Four parameters increase the sensitivity and specificity of the exon array analysis and disclose 25 novel aberrantly spliced exons in myotonic dystrophy

Abstract

Myotonic dystrophy type 1 (DM1) is an RNA gain-of-function disorder in which abnormally expanded CTG repeats of DMPK sequestrate a splicing trans-factor MBNL1 and upregulate another splicing trans-factor CUGBP1. To identify a diverse array of aberrantly spliced genes, we performed the exon array analysis of DM1 muscles. We analyzed 72 exons by RT-PCR and found that 27 were aberrantly spliced, whereas 45 were not. Among these, 25 were novel and especially splicing aberrations of LDB3 exon 4 and TTN exon 45 were unique to DM1. Retrospective analysis revealed that four parameters efficiently detect aberrantly spliced exons: (i) the signal intensity is high; (ii) the ratio of probe sets with reliable signal intensities (that is, detection above background P-value ¼ 0.000) is high within a gene; (iii) the splice index (SI) is high; and (iv) SI is deviated from SIs of the other exons that can be estimated by calculating the deviation value (DV). Application of the four parameters gave rise to a sensitivity of 77.8% and a specificity of 95.6% in our data set. We propose that calculation of DV, which is unique to our analysis, is of particular importance in analyzing the exon array data. INTRODUCTIONAlternative splicing regulates developmental stage-specific and tissuespecific gene expressions and markedly expands the proteome diversity with a limited number of genes. High-throughput sequencing of total mRNAs expressed in cells has revealed that 98% or more of multiexon genes are alternatively spliced, 1 with an average of seven alternative splicing per multiexon gene. 2 Alternative splicing is achieved by exonic/intronic splicing enhancers/silencers (ESE, ISE, ESS, ISS) in combination with spatial and temporal expression of trans-acting splicing factors, such as serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins. 3,4 Aberrations of alternative splicing are mediated by either mutations disrupting splicing cis-elements or dysregulation of splicing trans-factors. 5,6 Myotonic dystrophy is an autosomal dominant multisystem disorder affecting the skeletal muscles, eye, heart, endocrine system and central nervous system. The clinical symptoms include muscle weakness and wasting, myotonia, cataract, insulin resistance, hypogonadism, cardiac conduction defects, frontal balding and intellectual

RBFOX1 Cooperates with MBNL1 to Control Splicing in Muscle, Including Events Altered in Myotonic Dystrophy Type 1

“…We identified 50 mis-splicing events in the fetal DM1 myoblast cell cultures, including events known to be mis-spliced in adult tissues, such as BIN1, INSR, CLCC1, TTN.a and TTN.b [42]. We also confirmed a known misregulated splicing event at the 3′ end of the coding region of DMD (dystrophin), a gene in which mutations cause Duchenne and Becker muscular dystrophies.…”

“…More than 50 splicing alterations have been identified in skeletal and cardiac muscle of adult humans suffering from DM1 [8], [14], [42]. Overall, the defects indicate an incapacity to engage in a postnatal splicing transition [2], [22].…”

RNA Toxicity and Missplicing in the Common Eye Disease Fuchs Endothelial Corneal Dystrophy

“…Table 1 details another five splicing events found in corneal endothelial samples from patients with TNR repeat expansions that are also known to be differentially spliced in DM1 ( VEGFA , VPS39 , AKAP13 , SOS1 , and NFIX ), emphasizing the similarity in cellular phenotype to a known TNR expansion disease (29–32). …”

Exome sequencing of senescence-accelerated mice (SAM) reveals deleterious mutations in degenerative disease-causing genes

“…Mutations in the Ldb3 gene are responsible for myofibrillar myopathy and dilated cardiomyopathy in humans [37,38]. In addition, LDB3 exon 4 is aberrantly spliced in myotonic dystrophy type 1 [39]. Pathological changes in skeletal and cardiac muscles of SAMP strains, however, have not been fully analyzed.…”