrome is characterized clinically by initial behavioral disturbances, followed by cognitive decline that leads to dementia and death within a median of seven years from symptom onset. Similar to ALS and other neurodegenerative diseases, a large proportion (~60·0%) of these cases are categorized as sporadic, and the etiology of this idiopathic form of disease is largely unknown.⁷ There is growing recognition that ALS and FTD represent a continuum of neurological disease that share a common pathology consisting of TDP-43-positive inclusions within the CNS.⁸

We recently reported that a large hexanucleotide repeat expansion located within the non-coding portion of the C9ORF72 gene is the cause of chromosome 9-linked ALS and FTD.^9,10 This genetic lesion accounts for an unprecedented large portion (~40·0%) of both familial ALS and familial FTD cases. The same mutation was present in nearly one quarter of apparently sporadic ALS and FTD cases in the genetically homogeneous Finnish population, and in 4·1%of sporadic ALS cases and 3·0% of sporadic FTD cases from the United States. However, these estimates were based on relatively small cohorts drawn from a limited number of institutions.

These findings prompted us to more accurately estimate the frequency of this C9ORF72 hexanucleotide repeat expansion in a large cohort of European and US patients with sporadic ALS and sporadic FTD. We also examined the occurrence of this mutation in diverse, non-Caucasian populations around the world. To do this, we screened 4,448 patients diagnosed with ALS and 1,425 patients diagnosed with FTD drawn from nineteen distinct regions. Our findings have clinical implications for the diagnosis and management of ALS and FTD patients.

Methods

Role of the funding sources

The sponsors of the study had no role in study design, data collection, analysis, interpretation, writing of the report, or in the decision to submit the paper for publication. All authors had full access to all of the data and held final responsibility for the decision to submit for publication.

Results

Supplementary Figure 1 (webappendix p 11) and Table 1 show the frequency of the C9ORF72 hexanucleotide repeat expansion in patients diagnosed with sporadic ALS and sporadic FTD from different geographical regions of the world. The pathogenic expansion was found in 7·0% of Caucasians diagnosed with sporadic ALS (n = 236 of 3,377 samples from the United States, Europe, Middle East and Australia), 4·1% of African-Americans (n = 2 of 49), and 8·3% of Hispanic ALS samples (n = 6 of 72). The rate of pathogenic expansion was lower among sporadic FTD cases: 6·0% of Caucasian patients carried the mutation (n = 59 of 981 samples). In contrast, the GGGGCC repeat expansion on chromosome 9p21 was not present in sporadic patients of Native American (n = 3 ALS cases), Asian (n = 238 ALS and 10 FTD cases), or Pacific Islander origin (n = 90 ALS cases), though this may reflect the smaller size of the cohorts screened in these populations.

In addition to the sporadic cases, we screened 588 familial ALS cases and 403 familial FTD cases for the presence of the C9ORF72 repeat expansion (Table 2 and webappendix p 12). Of these, 373 familial ALS cases and 230 familial FTD cases were reported in our original Neuron paper and elsewhere.^10–12 All of the cohorts are presented here to provide a comprehensive assessment of the global frequency of the expansion. Overall, 37·6% of familial ALS cases and 25·1% of familial FTD cases carried the genetic lesion, confirming our previous findings that this mutation was responsible for an unparalleled proportion of these diseases. Notably, we identified a single Japanese individual diagnosed with familial ALS, who carried the hexanucleotide repeat expansion. We also found that a familial FTD case from Lund, Sweden, carried the expansion, suggesting that the chromosome 9p21 genetic lesion may be responsible for the geographical cluster of FTD patients observed in that region.¹⁹

Of the 2,585 neurologically normal control samples screened for the C9ORF72 repeat expansion, five (0·2%) were found to be carriers: two were previously reported elderly Finnish samples¹⁰, and the remaining three were German and American individuals under the age of 40 (webappendix p 9).

Within Europe, the highest mutation frequency was observed in the Finnish population (21·1% of sporadic ALS patients and 18·8% of sporadic FTD patients).¹⁰ Approximately 6·0% of sporadic ALS patients from Germany and England carried the expansion, whereas Mediterranean Italian ALS patients had a lower rate (4·1%). Interestingly, 7·8% of sporadic ALS patients from the isolated island population of Sardinia were found to have the mutation, and the Dutch population had the lowest rate observed among European countries (2·2% of sporadic FTD cases). Both the Australian and US white populations had an intermediate rate with ~5·0% of their sporadic ALS patients carrying the pathogenic repeat expansion, perhaps reflecting the population and immigration histories of these continents.

