Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS

Abstract

Several families have been reported with autosomal-dominant frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), genetically linked to chromosome 9p21. Here, we report an expansion of a noncoding GGGGCC hexanucleotide repeat in the gene C9ORF72 that is strongly associated with disease in a large FTD/ALS kindred, previously reported to be conclusively linked to chromosome 9p. This same repeat expansion was identified in the majority of our families with a combined FTD/ALS phenotype and TDP-43-based pathology. Analysis of extended clinical series found the C9ORF72 repeat expansion to be the most common genetic abnormality in both familial FTD (11.7%) and familial ALS (23.5%). The repeat expansion leads to the loss of one alternatively spliced C9ORF72 transcript and to formation of nuclear RNA foci, suggesting multiple disease mechanisms. Our findings indicate that repeat expansion in C9ORF72 is a major cause of both FTD and ALS.

Figure 1. Neuropathology in familial FTD/ALS linked to chromosome 9p (family VSM-20)

(A, B) FTLD-TDP characterized by TDP-43 immunoreactive neuronal cytoplasmic inclusions and neurites in (A) neocortex and (B) hippocampal dentate granule cell layer. (C) TDP-34 immunoreactive neuronal cytoplasmic inclusions in spinal cord lower motor neurons, typical of ALS. (D) Numerous neuronal cytoplasmic inclusions and neurites in cerebellar granular layer immunoreactive for ubiquitin but not TDP-43. Scale bar: (A) 15 μm, (B) 30 μm, (C) 100 μm, (D) 12 μm.

Figure 2. Expanded GGGGCC hexanucleotide repeat in C9ORF72 causes FTD and ALS linked to chromosome 9p in family VSM-20

(A) Segregation of GGGGCC repeat in C9ORF72 and flanking genetic markers in disguised linkage pedigree of family VSM-20. The arrowhead denotes the proband. For the GGGGCC repeat, numbers indicate hexanucleotide repeat units and the X denotes that the allele could not be detected. Black symbols represent patients affected with frontotemporal dementia (left side filled), amyotrophic lateral sclerosis (right side filled) or both. White symbols represent unaffected individuals or at-risk individuals with unknown phenotype. Haplotypes for individuals 20-1, 20-2 and 20-3 are inferred from genotype data of siblings and offspring. (B) Fluorescent fragment length analyses of a PCR fragment containing the GGGGCC repeat in C9ORF72. PCR products from the unaffected father (20-9), affected mother (2-10) and their offspring (20-16, 20-17 and 20-18) are shown illustrating the lack of transmission from the affected parent to affected offspring. Numbers under the peaks indicate number of GGGGCC hexanucleotide repeats. (C) PCR products of repeat-primed PCR reactions separated on an ABI3730 DNA Analyzer and visualized by GENEMAPPER software. Electropherograms are zoomed to 2000 relative fluorescence units to show stutter amplification. Two expanded repeat carriers (20-8 and 20-15) and one non-carrier (20-5) from family VSM-20 are shown. (D) Southern blotting of four expanded repeat carriers and one non-carrier from family member of VSM-20 using genomic DNA extracted from lymphoblast cell lines. Lane 1 shows DIG-labeled DNA Molecular Weight Marker II (Roche) with fragments of 2027, 2322, 4361, 6557, 9416, 23130 bp, lane 2 shows DIG-labeled DNA Molecular Weight Marker VII (Roche) with fragments of 1882, 1953, 2799, 3639, 4899, 6106, 7427, and 8576 bp. Patients with expanded repeats (lanes 3-6) show an additional allele from 6-12kb, while a normal relative (lane 7) only shows the expected 2.3kb wild-type allele.

Figure 3. Correlation of GGGGCC hexanucleotide repeat length with rs3849942, a surrogate marker for the previously published chromosome 9p ‘risk’ haplotype

Histograms of number of GGGGCC repeats in 505 controls homozygous for the rs3849942 G-allele and 49 controls homozygous for the rs3849942 A-allele.

Figure 4. Effect of expanded hexanucleotide repeat on C9ORF72 expression

(A) Overview of the genomic structure of the C9ORF72 locus (top panel) and the C9ORF72 transcripts produced by alternative pre-mRNA splicing (bottom panels). Boxes represent coding (white) and non-coding (grey) exons and the positions of the start codon (ATG) and stop codon (TAA) are indicated. The GGGGCC repeat is indicated with a red diamond. The position of rs10757668 is indicated with a green star. (B) Sequence traces of C9ORF72 exon 2 spanning rs10757668 in gDNA (top panel) and cDNA (bottom panels) prepared from frontal cortex of an FTLD-TDP patient carrying an expanded GGGGCC repeat. The arrow indicates the presence of the wild-type (G) and mutant (A) alleles of rs10757668 in gDNA. Transcript specific cDNAs were amplified using primers spanning the exon 1b/exon 2 boundary (variant 1) or exon 1a/exon 2 boundary (variant 2 and 3). Sequenced traces derived from cDNA transcripts indicate the loss of variant 1 but not variant 2 or 3 mutant RNA. Similar results were obtained for two unrelated FTLD-TDP mutation carriers. The bottom panel shows a non-expanded repeat carrier heterozygous for rs10757668 to confirm the presence of both alleles of transcript variant 1 validating the method. (C) mRNA expression analysis of C9ORF72 transcript variant 1 using a custom-designed Taqman expression assay. Top panel shows lymphoblast cell lines derived from expanded repeat carriers from family VSM-20 (n=7) and controls (n=7) and bottom panel shows RNA extracted from frontal cortex brain samples from FTLD-TDP patients with (n=7) and without (n=7) the GGGGCC repeat expansion. Data indicate mean ± s.e.m. ** indicates P<0.01. (D) mRNA expression analysis of all C9ORF72 transcripts encoding for C9ORF72 isoform a (variant 1 and 3) using inventoried ABI Taqman expression assay Hs_00945132. Top panel shows RNA extracted from lymphoblast cell lines derived from expanded repeat carriers from family VSM-20 (n=7) and controls (n=7) and bottom panel shows RNA extracted from frontal cortex brain samples from FTLD-TDP patients with (n=7) and without (n=7) the GGGGCC repeat expansion. Data indicate mean ± s.e.m. * indicates P<0.05.

Figure 5. Expanded GGGGCC hexanucleotide repeat forms nuclear RNA foci in human brain and spinal cord

(A) Multiple RNA foci in the nucleus (stained with DAPI, blue) of a frontal cortex neuron of the proband of family 63 (63-1) using a Cy3-labeled (GGCCCC)₄ oligonucleotide probe (red). (B) RNA foci observed in the nucleus of two lower motor neurons in FTD/ALS patient (13-7) carrying an expanded GGGGCC repeat using a Cy3-labeled (GGCCCC)₄ oligonucleotide probe. (C) Absence of RNA foci in the nucleus of cortical neuron from FTLD-TDP patient (44-1) without an expanded GGGGCC repeat in C9ORF72. (D) spinal cord tissue sections from patient 13-7 that showed RNA foci with the (GGCCCC)₄ oligonucleotide probe in (B) do not show any foci with a Cy3-labeled (CAGG)₆ oligonucleotide probe (negative control probe). Scale bar: 10 μm (A and C), 20 μm (B and D).