Screening a UK amyotrophic lateral sclerosis cohort provides evidence of multiple origins of the C9orf72 expansion

Abstract

An expanded hexanucleotide repeat in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Although 0-30 hexanucleotide repeats are present in the general population, expansions >500 repeats are associated with C9ALS/FTD. Large C9ALS/FTD expansions share a common haplotype and whether these expansions derive from a single founder or occur more frequently on a predisposing haplotype is yet to be determined and is relevant to disease pathomechanisms. Furthermore, although cases carrying 50-200 repeats have been described, their role and the pathogenic threshold of the expansions remain to be identified and carry importance for diagnostics and genetic counseling. We present clinical and genetic data from a UK ALS cohort and report the detailed molecular study of an atypical somatically unstable expansion of 90 repeats. Our results across different tissues provide evidence for the pathogenicity of this repeat number by showing they can somatically expand in the central nervous system to the well characterized pathogenic range. Our results support the occurrence of multiple expansion events for C9ALS/FTD.

Keywords: Amyotrophic lateral sclerosis; Frontotemporal dementia; Somatic instability.

Fig. 1

Case 61776 shows a somatically unstable repeat. (A) Map of the C9orf72 locus illustrating the probe and the restriction sites used for single-copy 1-kb probe Southern hybridization. The site of the hexanucleotide expansion is indicated with a circle. (B) Southern hybridization performed on a typical bimodal C9orf72 repeat expansion (Pos) and case 61776 with the following restriction digestions: EcoRI/BamHI (E/B), Bsu36I (Bs), and EcoRI (E). The somatic mosaicism in 61776 is evident in the E/B digest lane (marked with *).

Fig. 2

Immunohistochemical analysis of case 61776. Small p62-positive “star-like” inclusions were present in the granule cells of the dentate fascia (A) and the cerebellar cortex (B). Larger p62-positive inclusions were seen in the CA4 subregion of the hippocampus and also contained intranuclear inclusions (C). TDP-43-positive oligodendroglial inclusions were seen in the striatum (D) and substania nigra (F). TDP-43-positive neuronal cytoplasmic inclusions were present in the 12th nerve nucleus (E) and spinal cord (G). The “star-like” p62-positive inclusions were also positive with anti-glycine proline (H) and anti-glycine arginine dipeptide antibodies (I). Bar represents 50 μm in A and B; 20 μm in E, G, H, and I; and 10 μm in C, D, and F.

Fig. 3

Fluorescence in situ hybridization of case 61776. RNA foci containing sense (A and B) and antisense (C and D) C9orf72 expansion transcripts are present in the frontal cortex. Neurons are identified by NeuN staining in green (B) and in red (D). Scale bar = 5 μm.

Fig. 4

Single-copy probe Southern blotting of case 61776. (A) Southern blots of DNA from various tissues, DNA digested with BsU36I. The expansion to a large repeat has occurred in the frontal cortex, cerebellum, and spinal cord. (B) Blood and fibroblast DNA double-digested with BamHI/EcoRI. The fibroblasts only possess the 90 repeat allele and not the large expansion. (C) Southern blots following BamHI/EcoRI digestions performed on individuals with repeat sizes of, in order on the blot, 2+20, 2+22, 2+24, 5+26, 10+25, 2+23, 2+23, and 5+27 repeats (sized by PCR), and 2 independent blood DNA extractions from case 61776 (mosaic band marked with *) whose nonexpanded allele has 5 repeats. No mosaicism was shown in the cases with 20–27 repeats. The position of the bands did not reflect their size relative to each other in all cases: the electrophoresis of the bands where more DNA was loaded was retarded in the agarose gel. Abbreviations: Neg, negative control; PCR, polymerase chain reaction; Pos, positive control.