Rapid and inexpensive bedside diagnosis of RAN binding protein 2-associated acute necrotizing encephalopathy

Figures

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  Figure 1. RANBP2-specific RT eliminates the detection of RGPD. (A) Local alignment of RANBP2, RANBP2-T585M, and RGPD mRNAs illustrate the presence of the c.1880C>T genotype in 4 of the 7 RGPDs. Codon positions correspond to RANBP2. (B) Position of the primers that were used for RANBP2-specific RT and allele-specific qPCR of wild-type and mutant RANBP2. Common sequences between RANBP2 and RGPD are indicated in grey. (C–E) RNAs were extracted from THP-1 cells, and used for RANBP2-specific RT (with RANBP2-RT primer), total RT (with poly dT and random primers), or no RT (without reverse transcriptase). (C) Representative melting curves using RANBP2fwd/RANBP2rev primers show the amplification of multiple different transcripts with total RT and of a probable single transcript with RANBP2-specific RT. (D) qPCR was performed using RGPDfwd/RGPDrev (left) or RANBP2fwd/RANBP2rev (right) sets of primers. Ct values were normalized against the mean of RPL13a from the total RT. Results show 5 independent experiments and each dot corresponds to the mean of qPCR triplicates. Error bars represent a 95% confidence interval. (E) qPCR was performed using allele-specific primers on total RT or RANBP2-specific…

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  Figure 2. Allele-specific qPCR discriminates between WT and mutant RANBP2. (A–C) HEK293T cells were co-transfected with a RANBP2-targeting shRNA to knockdown endogenous expression and plasmids expressing either wild-type RANBP2, mutant RANBP2, or both. As a control, cells were transfected with a control shRNA or left untransfected. Cells were sorted by FACS (BD Aria) prior to RNA extraction. (A) qPCR amplification of total RANBP2 shows the degree of knockdown achieved and the equivalent expression levels of the WT and T585M RANBP2 constructs. The graph shows values normalized for RPL13a from a representative experiment performed in triplicate. (B,C) Allele-specific-qPCR detection of mutations at protein position 585 (B) or 653 (C) were performed and normalized against the amplification of total RANBP2 using RANBP2fwd/RANBP2rev primers 2−ΔCt . Values are the means from 2 independent experiments performed in triplicate ± SD. (B) Allele-specific qPCR was performed using primer 585fwd with either 585-WTrev or 585-MUTrev. (C) Allele-specific qPCR was performed using primer 653fwd with either 653-WTrev or 653-MUTrev. (D) RNA extracts from ANE1 subjects and healthy control subjects (Ctr) were…