Noncanonical and canonical splice sites: a novel mutation at the rare noncanonical splice-donor cut site (IVS4+1A>G) of SEDL causes variable splicing isoforms in X-linked spondyloepiphyseal dysplasia tarda

Abstract

X-linked spondyloepiphyseal dysplasia tarda can be caused by mutations in the SEDL gene. This study describes an interesting novel mutation (IVS4 þ 1A4G) located exactly at the rare noncanonical AT-AC consensus splicing donor point of SEDL, which regained the canonical GT-AG consensus splicing junction in addition to several other rarer noncanonical splice patterns. The mutation activated several cryptic splice sites and generated the production of seven erroneous splicing isoforms, which we confirmed by sequencing of RT-PCR products and resequencing of cDNA clones. All the practical splice donors/acceptors were further assessed using FSPLICE 1.0 and SPL(M) Platforms to predict potential splice sites in genomic DNA. Subsequently, the expression levels of SEDL among the affected patients, carriers and controls were estimated using real-time quantitative PCR. Expression analyses showed that the expression levels of SEDL in both patients and carriers were decreased. Taken together, these results illustrated how disruption of the AT donor site in a rare AT-AC intron, leading to a canonical GT donor site, resulted in a multitude of aberrant transcripts, thus impairing exon definition. The unexpected splicing patterns resulting from the special mutation provide additional challenges and opportunities for understanding splicing mechanisms and specificity.

Splicing mutations in human genetic disorders: examples, detection, and confirmation

“…The activation of cryptic acceptor splice sites is possible and leads to the generation of several alternative transcripts. In this case, bioinformatic software is not effective in identifying the cryptic splice acceptor sites because of the noncanonical ends Xiong et al ( 2009 ) Type V Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency ACADM (MCAD) c.362C>T (p.Thr96Ile) Missense mutation, abolish ESE Exon 5 skipping Mutation causes the loss of ESE site and abolishes SF2/ASF protein binding motif thus leading to exon skipping Ward and Cooper ( 2010 ) Neurofibromatosis type 1 NF1 c. 3362A>G (p.Glu1121Gly) Missense mutation, decreased the ratio of the ESE/ESS Two transcripts: (1) containing substitution and (2) exon 20 skipping Mutation results in the presence of two mRNA isoforms: one properly spliced contains missense change (p.Glu1121Gly) and the other one lacks exon 20 Xu et al ( 2014 ) Stickler syndrome COL2A1 192G>A (p.Cys64Ter) Nonsense mutation, abolish ESE Exon 2 skipping This is an example of class I-NAS. This nonsense mutation (p.Cys64Stop) causes exon 2 skipping by the disruption of ESE.…”

The significant other: splicing by the minor spliceosome

“…To date, only a few diseases have been linked to defects in minor splicing, some of them displaying tissue‐specific symptoms, while others have a wide range of system‐wide defects. Mutations in U12‐type 5′ss in the LKB1 and SEDL genes cause Peutz‐Jeghers syndrome and spondyloepiphyseal dysplasia tarda, respectively,83,92,93 and these diseases likely arise from the inactivity or insufficiency of the respective gene products.…”

McSplicer: a probabilistic model for estimating splice site usage from RNA-seq data

“…Point mutations in the consensus splice site sequence can affect the strength of a splice site and result in the skipping of the exon or even the skipping of multiple exons [47], or the activation of cryptic splice sites in monogenic disorders [3]. In fact, a single nucleotide substitution might produce multiple (erroneous) splicing isoforms at the same time, as has been observed, for example, for specific mutations in patients with cystic fibrosis (3 isoforms) [38], Ehlers-Danlos syndrome (4 isoforms) [47], Duchenne muscular dystrophy (3 isoforms) [17], and X-linked spondyloepiphyseal dysplasia tarda (7 isoforms) [53]. McSplicer does not attempt to reconstruct every single aberrant isoform, but similar to a weakening (strengthening) of a splice site as predicted from sequence alterations by, e.g., the Shapiro splice site probability score [41], the effect of a mutation will be reflected in a reduced or increased usage of the corresponding splice site estimated from RNA-seq reads.…”