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.

Mutation screening and phenotypic profiling of 2 amyotrophic lateral sclerosis-(ALS) and frontotemporal dementia-(FTD) associated genes, CHCHD10 and TUBA4A, were performed in a Belgian cohort of 459 FTD, 28 FTD-ALS, and 429 ALS patients. In CHCHD10, we identified a novel nonsense mutation (p.Gln108*) in a patient with atypical clinical FTD and pathology-confirmed Parkinson's disease (1/459, 0.22%) leading to loss of transcript. We further observed 3 previously described missense variants (p.Pro34Ser, p.Pro80Leu, and p.Pro96Thr) that were also present in the matched control series. In TUBA4A, we detected a novel frameshift mutation (p.Arg64Glyfs*90) leading to a truncated protein in 1 FTD patient (1/459 of 0.22%) with family history of Parkinson's disease and cognitive impairment, and a novel missense mutation (p.Thr381Met) in 2 sibs with familial ALS and memory problems (1 index patient/429, 0.23%) in whom we previously identified a pathogenic Chromosome 9 open reading frame 72 repeat expansion mutation. The present study confirms the role of CHCHD10 and TUBA4A in the FTD-ALS spectrum, although genetic variations in these 2 genes are extremely rare in the Belgian population and often associated with symptomatology of related neurodegenerative diseases including Parkinson's disease and Alzheimer's disease.

Knowledge of the molecular etiology of neurodegenerative brain diseases (NBD) has substantially increased over the past three decades. Early genetic studies of NBD families identified rare and highly penetrant deleterious mutations in causal genes that segregate with disease. Large genome-wide association studies uncovered common genetic variants that influenced disease risk. Major developments in next-generation sequencing (NGS) technologies accelerated gene discoveries at an unprecedented rate and revealed novel pathways underlying NBD pathogenesis. NGS technology exposed large numbers of rare genetic variants of uncertain significance (VUS) in coding regions, highlighting the genetic complexity of NBD. Since experimental studies of these coding rare VUS are largely lacking, the potential contributions of VUS to NBD etiology remain unknown. In this review, we summarize novel findings in NBD genetic etiology driven by NGS and the impact of rare VUS on NBD etiology. We consider different mechanisms by which rare VUS can act and influence NBD pathophysiology and discuss why a better understanding of rare VUS is instrumental for deriving novel insights into the molecular complexity and heterogeneity of NBD. New knowledge might open avenues for effective personalized therapies.

In a prospective study of dementia in Flanders (Belgium), we observed a substantial fraction of early-onset dementia patients who did not fulfill the criteria for a specific dementia subtype, leaving the patients without a precise clinical diagnosis. We selected 211 of these patients for genetic testing of causal genes linked to neurodegenerative brain diseases. In this group, the onset or inclusion age was 59.9 ± 8.2 years and 27.4% had a positive family history. We used a panel of 16 major genes linked to Alzheimer's disease, frontotemporal dementia, amyotrophic lateral sclerosis, Parkinson's disease, and prion diseases. In addition, we tested for the presence of a pathogenic C9orf72 repeat expansion. We identified 13 rare variants in 15 patients, including a carrier of variants in 2 different genes. Six patients (2.84%), carried a mutation in a Mendelian causal gene, that is, APP, MAPT, SOD1, TBK1, and C9orf72. In the other 7 patients, 7 variants were of uncertain significance, including a frameshift mutation in PSEN2, p.G359Lfs*74, in 2 patients sharing a common haplotype, and in LRRK2, p.L2063fs*. Expression studies showed reduced PSEN2 and a near complete loss of LRRK2, in lymphoblast cells or brain material of these patients. Overall, our study underscores the relevance of genetic testing of known causal genes in early-onset patients with symptomatology of neurodegenerative dementia but an unclear clinical diagnosis. A positive genetic result can help to obtain a precise diagnosis as well as a better understanding of the presence of multiple affected relatives in the family.

Objective: To investigate the molecular basis of a Belgian family with autosomal recessive adult-onset neuronal ceroid lipofuscinosis (ANCL or Kufs disease [KD]) with pronounced frontal lobe involvement and to expand the findings to a cohort of unrelated Belgian patients with frontotemporal dementia (FTD). Methods: Genetic screening in the ANCL family and FTD cohort (n = 461) was performed using exome sequencing and targeted massive parallel resequencing. Results: We identified a homozygous mutation (p.Ile404Thr) in the Cathepsin F (CTSF) gene cosegregating in the ANCL family. No other mutations were found that could explain the disease in this family. All 4 affected sibs developed motor symptoms and early-onset dementia with prominent frontal features. Two of them evolved to akinetic mutism. Disease presentation showed marked phenotypic variation with the onset ranging from 26 to 50 years. Myoclonic epilepsy in one of the sibs was suggestive for KD type A, while epilepsy was not present in the other sibs who presented with clinical features of KD type B. In a Belgian cohort of unrelated patients with FTD, the same heterozygous p.Arg245His mutation was identified in 2 patients who shared a common haplotype. Conclusions: A homozygous CTSF mutation was identified in a recessive ANCL pedigree. In contrast to the previous associations of CTSF with KD type B, our findings suggest that CTSF genetic testing should also be considered in patients with KD type A as well as in early-onset dementia with prominent frontal lobe and motor symptoms.

Background: Alzheimer’s disease (AD) mutations in amyloid precursor protein (APP) and presenilins (PSENs) could potentially lead to the production of longer amyloidogenic Aβ peptides. Amongst these, Aβ_1–43 is more prone to aggregation and has higher toxic properties than the long-known Aβ_1–42. However, a direct effect on Aβ_1–43 in biomaterials of individuals carrying genetic mutations in the known AD genes is yet to be determined. Methods: N = 1431 AD patients (n = 280 early-onset (EO) and n = 1151 late-onset (LO) AD) and 809 control individuals were genetically screened for APP and PSENs. For the first time, Aβ_1–43 levels were analysed in cerebrospinal fluid (CSF) of 38 individuals carrying pathogenic or unclear rare mutations or the common PSEN1 p.E318G variant and compared with Aβ_1–42 and Aβ_1–40 CSF levels. The soluble sAPPα and sAPPβ species were also measured for the first time in mutation carriers. Results: A known pathogenic mutation was identified in 5.7% of EOAD patients (4.6% PSEN1, 1.07% APP) and in 0.3% of LOAD patients. Furthermore, 12 known variants with unclear pathogenicity and 11 novel were identified. Pathogenic and unclear mutation carriers showed a significant reduction in CSF Aβ_1–43 levels compared to controls (p = 0.037; < 0.001). CSF Aβ_1–43 levels positively correlated with CSF Aβ_1–42 in both pathogenic and unclear carriers and controls (all p < 0.001). The p.E318G carriers showed reduced Aβ_1–43 levels (p < 0.001), though genetic association with AD was not detected. sAPPα and sAPPβ CSF levels were significantly reduced in the group of unclear (p = 0.006; 0.005) and p.E318G carriers (p = 0.004; 0.039), suggesting their possible involvement in AD. Finally, using Aβ_1–43 and Aβ_1–42 levels, we could re-classify as “likely pathogenic” 3 of the unclear mutations. Conclusion: This is the first time that Aβ_1–43 levels were analysed in CSF of AD patients with genetic mutations in the AD causal genes. The observed reduction of Aβ_1–43 in APP and PSENs carriers highlights the pathogenic role of longer Aβ peptides in AD pathogenesis. Alterations in Aβ_1–43 could prove useful in understanding the pathogenicity of unclear APP and PSENs variants, a critical step towards a more efficient genetic counselling.