The loss of variant 1 expression in the GGGGCC repeat carriers was further confirmed by real-time RT-PCR using a custom-designed Taqman assay specific to variant 1. In lymphoblast cell lines of patients from family VSM-20 and in frontal cortex samples from unrelated FTLD-TDP patients carrying expanded repeats, the level of C9ORF72 variant 1 was approximately 50% reduced compared to nonrepeat carriers ( Figure 4C). Since C9ORF72 variants 1 and 3, which each contain a different noncoding first exon, both encode C9ORF72 isoform a (NP_060795.1), we next determined the effect of the expanded repeats on the total levels of transcripts encoding this

isoform (variants 1 and 3 combined) using an inventoried ABI Taqman assay (Hs_00945132). Significant mRNA reductions were observed in both lymphoblast cells (34% reduction) and frontal cortex samples (38% reduction) JAK inhibitor from expanded repeat carriers ( Figure 4D). In contrast, no appreciable changes in total levels of C9ORF72 protein could be observed by western blot analysis of lymphoblast cell lysates or brain ( Figure S2), or by immunohistochemical

Romidepsin mw analysis of C9ORF72 in postmortem brain or spinal cord tissue from expanded repeat carriers ( Figure S2). These protein expression data should, however, be considered preliminary since they are based on a limited number of samples using relatively uncharacterized commercially obtained C9ORF72 antibodies without detailed quantitative analyses. In recent years, intracellular accumulation of expanded nucleotide repeats as RNA foci in the nucleus and/or cytoplasm of affected cells has emerged as an important disease mechanism for the growing class of noncoding repeat expansion disorders (Todd and Paulson, 2010). To determine whether GGGGCC repeat expansions in C9ORF72 result in the Carnitine dehydrogenase formation of RNA foci, we performed RNA fluorescence in situ

hybridization (FISH) in paraffin-embedded sections of postmortem frontal cortex and spinal cord tissue from FTLD-TDP patients. For each neuroanatomical region, sections from two patients with expanded GGGGCC repeats and two affected patients with normal repeat lengths were analyzed. Using a probe targeting the GGGGCC repeat (probe (GGCCCC)4), multiple RNA foci were detected in the nuclei of 25% of cells in both the frontal cortex and the spinal cord from patients carrying the expansion, whereas a signal was observed in only 1% of cells in tissue sections from noncarriers ( Figures 5A–5C). Foci were never observed in any of the samples using a probe targeting the unrelated CCTG repeat (probe (CAGG)6), implicated in myotonic dystrophy type 2 (DM2) ( Liquori et al., 2001), further supporting the specificity of the RNA foci composed of GGGGCC in these patients ( Figure 5D).