Novel clinical associations with specific C9ORF72 transcripts in patients with repeat expansions in C9ORF72

Abstract

The loss of chromosome 9 open reading frame 72 (C9ORF72) expression, associated with C9ORF72 repeat expansions, has not been examined systematically. Three C9ORF72 transcript variants have been described thus far; the GGGGCC repeat is located between two non-coding exons (exon 1a and exon 1b) in the promoter region of transcript variant 2 (NM_018325.4) or in the first intron of variant 1 (NM_145005.6) and variant 3 (NM_001256054.2). We studied C9ORF72 expression in expansion carriers (n = 56) for whom cerebellum and/or frontal cortex was available. Using quantitative real-time PCR and digital molecular barcoding techniques, we assessed total C9ORF72 transcripts, variant 1, variant 2, variant 3, and intron containing transcripts [upstream of the expansion (intron 1a) and downstream of the expansion (intron 1b)]; the latter were correlated with levels of poly(GP) and poly(GA) proteins aberrantly translated from the expansion as measured by immunoassay (n = 50). We detected a decrease in expansion carriers as compared to controls for total C9ORF72 transcripts, variant 1, and variant 2: the strongest association was observed for variant 2 (quantitative real-time PCR cerebellum: median 43 %, p = 1.26e-06, and frontal cortex: median 58 %, p = 1.11e-05; digital molecular barcoding cerebellum: median 31 %, p = 5.23e-10, and frontal cortex: median 53 %, p = 5.07e-10). Importantly, we revealed that variant 1 levels greater than the 25th percentile conferred a survival advantage [digital molecular barcoding cerebellum: hazard ratio (HR) 0.31, p = 0.003, and frontal cortex: HR 0.23, p = 0.0001]. When focusing on intron containing transcripts, analysis of the frontal cortex revealed an increase of potentially truncated transcripts in expansion carriers as compared to controls [digital molecular barcoding frontal cortex (intron 1a): median 272 %, p = 0.003], with the highest levels in patients pathologically diagnosed with frontotemporal lobar degeneration. In the cerebellum, our analysis suggested that transcripts were less likely to be truncated and, excitingly, we discovered that intron containing transcripts were associated with poly(GP) levels [digital molecular barcoding cerebellum (intron 1a): r = 0.33, p = 0.02, and (intron 1b): r = 0.49, p = 0.0004] and poly(GA) levels [digital molecular barcoding cerebellum (intron 1a): r = 0.34, p = 0.02, and (intron 1b): r = 0.38, p = 0.007]. In summary, we report decreased expression of specific C9ORF72 transcripts and provide support for the presence of truncated transcripts as well as pre-mRNAs that may serve as templates for RAN translation. We further show that higher C9ORF72 levels may have beneficial effects, which warrants caution in the development of new therapeutic approaches.

Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts

“…S1C). These data are consistent with the prior finding that the abundance of poly(GP) proteins correlate with levels of repeat-containing transcripts (11, 12). Importantly, deleting SPT4 in (GGGGCC) 40 -expressing yeast significantly decreased levels of RAN-translated poly(GP) (Fig.…”

“…The resulting dipeptide repeat (DPR) proteins could cause disease through proteotoxic mechanisms (11). Lastly, the expanded repeats reduce C9orf72 mRNA expression (12, 13), which may cause a loss of C9orf72 function. A therapeutic approach that uses antisense oligonucleotides (ASOs) to target GGGGCC repeat-containing RNA transcripts for degradation is being pursued (5, 14, 15).…”

ALS and FTD: an epigenetic perspective

“…The loss of function theory has been supported by several reports demonstrating decreased expression of one or multiple C9orf72 transcript variants in frontal cortex, motor cortex, cerebellum and cervical spinal cord of FTD or ALS C9orf72 HRE carriers (c9FTD/ALS) [15,36,57,50,64,126,178], as well as in lymphoblastoid cell lines generated from c9FTD/ALS patient blood [36] and neuronal cell lines differentiated from c9FTD/ALS induced pluripotent stem cells (iPSCs) [4,50]. Hypermethylation of the 5′ CpG island located in the C9orf72 promoter region has been shown by different groups to be present in about 10–30% of c9FTD/ALS subjects [14,104,187,191], possibly leading to reduced C9orf72 expression levels in these cases.…”

Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers

“…Affected patients exhibit expansions to upwards of 1,000 or more of the repeats. Various concepts have emerged concerning the molecular basis of disease pathophysiology, including impediments to expression of the C9orf72 gene itself (van Blitterswijk et al, 2015), expression of putatively toxic sense or anti-sense transcripts of the repeats (Donnelly et al, 2013; Haeusler et al, 2014; Lagier-Tourenne et al, 2013; Mizielinska et al, 2013), and expression of putatively toxic repeat-associated, non-ATG (RAN) translation products (Ash et al, 2013; Mori et al, 2013; Zu et al, 2013). …”