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.

Keywords: Amyotrophic lateral sclerosis; C9ORF72; Disease modifier; Frontotemporal dementia; Frontotemporal lobar degeneration; Motor neuron disease.

Fig. 1

Localization of C9ORF72 probes. The three known C9ORF72 transcripts are visualized; the repeat expansion in C9ORF72 is represented by a red triangle and located in the promoter region of variant 2 (NM_018325.4) or in the first intron of variant 1 (NM_145005.6) and variant 3 (NM_001256054.2). Whereas variant 1 is predicted to result in a 222 amino acid long protein (exon 2–5, isoform b), variant 2 and variant 3 appear to result in 481 amino acid long proteins (exon 2–11, isoform a). For our TaqMan assays, probes were used targeting total C9ORF72 transcripts, variant 2, and variant 3 (green). Our digital molecular barcoding experiment contained probes for total C9ORF72 transcripts, variant 1, and variant 2 as well as probes that target the region between exon 1a and the expansion (transcripts containing intron 1a) and between exon 1b and exon 2 (transcripts containing intron 1b, purple)

Fig. 2

Expression of specific C9ORF72 transcripts. C9Plus patients with C9ORF72 repeat expansions, C9Minus patients without C9ORF72 repeat expansions, and Control control subjects without neurological diseases. For each box plot, the median is represented by a solid line (values are specified between brackets below panels), and each box spans the IQR (25th percentile to 75th percentile). In the cerebellum, a reduction in total C9ORF72 transcripts is observed in patients with C9ORF72 repeat expansions as compared to patients without these expansions or to control subjects (a using TaqMan assays, Table 2). Additionally, a decrease in variant 2 is detected in the cerebellum of C9ORF72 expansion carriers as compared to patients without expansions or to control subjects (b using TaqMan assays, Table 2). In the cerebellum, there is an association between expression levels measured using TaqMan assays and NanoString technologies, both for total C9ORF72 transcripts (c Online Resource Table 6) and for variant 2 (d Online Resource Table 6). In the frontal cortex, levels of intron 1a containing transcripts are higher in expansion carriers than in patients without expansions or in control subjects (e using NanoString technologies, Table 3), and within our cohort of expansion carriers, the highest levels appear to be present in patients with FTLD or FTLD/MND (f using NanoString technologies, Table 4)

Fig. 3

Associations with clinical and pathological characteristics. Associations with clinical and pathological variables are shown for our overall cohort of C9ORF72 expansion carriers. Survival following disease onset, according to 25th percentile variant 1 levels, is displayed for the cerebellum (a using NanoString technologies, Table 5) and for the frontal cortex (b using NanoString technologies, Table 5). Additionally, associations between intron 1b containing transcripts and poly(GP) levels (c using NanoString technologies, Table 6) or poly(GA) levels (d using NanoString technologies, Table 6) are shown for the cerebellum