We previously reported the identification of a 42-SNP founder haplotype across the same 232kb block of chromosome 9p21 region where the pathogenic hexanucleotide expansion was ultimately found.^16,20 For the current paper, we analyzed genome-wide SNP chip data from 262 patients that carried the C9ORF72 hexanucleotide repeat expansion, including 107 Finnish ALS cases, 97 US cases, 34 Italian cases, 9 Australian cases, 7 German cases, 7 Israeli cases and 1 Japanese case. This analysis showed that each case carrying the pathogenic GGGGCC repeat expansion also shared the Finnish founder risk haplotype, at least in part (Figure 1A). Furthermore, sporadic and familial cases carried the same founder risk haplotype. These findings suggest that the pathogenic hexanucleotide repeat expansion in the C9ORF72 gene may have occurred on a single occasion in human history and subsequently disseminated throughout these populations. Analysis of haplotype sharing between these cases estimated the age of the C9ORF72 repeat expansion to be ~1,500 years old (median = 100·5 generations, interquartile range = 57·6 – 127·6 generations, assuming one generation represents 15 years, Figure 1B).

Age-related penetrance of the C9ORF72 hexanucleotide repeat expansion was calculated based on the ages of 603 mutant-gene carriers (Figure 2). The pathogenic expansion was non-penetrant in carriers below 35 years of age, increasing to 50·0% penetrance by 58 years of age, and was almost fully penetrant by 80 years of age. No difference was observed in disease penetrance according to familial status; by ALS or FTD diagnosis; by gender; or, according to symptom onset in ALS or FTD patients (webappendix p 13).

Clinical details of patients carrying the hexanucleotide repeat expansion are given in Table 3. ALS patients with the pathogenic repeat expansion were more likely to be female (χ²= 11·2, 1 degree of freedom (df), p-value = 0·0008), have a family history of disease (χ²= 589·0, 1 df, p-value < 0·0001), and to have bulbar-onset disease (χ²= 10·7, 1 df, p-value = 0·0011) compared to cases that did not carry the expansion. FTD patients were also more likely to have a family history of disease (χ²= 106·0, 1 df, p-value < 0·0001), and to present with behavioural variant FTD (χ²= 25·1, 2 df, p-value < 0·0001).

Discussion

Our data show that the C9ORF72 hexanucleotide repeat expansion is the most frequent cause of sporadic ALS and sporadic FTD identified to date, accounting for ~5·0–7·0% of all Caucasian cases in our large series of patients. These frequency rates were moderately higher than published estimates that were based on smaller cohorts obtained from a single institution.⁹ Prior to the identification of the genetic lesion underlying chromosome 9-linked ALS/FTD, the most common genetic cause of sporadic ALS was mutations in the SOD1 gene (accounting for 0·7% of cases in a population-based cohort)³, whereas mutations in the PGRN gene were the most common cause of sporadic FTD (3·0–4·0% in clinic referral series).²¹ The high frequency of the pathogenic expansion in our patient cohort is consistent with previous GWAS studies that identified the association signal on chromosome 9p21 as the only replicable locus in the sporadic form of ALS and FTD.^16,22–24 Our findings confirm the central importance of genetics in the pathogenesis of the idiopathic form of these fatal neurodegenerative diseases.

Our haplotype data suggest that the pathogenic GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene arose from a single mutational event^16,20 that occurred ~1,500 years ago. The geographical distribution of the mutation suggests that the mutation appeared in Northern Europe and spread from there. Alternatively, the high frequencies in Finland and other isolated populations could be explained by the history of these communities. Finland and Sardinia are isolated areas, and have genetically homogeneous populations that originated from a small number of founders.²⁵ Genetic drift has had a large influence on allele frequencies in these populations and could explain the high occurrence of the mutation in these geographical isolates.

Recognizing that all cases carrying the C9ORF72 repeat expansion share a common ancestor has important implications for the interpretation of global frequency data of this mutation. Though the hexanucleotide repeat expansion is commonest among European Caucasians, it is also present in African American, Hispanic, and Middle Eastern populations. This likely reflects the scale and nature of past human migration and intermarriage involving these ethnicities. Similarly, the relative absence of the pathogenic hexanucleotide repeat in the Indian subcontinent, Asia, and the Pacific Islands may be explained by the greater physical distances of these regions from Europe, and the consequent lack of admixture between these populations. Interestingly, the single Japanese patient that we identified as a carrier of the C9ORF72 expansion carried the Finnish risk haplotype, reinforcing the notion that the expansion occurred on a single occasion in the past.

