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.

Clinical features of TBK1 carriers compared with C9orf72, GRN and non-mutation carriers in a Belgian cohort.

We identified in a cohort of patients with frontotemporal dementia (n = 481) or amyotrophic lateral sclerosis (n = 147), 10 index patients carrying a TBK1 loss of function mutation reducing TBK1 expression by 50%. Here, we describe the clinical and pathological characteristics of the 10 index patients and six of their affected relatives carrying a TBK1 mutation. Six TBK1 carriers were diagnosed with frontotemporal dementia, seven with amyotrophic lateral sclerosis, one with both clinical phenotypes and two with dementia unspecified. The mean age at onset of all 16 TBK1 carriers was 62.1 ± 8.9 years (range 41-73) with a mean disease duration of 4.7 ± 4.5 years (range 1-13). TBK1 carriers with amyotrophic lateral sclerosis had shorter disease duration than carriers with frontotemporal dementia. Six of seven TBK1 carriers were diagnosed with the behavioural variant of frontotemporal dementia, presenting predominantly as disinhibition. Memory loss was an important associated symptom in the initial phase of the disease in all but one of the carriers with frontotemporal dementia. Three of the patients with amyotrophic lateral sclerosis exhibited pronounced upper motor neuron symptoms. Overall, neuroimaging displayed widespread atrophy, both symmetric and asymmetric. Brain perfusion single-photon emission computed tomography or fluorodeoxyglucose-positron emission tomography showed asymmetric and predominantly frontotemporal involvement. Neuropathology in two patients demonstrated TDP-43 type B pathology. Further, we compared genotype-phenotype data of TBK1 carriers with frontotemporal dementia (n = 7), with those of frontotemporal dementia patients with a C9orf72 repeat expansion (n = 65) or a GRN mutation (n = 52) and with frontotemporal dementia patients (n = 259) negative for mutations in currently known causal genes. TBK1 carriers with frontotemporal dementia had a later age at onset (63.3 years) than C9orf72 carriers (54.3 years) (P = 0.019). In clear contrast with TBK1 carriers, GRN carriers were more often diagnosed with the language variant than the behavioural variant, and presented in case of the diagnosis of behavioural variant, more often than TBK1 carriers with apathy as the predominant characteristic (P = 0.004). Also, TBK1 carriers exhibited more often extrapyramidal symptoms than C9orf72 carriers (P = 0.038). In conclusion, our study identified clinical differences between the TBK1, C9orf72 and GRN carriers, which allows us to formulate guidelines for genetic diagnosis. After a negative result for C9orf72, patients with both frontotemporal dementia and amyotrophic lateral sclerosis should be tested first for mutations in TBK1. Specifically in frontotemporal dementia patients with early memory difficulties, a relatively late age at onset or extrapyramidal symptoms, screening for TBK1 mutations should be considered.

TMEM106B is a genetic modifier of frontotemporal lobar degeneration with C9orf72 hexanucleotide repeat expansions.

Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72) have recently been linked to frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis, and may be the most common genetic cause of both neurodegenerative diseases. Genetic variants at TMEM106B influence risk for the most common neuropathological subtype of FTLD, characterized by inclusions of TAR DNA-binding protein of 43 kDa (FTLD-TDP). Previous reports have shown that TMEM106B is a genetic modifier of FTLD-TDP caused by progranulin (GRN) mutations, with the major (risk) allele of rs1990622 associating with earlier age at onset of disease. Here, we report that rs1990622 genotype affects age at death in a single-site discovery cohort of FTLD patients with C9orf72 expansions (n = 14), with the major allele correlated with later age at death (p = 0.024). We replicate this modifier effect in a 30-site international neuropathological cohort of FTLD-TDP patients with C9orf72 expansions (n = 75), again finding that the major allele associates with later age at death (p = 0.016), as well as later age at onset (p = 0.019). In contrast, TMEM106B genotype does not affect age at onset or death in 241 FTLD-TDP cases negative for GRN mutations or C9orf72 expansions. Thus, TMEM106B is a genetic modifier of FTLD with C9orf72 expansions. Intriguingly, the genotype that confers increased risk for developing FTLD-TDP (major, or T, allele of rs1990622) is associated with later age at onset and death in C9orf72 expansion carriers, providing an example of sign epistasis in human neurodegenerative disease.

