The Location of the Molecular Cause of Ehlers-Danlos Syndrome
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Classical Ehlers-Danlos Syndrome, a rare autosomal dominant disease, manifests itself in human connective tissues as hyperextensible skin and hypermobile joints. Research into the mechanism of the disease provides a basis for new treatments, and previous work showed that the most prevalent molecular cause was nonsense mediated mRNA decay (NMD) of type V collagen transcripts. This produces the genetic condition named haploinsufficiency. To delve into the disease mechanism, this study sought to sequence both COLS A1 transcripts in cDNA and exon one of COL5A1 in genomic DNA using PCR for one patient (P6). No effective conditions were found for exon one. To visualize splice site mutations, cDNA was amplified, but only normal sized bands appeared for the segments that were amplified successfully. As a result, no sequence was produced for P6. To determine whether P6had haploinsufficiency, single base extension was used to quantify the relative expression levels of COLS A1 transcripts produced from each allele using a single nucleotide polymorphism. Relative expression levels for patients with haploinsufficiency were dramatically different from control samples showing the assay detected the presence of NMD. In four of the five patients, a greater level of degradation occurred when the premature termination codon was followed by purine than a pyrimidine. When tested in this assay, P6 showed a dramatic change in expression ratios for COLS A1 implying that there was haploinsufficiency. In the future, this assay is useful to quickly determine which EDS patients have haploinsufficiency as the mechanism for their disease and which have a novel mechanism.