Urine mRNA to identify a novel pseudoexon causing dystrophinopathy

Abstract

In muscular dystrophies, identification of pathogenic pseudoexons involves sequencing of the target gene cDNA derived from muscle mRNA. Here we use a urine "liquid biopsy," droplet digital PCR, and sequencing of PCR products to identify a novel cryptic splice site in DMD intron 67 that causes dystrophinopathy. Pseudoexon inclusion is 35% in urine cells, 34% in urine extracellular RNA (exRNA), and 54% in muscle biopsy tissue, but absent in serum exRNA. Our results suggest that cryptic splice site use varies depending on the RNA source, and that urine RNA has the potential to substitute for muscle biopsies to identify DMD pseudoexons.

Conflict of interest statement

A patent application has been filed on the use of urine exRNA as markers of muscular dystrophies.

Figures

Figure 1

A novel point mutation in the 5′ splice site of intron 67 of the DMD gene causes dystrophinopathy. (A) Sequence of a normal DMD gene with an intact consensus 5′ splice site (5′ ss) in intron 67 (upper) and the location of the DMD intron 67 t‐to‐g substitution variant, shown in orange, that disrupts the 5′ss (lower). (B) Immunohistochemical analysis of biceps muscle biopsy cryosections from the individual with BMD (upper) using antibodies specific for the N‐terminus, rod domain, and C‐terminus portions of the dystrophin protein. A normal human muscle biopsy served as a control (lower). Hematoxylin and eosin (H&E) shows dystrophic muscle characterized by extensive loss of muscle fibers and replacement with fat and connective tissue. Bars = 50 μm.

Figure 2

Urine exRNA identifies a DMD cryptic splice site and frame‐shifting pseudoexon. (A) Examination of urine exRNA, urine cell total RNA, serum exRNA, and commercially available total RNA from UA muscle tissue using RT‐PCR and primers targeting DMD exons 66 and 68. (B) Sequencing of the PCR products from UA urine exRNA and muscle (right upper and lower) showed normal splicing of exons 67 and 68. By contrast, splice products in the urine exRNA and biceps muscle biopsy (left upper and lower) from the BMD patient both show inclusion of the first five nucleotides of intron 67. In addition, examination of the urine exRNA chromatogram also reveals a second splice product that shows the normal transition from exon 67 to exon 68, identical to UA urine exRNA and muscle. (C) Diagram of the cryptic splice site in intron 67 that shifts the reading frame to create a premature termination codon in exon 68, explaining the dystrophinopathy in this individual.

Figure 3

Quantification of intron 67 inclusion by droplet digital PCR (ddPCR). We used ddPCR to examine DMD intron 67 inclusion in serum exRNA, urine exRNA, urine cells, and biceps muscle biopsy tissue samples. Total RNA from UA muscle tissue served as a control. (A) Assay design using separate primer probe (PP) sets to identify intron 67 inclusion (DMD‐int‐67‐68) and intron 67 exclusion (DMD‐67‐68). The left primers targeting exon 67 and the right primers targeting exon 68 are identical in each PP set. Identification of intron 67 (orange)–containing transcripts uses a fluorescent FAM–labeled probe that targets the splice junction for exon 67‐intron 67‐exon 68, while identification of normally spliced transcripts uses a FAM–labeled probe that targets the splice junction for exons 67‐68. The DMD‐int67‐68 PP set amplifies only those transcripts that contain intron 67, and has an amplicon size of 102 base pairs (bp). The DMD‐67‐68 PP set amplifies only those transcripts that are spliced normally, lack intron 67, and have an amplicon size of 97 bp. (B) Representative droplet populations (blue) in BMD serum exRNA, urine exRNA, urine cells, and muscle, and UA muscle samples using each assay (DMD‐int67‐68 and DMD‐67‐68). (C) Quantification of DMD splice products that include intron 67 (left) and exclude intron 67 (right) as copies per microliter cDNA. Individual data points represent the mean of duplicate assays for each sample. Note that values for intron 67 inclusion in serum exRNA and UA muscle tissue RNA are zero. Error bars indicate mean ± s.e.m. (D) Calculation of DMD intron 67 inclusion using data in (C). Note that values for serum exRNA and UA muscle are zero.