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.

Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins.

Massive GGGGCC repeat expansion in the first intron of the gene C9orf72 is the most common known cause of familial frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Despite its intronic localization and lack of an ATG start codon, the repeat region is translated in all three reading frames into aggregating dipeptide-repeat (DPR) proteins, poly-(Gly-Ala), poly-(Gly-Pro) and poly-(Gly-Arg). We took an antibody-based approach to further validate the translation of DPR proteins. To test whether the antisense repeat RNA transcript is also translated, we raised antibodies against the predicted products, poly-(Ala-Pro) and poly-(Pro-Arg). Both antibodies stained p62-positive neuronal cytoplasmic inclusions throughout the cerebellum and hippocampus indicating that not only sense but also antisense strand repeats are translated into DPR proteins in the absence of ATG start codons. Protein products of both strands co-aggregate suggesting concurrent translation of both strands. Moreover, an antibody targeting the putative carboxyl terminus of DPR proteins can detect inclusion pathology in C9orf72 repeat expansion carriers suggesting that the non-ATG translation continues through the entire repeat and beyond. A highly sensitive monoclonal antibody against poly-(Gly-Arg), visualized abundant inclusion pathology in all cortical regions and some inclusions also in motoneurons. Together, our data show that the GGGGCC repeat is bidirectionally translated into five distinct DPR proteins that co-aggregate in the characteristic p62-positive TDP-43 negative inclusions found in FTLD/ALS cases with C9orf72 repeat expansion. Novel monoclonal antibodies against poly-(Gly-Arg) will facilitate pathological diagnosis of C9orf72 FTLD/ALS.

Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase.

Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR-DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescue GRN haploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.

Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21.

Frontotemporal dementia (FTD) with ubiquitin-immunoreactive neuronal inclusions (both cytoplasmic and nuclear) of unknown nature has been linked to a chromosome 17q21 region (FTDU-17) containing MAPT (microtubule-associated protein tau). FTDU-17 patients have consistently been shown to lack a tau-immunoreactive pathology, a feature characteristic of FTD with parkinsonism linked to mutations in MAPT (FTDP-17). Furthermore, in FTDU-17 patients, mutations in MAPT and genomic rearrangements in the MAPT region have been excluded by both genomic sequencing and fluorescence in situ hybridization on mechanically stretched chromosomes. Here we demonstrate that FTDU-17 is caused by mutations in the gene coding for progranulin (PGRN), a growth factor involved in multiple physiological and pathological processes including tumorigenesis. Besides the production of truncated PGRN proteins due to premature stop codons, we identified a mutation within the splice donor site of intron 0 (IVS0 + 5G > C), indicating loss of the mutant transcript by nuclear degradation. The finding was made within an extensively documented Belgian FTDU-17 founder family. Transcript and protein analyses confirmed the absence of the mutant allele and a reduction in the expression of PGRN. We also identified a mutation (c.3G > A) in the Met1 translation initiation codon, indicating loss of PGRN due to lack of translation of the mutant allele. Our data provide evidence that PGRN haploinsufficiency leads to neurodegeneration because of reduced PGRN-mediated neuronal survival. Furthermore, in a Belgian series of familial FTD patients, PGRN mutations were 3.5 times more frequent than mutations in MAPT, underscoring a principal involvement of PGRN in FTD pathogenesis.

Serum biomarker for progranulin-associated frontotemporal lobar degeneration.

