Clinical Evidence of Disease Anticipation in Families Segregating a C9orf72 Repeat Expansion.

Importance: Patients carrying a C9orf72 repeat expansion leading to frontotemporal dementia and/or amyotrophic lateral sclerosis have highly variable ages at onset of disease, suggesting the presence of modifying factors. Objective: To provide clinical-based evidence for disease anticipation in families carrying a C9orf72 repeat expansion by analyzing age at onset, disease duration, and age at death in successive generations. Design, Setting, and Participants: This cohort study was performed from June 16, 2000, to June 1, 2016, in 36 extended Belgian families in which a C9orf72 repeat expansion was segregating. The generational effect on age at onset, disease duration, and age at death was estimated using a mixed effects Cox proportional hazards regression model, including random-effects terms for within-family correlation and kinship. Time until disease onset or last examination, time from disease onset until death or last examination, or age at death was collected for for 244 individuals (132 proven or obligate C9orf72 carriers), of whom 147 were clinically affected (89 proven or obligate C9orf72 carriers). Main Outcomes and Measures: Generational effect on age at onset, disease duration, and age at death. Results: Among the 111 individuals with age at onset available (66 men and 45 women; mean [SD] age, 57.2 [9.1] years), the mean (SD) age at onset per generation (from earliest-born to latest-born generation) was 62.5 (8.3), 57.1 (8.2), 54.6 (10.2), and 49.3 (7.5) years. Censored regression analysis on all affected and unaffected at-risk relatives confirmed a decrease in age at onset in successive generations (P < .001). No generational effect was observed for disease duration or age at death. Conclusions and Relevance: The clinical data provide supportive evidence for the occurrence of disease anticipation in families carrying a C9orf72 repeat expansion by means of a decrease in age at onset across successive generations. This finding may help clinicians decide from which age onward it may be relevant to clinically follow presymptomatic individuals who carry a C9orf72 repeat expansion.

TBK1 Mutation Spectrum in an Extended European Patient Cohort with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis.

We investigated the mutation spectrum of the TANK-Binding Kinase 1 (TBK1) gene and its associated phenotypic spectrum by exonic resequencing of TBK1 in a cohort of 2,538 patients with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), or FTD plus ALS, ascertained within the European Early-Onset Dementia Consortium. We assessed pathogenicity of predicted protein-truncating mutations by measuring loss of RNA expression. Functional effect of in-frame amino acid deletions and missense mutations was further explored in vivo on protein level and in vitro by an NFκB-induced luciferase reporter assay and measuring phosphorylated TBK1. The protein-truncating mutations led to the loss of transcript through nonsense-mediated mRNA decay. For the in-frame amino acid deletions, we demonstrated loss of TBK1 or phosphorylated TBK1 protein. An important fraction of the missense mutations compromised NFκB activation indicating that at least some functions of TBK1 are lost. Although missense mutations were also present in controls, over three times more mutations affecting TBK1 functioning were found in the mutation fraction observed in patients only, suggesting high-risk alleles (P = 0.03). Total mutation frequency for confirmed TBK1 LoF mutations in the European cohort was 0.7%, with frequencies in the clinical subgroups of 0.4% in FTD, 1.3% in ALS, and 3.6% in FTD-ALS.

Loss of TBK1 is a frequent cause of frontotemporal dementia in a Belgian cohort.

OBJECTIVE: To assess the genetic contribution of TBK1, a gene implicated in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and FTD-ALS, in Belgian FTD and ALS patient cohorts containing a significant part of genetically unresolved patients. METHODS: We sequenced TBK1 in a hospital-based cohort of 482 unrelated patients with FTD and FTD-ALS and 147 patients with ALS and an extended Belgian FTD-ALS family DR158. We followed up mutation carriers by segregation studies, transcript and protein expression analysis, and immunohistochemistry. RESULTS: We identified 11 patients carrying a loss-of-function (LOF) mutation resulting in an overall mutation frequency of 1.7% (11/629), 1.1% in patients with FTD (5/460), 3.4% in patients with ALS (5/147), and 4.5% in patients with FTD-ALS (1/22). We found 1 LOF mutation, p.Glu643del, in 6 unrelated patients segregating with disease in family DR158. Of 2 mutation carriers, brain and spinal cord was characterized by TDP-43-positive pathology. The LOF mutations including the p.Glu643del mutation led to loss of transcript or protein in blood and brain. CONCLUSIONS: TBK1 LOF mutations are the third most frequent cause of clinical FTD in the Belgian clinically based patient cohort, after C9orf72 and GRN, and the second most common cause of clinical ALS after C9orf72. These findings reinforce that FTD and ALS belong to the same disease continuum.

