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. Author manuscript; available in PMC: 2012 Oct 20.
Published in final edited form as: Neuron. 2011 Sep 21;72(2):245–256. doi: 10.1016/j.neuron.2011.09.011

Figure 4. Effect of expanded hexanucleotide repeat on C9ORF72 expression.

Figure 4

(A) Overview of the genomic structure of the C9ORF72 locus (top panel) and the C9ORF72 transcripts produced by alternative pre-mRNA splicing (bottom panels). Boxes represent coding (white) and non-coding (grey) exons and the positions of the start codon (ATG) and stop codon (TAA) are indicated. The GGGGCC repeat is indicated with a red diamond. The position of rs10757668 is indicated with a green star. (B) Sequence traces of C9ORF72 exon 2 spanning rs10757668 in gDNA (top panel) and cDNA (bottom panels) prepared from frontal cortex of an FTLD-TDP patient carrying an expanded GGGGCC repeat. The arrow indicates the presence of the wild-type (G) and mutant (A) alleles of rs10757668 in gDNA. Transcript specific cDNAs were amplified using primers spanning the exon 1b/exon 2 boundary (variant 1) or exon 1a/exon 2 boundary (variant 2 and 3). Sequenced traces derived from cDNA transcripts indicate the loss of variant 1 but not variant 2 or 3 mutant RNA. Similar results were obtained for two unrelated FTLD-TDP mutation carriers. The bottom panel shows a non-expanded repeat carrier heterozygous for rs10757668 to confirm the presence of both alleles of transcript variant 1 validating the method. (C) mRNA expression analysis of C9ORF72 transcript variant 1 using a custom-designed Taqman expression assay. Top panel shows lymphoblast cell lines derived from expanded repeat carriers from family VSM-20 (n=7) and controls (n=7) and bottom panel shows RNA extracted from frontal cortex brain samples from FTLD-TDP patients with (n=7) and without (n=7) the GGGGCC repeat expansion. Data indicate mean ± s.e.m. ** indicates P<0.01. (D) mRNA expression analysis of all C9ORF72 transcripts encoding for C9ORF72 isoform a (variant 1 and 3) using inventoried ABI Taqman expression assay Hs_00945132. Top panel shows RNA extracted from lymphoblast cell lines derived from expanded repeat carriers from family VSM-20 (n=7) and controls (n=7) and bottom panel shows RNA extracted from frontal cortex brain samples from FTLD-TDP patients with (n=7) and without (n=7) the GGGGCC repeat expansion. Data indicate mean ± s.e.m. * indicates P<0.05.