January 3, 2018  |  Human genetics research

Alternative splicing analysis reveals isoforms associated with FMR1 repeat expansion

In a recent publication, scientists from the University of California, Davis, and PacBio reported results from an investigation of alternative splicing associated with a repeat expansion in the gene linked to fragile X syndrome. They used SMRT Sequencing to detect full-length isoforms (Iso-Seq analysis) associated with individuals at risk of FXTAS, an adult-onset neurodegenerative disorder.

Altered expression of the FMR1 splicing variants landscape in premutation carriers” comes from lead author Elizabeth Tseng, senior author Flora Tassone, and collaborators. Previous studies from the Tassone lab had used SMRT Sequencing to detect full-length isoforms in samples from premutation carriers (individuals with more CGG repeats than a healthy person, but not enough to cause fragile X syndrome) and had identified a number of known isoforms. In this study, the scientists aimed for a more comprehensive analysis of alternative splicing of the FMR1 gene. “Although evidence suggests a strong role for regulation of the FMR1 gene expression in clinical outcomes,” the team reports, “there have been no detailed molecular characterizations on the role of alternative splicing in the development of FMR1 premutation associated disorders.”

To tackle this challenge, they deployed SMRT Sequencing to characterize transcript isoforms of the FMR1 gene in tissue samples from three premutation carriers and three matched controls, plus blood samples from 30 premutation carriers and 15 controls. The tissue samples were collected from cerebellum, testis, muscle, and heart. Iso-Seq analysis yielded up to 28,000 full-length transcript reads from the premutation carrier tissue samples. In total, the authors identified 49 unique FMR1 isoforms, including 16 previously characterized isoforms and a number of novel ones. This study has revealed new splicing patterns and a novel 140-bp exon that were shown to have elevated expression in premutation and FXTAS samples compared to normal controls. Of the 49 FMR1 isoforms, the scientists note, “30 of them were exclusively detected in premutation carriers based on sequencing results.”

This study underscores the power of Iso-Seq analysis in comprehensively characterizing  full-length transcript isoforms for a gene of interest.  By eliminating the need for sequence assembly, as is required using short-read sequencing methods, Iso-Seq analysis returns each isoform sequence in its entirety in a single read, thereby enabling the discovery of novel exons, intron retention, fusion transcripts and, ultimatel, previously undetected novel isoforms.

“Our findings suggest that an abnormal alternative splicing process is present in individuals with premutation alleles,” the team concludes. “The characterization of the expression levels of the different FMR1 isoforms is fundamental for understanding the regulation of the FMR1 gene as imbalance in their expression could lead to an altered functional diversity with neurotoxic consequences.”

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