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. 2012 Aug 10;91(2):313–319. doi: 10.1016/j.ajhg.2012.07.002

Figure 2.

Figure 2

Expression of FUS mRNA Carrying ET and ALS Mutations

(A) Lymphoblastoid cells derived from five ET-affected individuals (individuals II:13, III:14, IV:6, IV:11, and IV:12 in Figure S1) who carry the FUS c.868C>T (p.Gln290∗) mutation and from ALS-affected individuals expressing three different FUS mutations (c.1555C>T [p.Gln519∗], c.1562G>A [p.Arg521His], and c.1542-2A>C) were treated with the protein-synthesis inhibitor puromycin (300 μg for 6 hr). After this, treated cells were harvested, and total RNA was prepared (Trizol extraction, Invitrogen) alongside the total RNA of untreated cells. Quantitative RT-PCR reactions were then performed with a Taqman probe specific to FUS (∗∗∗p value = 6.47 × 10−5 [comparing the difference in mRNA expression in ET and ALS untreated cells]; ∗∗p value = 0.03 [comparing the difference in mRNA expression in ET and ALS treated cells]). For each group of affected (ET and ALS) individuals, the average levels of expression are plotted. t tests were used for statistics, and error bars correspond to the standard error of the mean (SEM).

(B) Standard nonquantitative RT-PCR reactions were prepared with the RNA used in (A) and primers in the flanking exons (or UTRs) of each mutation. Amplified cDNA was sequenced, and whereas the ALS mutations were seen independently of a puromycin treatment, the ET mutation could only be seen in puromycin-treated cells.

(C) Quantitative allele-specific expression measurements of the same five FUS c.868C>T ET individuals from above were made with a set of Taqman custom-designed probes and primers for the wild-type (c.868C) and mutant (c.868T) alleles. After the puromycin treatment, the expression of the mutant allele became detectable. The specificity of the custom probe for the c.868C>T transcript is demonstrated in Figure S5. All expression levels were normalized with the human 18S ribosomal RNA (rRNA) gene and were calculated in comparison to the average level of expression of two healthy controls. t tests were used for statistics, and error bars correspond to SEM.