OBJECTIVE: Mutations that lead to a loss of progranulin (PGRN) explain a considerable portion of the occurrence of frontotemporal lobar degeneration. We tested a biomarker allowing rapid detection of a loss of PGRN. METHODS: We used an enzyme-linked immunosorbent assay to measure in serum the PGRN protein levels of six affected and eight unaffected carriers from within an extended Belgian founder family segregating the null mutation IVS1+5G>C. Further, we measured serum PGRN levels in 2 patients with another null mutation (a Met1 and a frameshift mutation), in 4 patients carrying a predicted pathogenic missense mutation and in 5 patients carrying a benign missense polymorphism, in 9 unaffected noncarrier relatives, and in 22 community controls. RESULTS: Serum PGRN levels were reduced in both affected and unaffected null mutation carriers compared with noncarrier relatives (p(exact) < 0.0001), and allowed perfect discrimination between carriers and noncarriers (sensitivity: 1.0; 1 - specificity: 0.0). Serum PGRN levels in Cys139Arg and Arg564Cys mutation carriers were significantly lower than in controls, but greater than in null mutation carriers, fitting the hypothesis of partial loss of function caused by these missense mutations. As expected, levels for carriers of benign missense polymorphisms were not significantly different from controls. INTERPRETATION: Our results indicate that the serum PGRN level is a reliable biomarker for diagnosing and early detection of frontotemporal lobar degeneration caused by PGRN null mutations, and provided the first in vivo evidence that at least some missense mutations in PGRN may lead to a (partial) loss of PGRN.

The Genetics of C9orf72 Expansions.

Repeat expansions in the promoter region of C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and related disorders of the ALS/frontotemporal lobar degeneration (FTLD) spectrum. Remarkable clinical heterogeneity among patients with a repeat expansion has been observed, and genetic anticipation over different generations has been suggested. Genetic factors modifying the clinical phenotype have been proposed, including genetic variation in other known disease genes, the genomic context of the C9orf72 repeat, and expanded repeat size, which has been estimated between 45 and several thousand units. The role of variability in normal and expanded repeat sizes for disease risk and clinical phenotype is under debate. Different pathogenic mechanisms have been proposed, including loss of function, RNA toxicity, and dipeptide repeat (DPR) protein toxicity resulting from abnormal translation of the expanded repeat, but the major mechanism is yet unclear.

Common and rare TBK1 variants in early-onset Alzheimer disease in a European cohort.

TANK-binding kinase 1 (TBK1) loss-of-function (LoF) mutations are known to cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), often combined with memory deficits early in the disease course. We performed targeted resequencing of TBK1 in 1253 early onset Alzheimer's disease (EOAD) patients from 8 European countries to investigate whether pathogenic TBK1 mutations are enriched among patients with clinical diagnosis of EOAD. Variant frequencies were compared against 2117 origin-matched controls. We identified only 1 LoF mutation (p.Thr79del) in a patient clinically diagnosed with Alzheimer's disease and a positive family history of ALS. We did not observe enrichment of rare variants in EOAD patients compared to controls, nor of rare variants affecting NFκB induction. Of 3 common coding variants, rs7486100 showed evidence of association (OR 1.46 [95% CI 1.13-1.9]; p-value 0.01). Homozygous carriers of the risk allele showed reduced expression of TBK1 (p-value 0.03). Our findings are not indicative of a significant role for TBK1 mutations in EOAD. The association between common variants in TBK1, disease risk and reduced TBK1 expression warrants follow-up in FTD/ALS cohorts.

Mutations other than null mutations producing a pathogenic loss of progranulin in frontotemporal dementia.

Null mutations in the progranulin gene (GRN, PGRN) were recently identified as the causal mechanism underlying frontotemporal dementia (FTD) with ubiquitin-positive brain pathology linked to chromosome 17 (FTDU-17). In a Belgian and French FTD series comprising 332 patients, we reported 13 PGRN null mutations which were mainly nonsense and frameshift mutations resulting in premature stop codons. Here we report in the same patient series three missense mutations of which two (c.743C>T, p.Pro248Leu and c.1294C>T, p.Arg432Cys) were predicted in silico to severely affect protein folding and/or processing leading to PGRN protein haploinsufficiency. In addition, we observed three sequence variations in the 5' regulatory region that might potentially affect PGRN transcription activity. Our findings extend the mutation spectrum in PGRN leading to loss of functional PGRN as the basis for FTD.

