A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD

Abstract

The chromosome 9p21 amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD) locus contains one of the last major unidentified autosomal-dominant genes underlying these common neurodegenerative diseases. We have previously shown that a founder haplotype, covering the MOBKL2b, IFNK, and C9ORF72 genes, is present in the majority of cases linked to this region. Here we show that there is a large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72 on the affected haplotype. This repeat expansion segregates perfectly with disease in the Finnish population, underlying 46.0% of familial ALS and 21.1% of sporadic ALS in that population. Taken together with the D90A SOD1 mutation, 87% of familial ALS in Finland is now explained by a simple monogenic cause. The repeat expansion is also present in one-third of familial ALS cases of outbred European descent, making it the most common genetic cause of these fatal neurodegenerative diseases identified to date.

Figure 1. Pedigrees of patients carrying the C9ORF72 GGGGCC hexanucleotide repeat expansion

(A–E) Pedigrees of patients with the hexanucleotide repeat expansion. Mutant alleles are shown by mt, whereas wild-type alleles are indicated by wt. Inferred genotypes are in brackets. Blue diamonds represent a diagnosis of ALS, orange diamonds represent FTD, and green diamonds represent ALS-FTD. Probands are indicated by arrows. Sex of the pedigree members is obscured to protect privacy.

Figure 2. GGGGCC hexanucleotide repeat expansion in the first intron and promoter of C9ORF72

(A) Physical map of the chromosome 9p21 ALS/FTD locus showing the p-values for SNPs genotyped in the previous GWAs (Laaksovirta et al., 2010), the location of the GWAs association signal within a 232kb block of linkage disequilibrium, the MOBKL2B, IFNK and C9ORF72 genes within this region, and the position of the GGGGCC hexanucleotide repeat expansion within the two main transcripts of C9ORF72 (RefSeq accession numbers NM_018325.2 and NM_145005.4, see online www.ncbi.nlm.nih.gov/RefSeq/ for further details; GenBank accession numbers GI:209863035 and GI:209863036, see online www.ncbi.nlm.nih.gov/genbank/ for further details); (B) A graphical representation of primer binding for repeat-primed PCR analysis is shown in the upper panel. In the lower panel, capillary-based sequence traces of the repeat-primed PCR are shown. Orange lines indicate the size markers, and the vertical axis represents fluorescence intensity. A typical saw tooth tail pattern that extends beyond the 300 bp marker with a 6 bp periodicity is observed in the case carrying the GGGGCC repeat expansion; (C) Detection of the repeat expansion in the lymphoblastoid cell line from the affected proband of the GWENT#1 kindred (ND06769) by FISH using Alexa Fluor 488 - labeled oligonucleotide probe seen as a green fluorescence signal on one of the homologues of chromosome 9p (i) consistent with a repeat expansion size of more than 1.5kb. DAPI-inverted image (ii & iv). No hybridization signal was detected on metaphase cells or interphase nuclei from the lymphoblastoid cell line of control individual ND 11463 (iii) and 5 other normal control individuals (data not shown). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, red color), x60 objective.

Figure 3. Repeat-primed PCR assay distinguishes samples carrying a pathogenic GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene from wild-type samples

A bimodal distribution is evident with samples carrying the repeat expansion showing 30 or more repeats and control samples having less than 20 repeats. The repeat-primed PCR assay determines whether or not a sample carries a large pathogenic expansion, but does not measure the actual number of repeats in a large pathogenic expansion. (A) Histogram of repeat lengths based on the repeat-primed PCR assay observed in Finnish cases (n = 402); (B) Histogram of repeat lengths based on the repeat-primed PCR assay observed in Finnish controls (n = 478); (C) Histogram of repeat lengths based on the repeat-primed PCR assay in familial ALS cases of general European (non-Finnish) descent (n = 268); (D) Histogram of repeat lengths based on the repeat-primed PCR assay in control samples of European descent (n = 409) and Human Gene Diversity Panel samples (n = 300).

Figure 4. Expression analysis of C9ORF72 RNA

Expression array analysis of C9ORF72 in various human CNS regions obtained from neuropathologically normal individuals (n = 137).

Figure 5. Preliminary analysis of C9ORF72 protein levels in control cell lines and cell lines derived from ALS patients

(A) Immunocytochemistry of C9ORF72 protein in human-derived primary fibroblasts obtained from a healthy individual (Ctrl fibr.) and from ALS patients (ALS-75 and ALS-50). Green signals represent C9ORF72 (Santa Cruz antibody). Scale bars 20 μm; (B) Nuclear staining pattern of C9ORF72 protein in control and ALS fibroblasts. Green signals represent C9ORF72 protein (Santa Cruz) and red signals represent propidium iodide (PI) (nuclear stain). Scale bars 20 μm; (C) Immunocytochemistry of C9ORF72 protein in mouse-derived NSC-34 motor neuron cell line. Green signals represent C9ORF72 protein (Santa Cruz), red signals represent propidium iodide (PI) (nuclear stain), and blue signals represent wheat germ agglutinin (WGA) (membrane stain). Scale bar 20 μm. (D) Nuclear staining pattern of C9ORF72 protein in NSC-34 mouse motor neuron cell line. Green signals represent C9ORF72 protein, and red signals represent propidium iodide (PI) (nuclear stain). Scale bars 20 μm.