Expanding the Boundaries of RNA Sequencing as a Diagnostic Tool for Rare Mendelian Disease.

Gene-panel and whole-exome analyses are now standard methodologies for mutation detection in Mendelian disease. However, the diagnostic yield achieved is at best 50%, leaving the genetic basis for disease unsolved in many individuals. New approaches are thus needed to narrow the diagnostic gap. Whole-genome sequencing is one potential strategy, but it currently has variant-interpretation challenges, particularly for non-coding changes. In this study we focus on transcriptome analysis, specifically total RNA sequencing (RNA-seq), by using monogenetic neuromuscular disorders as proof of principle. We examined a cohort of 25 exome and/or panel "negative" cases and provided genetic resolution in 36% (9/25). Causative mutations were identified in coding and non-coding exons, as well as in intronic regions, and the mutational pathomechanisms included transcriptional repression, exon skipping, and intron inclusion. We address a key barrier of transcriptome-based diagnostics: the need for source material with disease-representative expression patterns. We establish that blood-based RNA-seq is not adequate for neuromuscular diagnostics, whereas myotubes generated by transdifferentiation from an individual's fibroblasts accurately reflect the muscle transcriptome and faithfully reveal disease-causing mutations. Our work confirms that RNA-seq can greatly improve diagnostic yield in genetically unresolved cases of Mendelian disease, defines strengths and challenges of the technology, and demonstrates the suitability of cell models for RNA-based diagnostics. Our data set the stage for development of RNA-seq as a powerful clinical diagnostic tool that can be applied to the large population of individuals with undiagnosed, rare diseases and provide a framework for establishing minimally invasive strategies for doing so.

Responses of rainbow trout intestinal epithelial cells to different kinds of nutritional deprivation.

In order to develop an in vitro system to study the cell biology of starvation in the fish intestine, rainbow trout intestinal epithelial cells were subjected to three kinds of nutrient deprivation and evaluated for 7 days. The RTgutGC cell line was grown into monolayers in Leibovitz's basal medium supplemented with fetal bovine serum (L15/FBS) and then subjected to deprivation of serum (L15); of serum, amino acids, and vitamin (L15/ex); and of all nutrients (L15/salts). After 7 days of nutrient deprivation, the cells remained attached to the plastic surface as monolayers but changes were seen in shape, with the cells becoming more polygonal, actin and α-tubulin cytoskeleton organization, and in tight junction protein-1 (ZO-1) localization. Two barrier functions, transepithelial electrical resistance (TEER) and Lucifer Yellow (LY) retention, were impaired by nutrient deprivation. In L15/FBS, cells rapidly healed a gap or wound in the monolayer. In L15 and L15/ex, some cells moved into the gap, but after 7 days, the wound remained unhealed, whereas in L15/salts, cells did not even migrate into the gap. Upon nutrient replenishment (L15/FBS) after 7 days in L15, L15/ex, or L15/salts, cells proliferated again and healed a wound. After 7 days of nutrient deprivation, monolayers were successfully passaged with trypsin and cells in L15/FBS grew to again form monolayers. Therefore, rainbow trout intestinal epithelial cells survived starvation, but barrier and wound healing functions were impaired.