We assessed the geographical distribution of C9orf72 G4C2 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 G4C2 repeat in C9orf72 expansion carriers (P < 0.001) with the most common indel creating one long contiguous imperfect G4C2 repeat, which is likely more prone to replication slippage and pathological expansion.

The established hypothesis for the increase on erythrocyte aggregation associated with a higher incidence of cardiovascular pathologies is based on an increase on plasma adhesion proteins concentration, particularly fibrinogen. Fibrinogen-induced erythrocyte aggregation has been considered to be caused by its nonspecific binding to erythrocyte membranes. In contrast, platelets are known to have a fibrinogen integrin receptor expressed on the membrane surface (the membrane glycoprotein complex alpha(IIb)beta(3)). We demonstrate, by force spectroscopy measurements using an atomic force microscope (AFM), the existence of a single molecule interaction between fibrinogen and an unknown receptor on the erythrocyte membrane, with a lower but comparable affinity relative to platelet binding (average fibrinogen--erythrocyte and --platelet average (un)binding forces were 79 and 97 pN, respectively). This receptor is not as strongly influenced by calcium and eptifibatide (an alpha(IIb)beta(3) specific inhibitor) as the platelet receptor. However, its inhibition by eptifibatide indicates that it is an alpha(IIb)beta(3)-related integrin. Results obtained for a Glanzmann thrombastenia (a rare hereditary bleeding disease caused by alpha(IIb)beta(3) deficiency) patient show (for the first time) an impaired fibrinogen--erythrocyte binding. Correlation with genetic sequencing data demonstrates that one of the units of the fibrinogen receptor on erythrocytes is a product of the expression of the beta(3) gene, found to be mutated in this patient. This work demonstrates and validates the applicability of AFM-based force spectroscopy as a highly sensitive, rapid and low operation cost nanotool for the diagnostic of genetic mutations resulting in hematological diseases, with an unbiased functional evaluation of their severity.

Mutations in the gene coding for Sequestosome 1 (SQSTM1) have been genetically associated with amyotrophic lateral sclerosis (ALS) and Paget disease of bone. In the present study, we analyzed the SQSTM1 coding sequence for mutations in an extended cohort of 1,808 patients with frontotemporal lobar degeneration (FTLD), ascertained within the European Early-Onset Dementia consortium. As control dataset, we sequenced 1,625 European control individuals and analyzed whole-exome sequence data of 2,274 German individuals (total n = 3,899). Association of rare SQSTM1 mutations was calculated in a meta-analysis of 4,332 FTLD and 10,240 control alleles. We identified 25 coding variants in FTLD patients of which 10 have not been described. Fifteen mutations were absent in the control individuals (carrier frequency <0.00026) whilst the others were rare in both patients and control individuals. When pooling all variants with a minor allele frequency <0.01, an overall frequency of 3.2 % was calculated in patients. Rare variant association analysis between patients and controls showed no difference over the whole protein, but suggested that rare mutations clustering in the UBA domain of SQSTM1 may influence disease susceptibility by doubling the risk for FTLD (RR = 2.18 [95 % CI 1.24–3.85]; corrected p value = 0.042). Detailed histopathology demonstrated that mutations in SQSTM1 associate with widespread neuronal and glial phospho-TDP-43 pathology. With this study, we provide further evidence for a putative role of rare mutations in SQSTM1 in the genetic etiology of FTLD and showed that, comparable to other FTLD/ALS genes, SQSTM1 mutations are associated with TDP-43 pathology.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-014-1298-7) contains supplementary material, which is available to authorized users.