Large C9orf72 hexanucleotide repeat expansions are seen in multiple neurodegenerative syndromes and are more frequent than expected in the UK population

Abstract

Hexanucleotide repeat expansions in C9orf72 are a major cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Understanding the disease mechanisms and a method for clinical diagnostic genotyping have been hindered because of the difficulty in estimating the expansion size. We found 96 repeat-primed PCR expansions: 85/2,974 in six neurodegenerative diseases cohorts (FTLD, ALS, Alzheimer disease, sporadic Creutzfeldt-Jakob disease, Huntington disease-like syndrome, and other nonspecific neurodegenerative disease syndromes) and 11/7,579 (0.15%) in UK 1958 birth cohort (58BC) controls. With the use of a modified Southern blot method, the estimated expansion range (smear maxima) in cases was 800-4,400. Similarly, large expansions were detected in the population controls. Differences in expansion size and morphology were detected between DNA samples from tissue and cell lines. Of those in whom repeat-primed PCR detected expansions, 68/69 were confirmed by blotting, which was specific for greater than 275 repeats. We found that morphology in the expansion smear varied among different individuals and among different brain regions in the same individual. Expansion size correlated with age at clinical onset but did not differ between diagnostic groups. Evidence of instability of repeat size in control families, as well as neighboring SNP and microsatellite analyses, support multiple expansion events on the same haplotype background. Our method of estimating the size of large expansions has potential clinical utility. C9orf72-related disease might mimic several neurodegenerative disorders and, with potentially 90,000 carriers in the United Kingdom, is more common than previously realized.

Figure 1

Histogram of C9orf72 Repeat Size in the Healthy Population Histogram showing frequency of C9orf72 repeat sizes from 1–32 in 58BC UK controls and the entire CEPH collection. rs3849942G-associated repeats are shown in green, and those associated with rs3849942A (“risk” haplotype marker) are shown in red. The phase of genotypes with repeat size was calculated for the CEPH individuals, and frequencies were then applied to the 58BC data.

Figure 2

Schematic of Southern Blotting Data for Large C9orf72 Repeat Expansions A schematic of Southern blot data for 57 cases and 11 controls shows C9orf72 repeats sizes across seven cohorts. Individual blot data are represented by colored bars, modes are indicated by similarly colored dots, and the midpoint of size is represented by a vertical black bar. Ages of onset, when available, are given in years at the right-hand end of individual bars. DNA was extracted from tissues, as shown on the left. In three controls, data are shown for DNA extracted from a lymphocyte cell line (LCL) and peripheral blood (blood). Brackets indicate that two adjacent bars are from the same individual. A single asterisk indicates an unusual ALS case with a doublet of bands of relatively low size, and double asterisks indicate a single 58BC individual with a large repeat size from LCL DNA and a diagnosis of ALS.

Figure 3

Example Southern Blots of Various Large C9orf72 Repeat Expansions A Southern blot showing C9orf72 repeat expansions in eight cases and one ECACC control and two 58BC controls demonstrates typical banding patterns and lower size in LCL DNA than in peripheral-blood DNA. Control DNA without an expansion is also shown. Cases 1 and 2 show Southern blotting of DNA from three different brain regions. The asterisk indicates GGGGCC containing a short-tandem-repeat genome motif unrelated to C9orf72. The missing bands in case 1 (brain stem) is most likely due to a reduced amount of gDNA.

Figure 4

Marked Difference in Observed Expansion Size for LCL and Peripheral-Blood DNA Southern blot showing data from three 58BC controls with C9orf72 expansions for both peripheral-blood DNA and LCL DNA. Typical LCL banding patterns can be seen and might represent pauciclonality of LCL DNA. Repeats associated with LCL DNA are smaller in size than repeats seen in peripheral-blood DNA; the latter repeats are similar in size to those in case DNA. The asterisk indicates GGGGCC containing a short-tandem-repeat genome motif unrelated to C9orf72.