Semi-automated quantification of C9orf72 expansion size reveals inverse correlation between hexanucleotide repeat number and disease duration in frontotemporal degeneration

Abstract

We investigated whether chromosome 9 open reading frame 72 hexanucleotide repeat expansion (C9orf72 expansion) size in peripheral DNA was associated with clinical differences in frontotemporal degeneration (FTD) and amyotrophic lateral sclerosis (ALS) linked to C9orf72 repeat expansion mutations. A novel quantification workflow was developed to measure C9orf72 expansion size by Southern blot densitometry in a cross-sectional cohort of C9orf72 expansion carriers with FTD (n= 39), ALS (n= 33), both (n=35), or who are unaffected (n= 21). Multivariate linear regressions were performed to assess whether C9orf72 expansion size from peripheral DNA was associated with clinical phenotype, age of disease onset, disease duration and age at death. Mode values of C9orf72 expansion size were significantly shorter in FTD compared to ALS (p=0.0001) but were not associated with age at onset in either FTD or ALS. A multivariate regression model correcting for patient’s age at DNA collection and disease phenotype revealed that C9orf72 expansion size is significantly associated with shorter disease duration (p=0.0107) for individuals with FTD, but not with ALS. Despite considerable somatic instability of the C9orf72 expansion, semi-automated expansion size measurements demonstrated an inverse relationship between C9orf72 expansion size and disease duration in patients with FTD. Our finding suggests that C9orf72 repeat size may be a molecular disease modifier in FTD linked to hexanucleotide repeat expansion.

Pathogenic determinants and mechanisms of ALS/FTD linked to hexanucleotide repeat expansions in the C9orf72 gene

“…Similar to what was observed in other repeat expansion diseases, the repeat size was also found to be associated with sample collection time [22, 23]. C9orf72 repeat expansion sizes have been widely reported to vary in different tissue types and cell types [19, 22–24], indicating the expansion of repeats might be caused by some cell division-independent mechanism in addition to somatic instability.…”

“…However, variable ranges of repeat number have been reported for controls and patients: fewer than 14 repeats in controls as 20–22 repeats were associated with FTD [20]; fewer than 23 repeats in controls [15]; 800–4400 repeats in patients [19]; 0–30 repeats in controls and more than 500 repeats in patients [21]. And finally a more recent study employing a southern blot based semi-automated quantification showed that most control carry fewer than 10 repeats, while patients carry 300–3800 repeats [22]. …”

“…C9orf72 repeat expansion sizes have been widely reported to vary in different tissue types and cell types [19, 22–24], indicating the expansion of repeats might be caused by some cell division-independent mechanism in addition to somatic instability. The repeat size variation among different tissues within a same carrier was larger than that between different carriers, suggesting that intrinsic properties of specific tissue might affect the stability of the expanded repeats.…”

Decoding ALS: from genes to mechanism

“…Working in parallel, Brian Traynor and colleagues independently discovered pathogenic GGGGCC repeat expansion in C9ORF72 79 . Similar to numerous other neurodegenerative diseases caused by microsatellite expansion, there appears to be an inverse relationship between repeat expansion length and disease severity 102,103 . Affected individuals apparently inherit hundreds of repeat copies, but these undergo expansion to 3000–5000 copies in cells of the nervous system 104 .…”

Expansion of the classification of FTLD-TDP: distinct pathology associated with rapidly progressive frontotemporal degeneration

“…Alignment of sequence reads and variant calling were assessed by SureCall software (Agilent). In addition, cases were tested for the C9orf72 hexanucleotide repeat expansion as previously described [32]. …”