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+1A>G) 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.

Figure 1

Sequence chromatograms showing mutation in SEDL of affected males and its consequences on splicing junctions. (a) gDNA sequence denotes the IVS4+1A>G mutation. The arrow indicates where the mutation occurred. Exonic sequence is shown in capital letters, intronic sequence in lower case; (b) gDNA sequence of normal control (c) cDNA sequence of RT-PCR product in the reverse-sequencing reaction of patients; (d) cDNA sequence of normal control (GGGGC–ATATGAGGTTT splicing junction); (e) del ATATGAG splice pattern in either patients or carriers; (f) del AT splice pattern in either patients or carriers.

Figure 2

Schematic figures showing alternative splicing events caused by the IVS4+1A>G mutation. Wt: denotes the wild-type SEDL gene with alternative splicing Exon 2. The coding region is shaded black. Hatched regions represent the 5′- and 3′-untranslated regions (UTRs). White squares stand for the erroneous deleted parts of coding regions. Gray squares indicate the excessive sequences in the mature transcript. (a) Erroneous splice patterns of ‘del ATATGAG', ‘del AT' and ‘del ATAT'; (b) Erroneous splice pattern of ‘E1+E3+E4+partial intron 4+E5+E6'; (c) ‘E1+E4+broken Intron4+E5+E6'; (d) ‘E1+partial intron 2+E3, 4+partial intron4 +E5, 6.'

Figure 3

Relative expression levels of SEDL genes in controls, carriers and patients.