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

Abstract

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.

Figure 1

Genotyping assays to characterize the C9orf72 region and G₄C₂ repeat. The C9orf72 G₄C₂ repeat (yellow box) is located upstream of the first exon of isoform NM_018325.3 (dark blue arrow) and adjacent to a GC-rich low-complexity sequence (LCS; light grey box) with their nucleotide sequences shown above. The sequence of the recurrent 10-bp deletion g.26747_26756delGTGGTCGGGG (Table 4, Supp. Figure S1), we observed in the LCS, is indicated in blue. Below the sequence, the primers with their corresponding PCR amplicons are shown for each of the PCR genotyping assays: STR-PCR in pink, forward RP-PCR in green, reverse RP-PCR in red and RP-PCR for sequencing in blue.

Figure 2

A: Distribution of normal repeat lengths in the Flanders-Belgian patients and control individuals. Histograms of G₄C₂ repeat units sized <60 repeats in Flanders-Belgian patients, excluding patients with mutations in known causal genes or with a pathological G₄C₂ expansion, compared with control individuals. B: Correlation of normal repeat lengths with rs2814707 alleles. Histograms of G₄C₂ repeat units in 610 control individuals homozygous for the rs2814707 C-allele and 53 homozygous for the rs2814707 T-allele.

Figure 3

Transcriptional activity of C9orf72 promoter with alleles of different repeat length. Bars represent relative Gaussia/Cypridina luciferase activities (RLA) for the different C9orf72 constructs compared with the wild-type allele of 2 units, for an increasing amount of repeat units. Values represent the mean (±SDEV) of 36 independent measurements relative to the 2 units wild-type allele. The significance of differences in expression was calculated using the Mann–Whitney U test. P values are presented above the bars.