C9orf72 G4C2 repeat expansions in Alzheimer's disease and mild cognitive impairment.

C9orf72 G4C2 repeat expansion is a major cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Its role in Alzheimer's disease (AD) is less clear. We assessed the prevalence of G4C2 pathogenic repeat expansions in Flanders-Belgian patients with clinical AD or mild cognitive impairment (MCI). In addition, we studied the effect of non-pathogenic G4C2 repeat length variability on susceptibility to AD, and on AD cerebrospinal fluid (CSF) biomarker levels. A pathogenic repeat expansion was identified in 5 of 1217 AD patients (frequency <1%). No pathogenic expansions were observed in patients with MCI (n = 200) or control individuals (n = 1119). Nonpathogenic repeat length variability was not associated with AD, risk of conversion to AD in MCI individuals, or CSF biomarker levels. We conclude that pathogenic C9orf72 G4C2 repeat expansions can be detected in clinical AD patients and could act as a contributor to AD pathogenesis. Non-pathogenic repeat length variability did not affect risk of AD or MCI, nor AD biomarker levels in CSF, indicating that C9orf72 is not a direct AD risk factor.

Distinct clinical characteristics of C9orf72 expansion carriers compared with GRN, MAPT, and nonmutation carriers in a Flanders-Belgian FTLD cohort.

OBJECTIVE: To characterize patients with frontotemporal lobar degeneration (FTLD) with a repeat expansion mutation in the gene C9orf72, and to determine whether there are differences in the clinical presentation compared with FTLD carriers of a mutation in GRN or MAPT or with patients with FTLD without mutation. DESIGN: Patient series. SETTING: Dementia clinics in Flanders, Belgium. PATIENTS: Two hundred seventy-five genetically and phenotypically thoroughly characterized patients with FTLD. MAIN OUTCOME MEASURES: Clinical and demographic characteristics of 26 C9orf72 expansion carriers compared with patients with a GRN or MAPT mutation, as well as patients with familial and sporadic FTLD without mutation. RESULTS: C9orf72 expansion carriers developed FTLD at an early age (average, 55.3 years; range, 42-69 years), significantly earlier than in GRN mutation carriers or patients with FTLD without mutation. Mean survival (6.2 years; range, 1.5-17.0 years) was similar to other patient groups. Most developed behavioral variant frontotemporal dementia (85%), with disinhibited behavior as the prominent feature. Concomitant amyotrophic lateral sclerosis is a strong distinguishing feature for C9orf72 -associated FTLD. However, in most patients (73%), amyotrophic lateral sclerosis symptoms were absent. Compared with C9orf72 expansion carriers, nonfluent aphasia and limb apraxia were significantly more common in GRN mutation carriers. CONCLUSIONS: C9orf72 -associated FTLD most often presents with early-onset behavioral variant frontotemporal dementia with disinhibition as the prominent feature, with or without amyotrophic lateral sclerosis. Based on the observed genotype-phenotype correlations between the different FTLD syndromes and different genetic causes, we propose a decision tree to guide clinical genetic testing in patients clinically diagnosed as having FTLD.

A pan-European study of the C9orf72 repeat associated with FTLD: geographic prevalence, genomic instability, and intermediate repeats.

