Clinico-pathological features in amyotrophic lateral sclerosis with expansions in C9ORF72

Abstract

Intronic expansion of the GGGGCC hexanucleotide repeat within the C9ORF72 gene causes frontotemporal dementia and amyotrophic lateral sclerosis/motor neuron disease in both familial and sporadic cases. Initial reports indicate that this variant within the frontotemporal dementia/amyotrophic lateral sclerosis spectrum is associated with transactive response DNA binding protein (TDP-43) proteinopathy. The amyotrophic lateral sclerosis/motor neuron disease phenotype is not yet well characterized. We report the clinical and pathological phenotypes associated with pathogenic C9ORF72 mutations in a cohort of 563 cases from Northern England, including 63 with a family history of amyotrophic lateral sclerosis. One hundred and fifty-eight cases from the cohort (21 familial, 137 sporadic) were post-mortem brain and spinal cord donors. We screened DNA for the C9ORF72 mutation, reviewed clinical case histories and undertook pathological evaluation of brain and spinal cord. Control DNA samples (n = 361) from the same population were also screened. The C9ORF72 intronic expansion was present in 62 cases [11% of the cohort; 27/63 (43%) familial, 35/500 (7%) cases with sporadic amyotrophic lateral sclerosis/motor neuron disease]. Disease duration was significantly shorter in cases with C9ORF72-related amyotrophic lateral sclerosis (30.5 months) compared with non-C9ORF72 amyotrophic lateral sclerosis/motor neuron disease (36.3 months, P < 0.05). C9ORF72 cases included both limb and bulbar onset disease and all cases showed combined upper and lower motor neuron degeneration (amyotrophic lateral sclerosis). Thus, clinically, C9ORF72 cases show the features of a relatively rapidly progressive, but otherwise typical, variant of amyotrophic lateral sclerosis associated with both familial and sporadic presentations. Dementia was present in the patient or a close family member in 22/62 cases with C9ORF72 mutation (35%) based on diagnoses established from retrospective clinical case note review that may underestimate significant cognitive changes in late disease. All the C9ORF72 mutation cases showed classical amyotrophic lateral sclerosis pathology with TDP-43 inclusions in spinal motor neurons. Neuronal cytoplasmic inclusions and glial inclusions positive for p62 immunostaining in non-motor regions were strongly over-represented in the C9ORF72 cases. Extra-motor pathology in the frontal cortex (P < 0.0005) and the hippocampal CA4 subfield neurons (P < 0.0005) discriminated C9ORF72 cases strongly from the rest of the cohort. Inclusions in CA4 neurons were not present in non-C9ORF72 cases, indicating that this pathology predicts mutation status.

Figure 1

Graphical representation of fragments generated in repeat primed polymerase chain reaction displayed using Peak Scanner Software. Individual peaks represent fragments of increasing length at 6 bp intervals, corresponding to single GGGGCC repeats; 280 bp is minimum polymerase chain reaction product length. (A) Graph from a patient with ALS with a pathological expansion in C9ORF72 showing exponentially tailing series of peaks consistent with a repeat length >30. (B) Graph from a patient with ALS without pathological expansion showing repeat length of approximately four repeats.

Figure 2

Graph showing the lengths of the GGGGCC hexanucleotide repeats in ALS cases (n = 563) and controls (n = 361); 62/563 (11%) of cases with ALS and 2/361 (0.6%) of control cases had repeat lengths of >30.

Figure 3

Family tree from a familial ALS case with the hexanucelotide repeat sequence in C9ORF72. The expansion segregated with disease in two cases and was present in an obligate carrier. + = carrier of expansion, − = confirmed absence of the expansion. Current age or age of death is shown where information was available.

Figure 4

Histological examination of cases with the C9ORF72 hexanucleotide repeat expansion reveals: depletion of motor neurons from the anterior horns of the spinal cord (A) with Bunina bodies (arrow) in residual neurons (B); Microglial activation in lateral corticospinal tracts (arrowheads, C); ubiquitylated neuronal (skein-like in D and compact in E) and glial (F) cytoplasmic inclusions in the anterior horns of the spinal cord; (G) TDP-43 positive skein-like neuronal cytoplasmic inclusions (arrows) and pre-inclusions (arrowhead) in the anterior horns of the spinal cord; (H) OPTN positive neuronal cytoplasmic inclusion in the anterior horns of the spinal cord; (I) ubiquitylated neuronal (arrow) and glial (arrowhead) cytoplasmic inclusions in the motor cortex; TDP-43 positive neuronal cytoplasmic inclusion in the motor cortex (arrow, J); ubiquitylated neuronal cytoplasmic inclusions (arrows) in the CA4 subfield of the hippocampus (K) and frontal neocortex (L). Lower power view of hippocampal CA4 subfield with adjacent dentate gyrus granule cells (left) reveals neuronal cytoplasmic inclusions (arrows) in CA4 of a case of ALS with C9ORF72 hexanucleotide repeat expansion (M) and none in a case without this expansion (N). Preparations: haematoxylin and eosin, A and B; CD68, C; p62, D–F, I, K–N; TDP-43, G and J; optineurin, H. Scale bar = 20 µm (B and F); 30 µm (D, E, G and H–L); 60 µm (A, M and N); 500 µm (C).

Figure 5

Immunohistochemistry for C9ORF72 shows similar features in neurologically healthy controls and individuals with motor neuron disease with and without the hexanucleotide repeat expansion. The spinal cord stained with C9ORF72 antibody shows: (A) Coarse granular staining of the spinal cord anterior horn neuropil with variable cytoplasmic labelling of motor neurons (arrow) and perinuclear labelling of small glial cells (e.g. arrowhead); patches of more intense labelling (arrowheads) on the surfaces of some motor neurons (B) that is often more marked in neuronal processes (C). In white matter (dorsal column) there is staining around axons corresponding to portions of myelin sheath (D). Granular staining of the neuropil with more prominent ‘streaks’ and only pale neuronal expression is seen in the hippocampus (CA4 subregion with dentate gyrus granule cell layer top right; E). Granular neuropil labelling with lesser staining of neurons is also seen in the neocortex (frontal cortex; F). Scale bar = 20 µm (B and F); 30 µm (A–D); 60 µm (E and F).