Abstract
Identical G+1 mutations in three different introns of the gene for type III procollagen (COL3A1) that cause aberrant splicing of RNA were found in three probands with life-threatening variants of Ehlers-Danlos syndrome. Because the three mutations were in a gene with multiple and homologous exons, they provided an interesting test for factors that influence aberrant splicing. The G+1 to A mutation in intron 16 caused extensive exon skipping, the G+1 to A mutation in intron 20 caused both use of a cryptic splice site and retention of all the intron sequences, and the G+1 to A mutation in intron 42 caused efficient use of a single cryptic splice site. The different patterns of RNA splicing were not explained by evaluation of potential cryptic splice sites in the introns by either their homology with 5'-splice sites from other genes or by their delta G(0)37 values for binding to U1 RNA. Instead, the results suggested that the patterns of aberrant RNA splicing were primarily determined by the relative rates at which adjacent introns were normally spliced.
Highlights
Identical G’l to A Mutations in Three Different Introns of the Type III Procollagen Gene (COL3Al) Produce Different Patterns of RNA
Identical G+’ mutations in three different. introns of the gene for type III procollagen (COL3Al) that cause aberrant splicing of RNA were found in three probands with life-threatening variants of Ehlers-Danlos syndrome
Because the three mutations were in a gene with multiple and homologous exons, they provided an interesting test for factors that influence aberrant splicing
Summary
I (+l IVS16) was a 36-year-old pregnant female who had thin skin with abnormally prominent superficial blood vessels. RNA Splicing Mutations in Type III Procollagen Gene and transparent skin with abnormally prominent blood vessels She had mild hypermobility of the joints, congenital dislocation of the hips, and a torn knee ligament. The ligated product was used as a template for the PCR reaction using a 5’ primer identical to sequences in exon 6 [37] and a 3’ primer complementary to codons in exon 17 [35]’ The PCR product was cloned into Ml3 to prepare the single-stranded probe as described above except that the two probes generated were end-labeled with la-32PldCTP and the Klenow fragment of DNA nolvmerase I after digestion with AccI or NcoI. The ligated DNA was used as a template for a PCR with 5’ primer identical to coding sequences in exon 34 and 3’ primer complementary to sequences ending with nucleotide +34 of intron 43. 5’-splice sites, the free energy parameters of Freier et al [39] were used
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