We assessed the geographical distribution of C9orf72 G(4) C(2) expansions in a pan-European frontotemporal lobar degeneration (FTLD) cohort (n = 1,205), ascertained by the European Early-Onset Dementia (EOD) consortium. Next, we performed a meta-analysis of our data and that of other European studies, together 2,668 patients from 15 Western European countries. The frequency of the C9orf72 expansions in Western Europe was 9.98% in overall FTLD, with 18.52% in familial, and 6.26% in sporadic FTLD patients. Outliers were Finland and Sweden with overall frequencies of respectively 29.33% and 20.73%, but also Spain with 25.49%. In contrast, prevalence in Germany was limited to 4.82%. In addition, we studied the role of intermediate repeats (7-24 repeat units), which are strongly correlated with the risk haplotype, on disease and C9orf72 expression. In vitro reporter gene expression studies demonstrated significantly decreased transcriptional activity of C9orf72 with increasing number of normal repeat units, indicating that intermediate repeats might act as predisposing alleles and in favor of the loss-of-function disease mechanism. Further, we observed a significantly increased frequency of short indels in the GC-rich low complexity sequence adjacent to the G(4) C(2) repeat in C9orf72 expansion carriers (P < 0.001) with the most common indel creating one long contiguous imperfect G(4) C(2) repeat, which is likely more prone to replication slippage and pathological expansion.

A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are extremes of a clinically, pathologically, and genetically overlapping disease spectrum. A locus on chromosome 9p21 has been associated with both disorders, and we aimed to identify the causal gene within this region. METHODS: We studied 305 patients with FTLD, 137 with ALS, and 23 with concomitant FTLD and ALS (FTLD-ALS) and 856 controls from Flanders (Belgium); patients were identified from a hospital-based cohort and were negative for mutations in known FTLD and ALS genes. We also examined the family of one patient with FTLD-ALS previously linked to 9p21 (family DR14). We analysed 130 kbp at 9p21 in association and segregation studies, genomic sequencing, repeat genotyping, and expression studies to identify the causal mutation. We compared genotype-phenotype correlations between mutation carriers and non-carriers. FINDINGS: In the patient-control cohort, the single-nucleotide polymorphism rs28140707 within the 130 kbp region of 9p21 was associated with disease (odds ratio [OR] 2·6, 95% CI 1·5-4·7; p=0·001). A GGGGCC repeat expansion in C9orf72 completely co-segregated with disease in family DR14. The association of rs28140707 with disease in the patient-control cohort was abolished when we excluded GGGGCC repeat expansion carriers. In patients with familial disease, six (86%) of seven with FTLD-ALS, seven (47%) of 15 with ALS, and 12 (16%) of 75 with FTLD had the repeat expansion. In patients without known familial disease, one (6%) of 16 with FTLD-ALS, six (5%) of 122 with ALS, and nine (4%) of 230 with FTLD had the repeat expansion. Mutation carriers primarily presented with classic ALS (10 of 11 individuals) or behavioural variant FTLD (14 of 15 individuals). Mean age at onset of FTLD was 55·3 years (SD 8·4) in 21 mutation carriers and 63·2 years (9·6) in 284 non-carriers (p=0·001); mean age at onset of ALS was 54·5 years (9·9) in 13 carriers and 60·4 years (11·4) in 124 non-carriers. Postmortem neuropathological analysis of the brains of three mutation carriers with FTLD showed a notably low TDP-43 load. In brain at postmortem, C9orf72 expression was reduced by nearly 50% in two carriers compared with nine controls (p=0·034). In familial patients, 14% of FTLD-ALS, 50% of ALS, and 62% of FTLD was not accounted for by known disease genes. INTERPRETATION: We identified a pathogenic GGGGCC repeat expansion in C9orf72 on chromosome 9p21, as recently also reported in two other studies. The GGGGCC repeat expansion is highly penetrant, explaining all of the contribution of chromosome 9p21 to FTLD and ALS in the Flanders-Belgian cohort. Decreased expression of C9orf72 in brain suggests haploinsufficiency as an underlying disease mechanism. Unidentified genes probably also contribute to the FTLD-ALS disease spectrum. FUNDING: Full funding sources listed at end of paper (see Acknowledgments).