Progranulin null mutations in both sporadic and familial frontotemporal dementia.

Frontotemporal dementia (FTD) is the second most frequent type of neurodegenerative dementias. Mutations in the progranulin gene (GRN, PGRN) were recently identified in FTDU-17, an FTD subtype characterized by ubiquitin-immunoreactive inclusions and linkage to chromosome 17q21. We looked for PGRN mutations in a large series of 210 FTD patients (52 familial, 158 sporadic) to accurately evaluate the frequency of PGRN mutations in both sporadic and familial FTD, and FTD with associated motoneuron disease (FTD-MND), as well as to study the clinical phenotype of patients with a PGRN mutation. We identified nine novel PGRN null mutations in 10 index patients. The relative frequency of PGRN null mutations in FTD was 4.8% (10/210) and 12.8% (5/39) in pure familial forms. Interestingly, 5/158 (3.2%) apparently sporadic FTD patients carried a PGRN mutation, suggesting the possibility of de novo mutations or incomplete penetrance. In contrast, none of the 43 patients with FTD-MND had PGRN mutations, supporting that FTDU-17 and FTD-MND are genetically distinct. The clinical phenotype of PGRN mutation carriers was particular because of the wide range in onset age and the frequent occurrence of early apraxia (50%), visual hallucinations (30%), and parkinsonism (30%) during the course of the disease. This study supports that PGRN null mutations represent a more frequent cause of FTD than MAPT mutations (4.8% vs. 2.9%) but are not responsible for FTD-MND. It also demonstrates that half of the patients with a PGRN mutation in our series had no apparent family history of dementia. Taking this into account, genetic testing should be now considered more systematically, even in patients without obvious familial history of FTD.

Alzheimer and Parkinson diagnoses in progranulin null mutation carriers in an extended founder family.

BACKGROUND: Progranulin gene (PGRN) haploinsufficiency was recently associated with ubiquitin-positive frontotemporal lobar degeneration linked to chromosome 17q21 (FTLDU-17). OBJECTIVE: To assess whether PGRN genetic variability contributed to other common neurodegenerative brain diseases, such as Alzheimer disease (AD) or Parkinson disease (PD). DESIGN: Mutation analysis of PGRN. SETTING: Memory Clinic of the Middelheim General Hospital. Patients We analyzed 666 Belgian patients with AD and 255 with PD. MAIN OUTCOME MEASURES: Results of PGRN sequencing, PGRN transcript analysis, short tandem repeat genotyping, and neuropathologic analysis. RESULTS: We identified 2 patients with AD and 1 patient with PD who carried the null mutation IVS0 + 5G>C, which we reported earlier in an extensively characterized Belgian founder family, DR8, segregating FTLDU. Postmortem pathologic diagnosis of the patient with PD revealed both FTLDU and Lewy body pathologic features. In addition, we identified in PGRN only 1 other null mutation, the nonsense mutation p.Arg535X, in 1 patient with probable AD. However, in vitro analysis predicted a PGRN C-truncated protein, although it remains to be elucidated if this shortened transcript leads to haploinsufficiency. CONCLUSIONS: Our mutation data indicated that null mutations are rare in patients with AD (3/666 = 0.45%) and PD (1/255 = 0.39%). Also, AD and PD clinical diagnoses in patients who carry PGRN null mutations likely result from etiologic heterogeneity rather than PGRN haploinsufficiency.

APP duplication is sufficient to cause early onset Alzheimer's dementia with cerebral amyloid angiopathy.