The sharing of a common risk haplotype across the C9ORF72 gene in both sporadic and familial ALS cases indicates that these apparently sporadic cases are in fact cryptically related familial cases. This scenario can occur for many reasons: first, lack of knowledge of the pedigree on the part of the patient or neurologist; second, previous generations may have died at a young age prior to the onset of neurological symptoms. The median age at onset among patients with the expansion was 57 years of age, and life expectancy in the United States only exceeded this point in the early 1940’s²⁶; and finally, decreased penetrance of the mutation, where not all individuals carrying the expansion manifest a clinical phenotype, may be a contributing factor in apparently sporadic disease. Indeed, we have observed symptom onset in the ninth decade of life among cases carrying the expansion, and we have encountered a small number of elderly, neurologically normal individuals carrying the expansion. Thus, the penetrance of this mutation appears to be complete only at a late stage of life, an observation that is particularly relevant to genetic counseling of healthy individuals carrying the expansion. The molecular biological substrate underlying this variability in age at onset is currently unclear: it may be driven by differences in expansion lengths between patients, by age-related methylation across the locus, or by genetic factors elsewhere in the genome.

We compared our results with those of previous studies that reported the frequency of the C9ORF72 hexanucleotide repeat expansion in the pathogenesis of ALS and FTD (panel). Data were available from seven studies (webappendix p 10). Our study represents one of the largest cohort of ALS and FTD cases screened to date, and also provides an initial report of the frequency of the pathogenic repeat expansion in non-Caucasian patients, a detailed examination of the haplotype across the locus, and aninitial estimate of age-related disease penetrance in a large group of individuals carrying the expansion.

Our data have important implications for the clinical care of patients diagnosed with ALS and FTD. The current clinical standard is to offer genetic testing to patients reporting a family history of ALS and/or FTD²⁷, and to reassure patients classified as having sporadic disease that their relatives are not at increased risk of neurodegeneration. A paper recently published in this journal suggested, based on analysis of 191 Irish ALS patients, that genetic testing for the C9ORF72 repeat expansion is unnecessary in affected individuals without a family history of disease or significant cognitive impairment.²⁸ In direct contradiction to this, it is the opinion of this article’s authors that genetic testing is a valuable tool in the accurate diagnosis of both conditions and in the decision making process for patients and their families. The discrepancy between the Byrne et al and our study may stem from differences in how sporadic and familial disease were defined in the two studies. Accumulating data, such as for the 5,873 ALS and FTD cases from diverse populations reported here, is an important step towards answering this key question in patient management. Given the large number of patients that carry the repeat expansion, the field should, at the very least, consider a focused debate on this issue.

Our paper has some limitations. First, the number of patients from certain geographical regions was small, and the mutational frequencies may change for those ethnicities as additional cases are screened. Nevertheless, our data on more than 5,000 cases provides a reasonable estimation of C9ORF72 global frequency. Second, although we have examined the chromosome 9p21 haplotype in a large and diverse cohort of individuals carrying the pathogenic expansion, additional testing of carriers may reveal other haplotypes, thereby indicating that the expansion arose on more than one occasion. Even in this instance, our data indicates that the vast majority of expansion carriers share a common ancestor.^16,20 Third, age-related penetrance estimates were generated based on data from retrospective cohorts, which potentially leads to over-estimation of penetrance. Additional prospective studies are necessary to confirm these estimates. Finally, case classification as familial or sporadic was based on clinical questioning at sample collection. The level of scrutiny may have varied across centers and countries, but it was not feasible to re-collect this information for these existing cohorts.

In conclusion, the hexanucleotide C9ORF72 repeat expansion causes 5·0–7·0% of apparently sporadic ALS and FTD cases in Caucasians, making it by far the most common cause of the sporadic form of these fatal neurodegenerative diseases identified to date. In addition, our data indicate that the pathogenic expansion on chromosome 9p21 may have occurred on a single occasion, possibly in Scandinavia, and that the modern-day global distribution of the mutation perhaps reflects the genetic legacy of their descendants. Furthermore, the finding of the C9ORF72 pathogenic hexanucleotide repeat expansion in nearly one in fifteen apparently sporadic patients raises the question as to whether genetic testing should be offered to patients regardless of family history of disease.

Supplementary Material