Lack of unique neuropathology in amyotrophic lateral sclerosis associated with p.K54E angiogenin (ANG) mutation

Abstract

Aims: Five to 10% of cases of amyotrophic lateral sclerosis are familial, with the most common genetic causes being mutations in the C9ORF72, SOD1, TARDBP and FUS genes. Mutations in the angiogenin gene, ANG, have been identified in both familial and sporadic patients in several populations within Europe and North America. The aim of this study was to establish the incidence of ANG mutations in a large cohort of 517 patients from Northern England and establish the neuropathology associated with these cases.

Methods: The single exon ANG gene was amplified, sequenced and analysed for mutations. Pathological examination of brain, spinal cord and skeletal muscle included conventional histology and immunohistochemistry.

Results: Mutation screening identified a single sporadic amyotrophic lateral sclerosis case with a p.K54E mutation, which is absent from 278 neurologically normal control samples. The clinical presentation was of limb onset amyotrophic lateral sclerosis, with rapid disease progression and no evidence of cognitive impairment. Neuropathological examination established the presence of characteristic ubiquitinated and TDP-43-positive neuronal and glial inclusions, but no abnormality in the distribution of angiogenin protein.

Discussion: There is only one previous report describing the neuropathology in a single case with a p.K17I ANG mutation which highlighted the presence of eosinophilic neuronal intranuclear inclusions in the hippocampus. The absence of this feature in the present case indicates that patients with ANG mutations do not always have pathological changes distinguishable from those of sporadic amyotrophic lateral sclerosis.

Keywords: amyotrophic lateral sclerosis; angiogenin; glial inclusions; intranuclear inclusions; neuronal inclusions; neuropathology.

Figure 1

The p.K54E mutation identified in ANG. The non-synonymous A>G nucleotide substitution at position c.232 gives rise to the amino acid substitution, p.K54E. Chromatograms are shown for wild-type sequence (a), and the heterozygous c.232A>G case (b). Screening for this change in control samples was conducted by digestion of the ANG PCR product with Taq^αI. Presence of the G allele introduces a Taq^αI digest site, resulting in the production of 287 bp and 263 bp fragments from the 550 bp PCR product (c).

Figure 2

Schematic diagram of the ANG protein and location of the p.K54E mutation. The protein domains and secondary structure are derived from information provided on the ANG protein (P03950) found on the UniProt database.

Figure 3

Immunohistochemistry of sporadic amyotrophic lateral sclerosis (SALS) case with p.K54E ANG mutation. Images show p62 (a–d), TDP-43 (e–g), FUS (h), angiogenin (i), H&E (j), CD68 (k) and α2 actinin (l) showing: neuronal (a, b, d, e and g) and glial (c and f) cytoplasmic inclusions in the spinal cord; neuronal cytoplasmic inclusions in the motor cortex (d and g); normal, predominantly nuclear labelling of FUS in the spinal cord (h), granular cytoplasmic staining in a motor neurone with the appearance of lipofuscin (i); Bunina bodies (arrowheads; j); a microglial reaction that is most marked in the lateral descending tract of the spinal cord and least marked in the dorsal columns (k); normal labelling of Z-disc in skeletal muscle by α2 actinin (l). Scale: a–i and l, bar = 20 μm; j, bar = 20 μm; k, bar = 1 mm.