We assessed the impact of amyloid precursor protein (APP) gene locus duplications in early onset Alzheimer's disease in a Dutch population-based sample. Using real-time PCR and an in-house-developed multiplex amplicon quantification assay, we identified a genomic APP duplication in 1 out of 10 multigenerational families segregating early onset Alzheimer's disease. In this family, cerebral amyloid angiopathy (CAA) coincided with this disease. The duplicated genomic region included no other genes than APP and extended maximally over 0.7 Mb. In a sample of 65 familial early onset patients, we observed the same APP genomic duplication in one patient (1.7%), while in 36 isolated patients duplications in the APP locus were absent. This indicated that APP locus duplications explained <2% of familial, non-autosomal dominant Alzheimer's disease and are an infrequent cause of de novo mutation. Our findings corroborated a recent French study, and indicated that investigating genomic duplications in the APP locus in families segregating Alzheimer's disease and CAA should be considered.

A Belgian ancestral haplotype harbours a highly prevalent mutation for 17q21-linked tau-negative FTLD.

Among patients with frontotemporal lobar degeneration (FTLD), the respective frequencies of dominant 17q21-linked tau-negative FTLD (with unidentified molecular defect) and 17q21-linked tau-positive FTLD (due to MAPT mutations) remain unknown. Here, in a series of 98 genealogically unrelated Belgian FTLD patients, we identified an ancestral 8 cM MAPT containing haplotype in two patients belonging to multiplex families DR2 and DR8, without demonstrable MAPT mutations, in which FTLD was conclusively linked to 17q21 [maximum summed log of the odds (LOD) score of 5.28 at D17S931]. Interestingly, the same DR2-DR8 ancestral haplotype was observed in five additional familial FTLD patients, indicative of a founder effect. In the FTLD series, the DR2-DR8 ancestral haplotype explained 7% (7 out of 98) of FTLD and 17% (7 out of 42) of familial FTLD and was seven times more frequent than MAPT mutations (1 out of 98 or 1%). Clinically, DR2-DR8 haplotype carriers presented with FTLD often characterized by language impairment, and in one carrier the neuropathological diagnosis was FTLD with rare tau-negative ubiquitin-positive inclusions. Together, these results strongly suggest that the DR2-DR8 founder haplotype at 17q21 harbours a tau-negative FTLD causing mutation that is a much more frequent cause of FTLD in Belgium than MAPT mutations.

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.

Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD.

Hexanucleotide repeat expansions in C9orf72 are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) (c9ALS/FTD). Unconventional translation of these repeats produces dipeptide repeat proteins (DPRs) that may cause neurodegeneration. We performed a modifier screen in Drosophila and discovered a critical role for importins and exportins, Ran-GTP cycle regulators, nuclear pore components, and arginine methylases in mediating DPR toxicity. These findings provide evidence for an important role for nucleocytoplasmic transport in the pathogenic mechanism of c9ALS/FTD.

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.

Investigating the role of rare heterozygous TREM2 variants in Alzheimer's disease and frontotemporal dementia.

Homozygous mutations in exon 2 of TREM2, a gene involved in Nasu-Hakola disease, can cause frontotemporal dementia (FTD). Moreover, a rare TREM2 exon 2 variant (p.R47H) was reported to increase the risk of Alzheimer's disease (AD) with an odds ratio as strong as that for APOEε4. We systematically screened the TREM2 coding region within a Belgian study on neurodegenerative brain diseases (1216 AD patients, 357 FTD patients, and 1094 controls). We observed an enrichment of rare variants across TREM2 in both AD and FTD patients compared to controls, most notably in the extracellular IgV-set domain (relative risk = 3.84 [95% confidence interval = 1.29-11.44]; p = 0.009 for AD; relative risk = 6.19 [95% confidence interval = 1.86-20.61]; p = 0.0007 for FTD). None of the rare variants individually reached significant association, but the frequency of p.R47H was increased ~ 3-fold in both AD and FTD patients compared to controls, in line with previous reports. Meta-analysis including 11 previously screened AD cohorts confirmed the association of p.R47H with AD (p = 2.93×10(-17)). Our data corroborate and extend previous findings to include an increased frequency of rare heterozygous TREM2 variations in AD and FTD, and show that TREM2 variants may play a role in neurodegenerative diseases in general.