Molecular mechanisms of classical Ehlers-Danlos syndrome (EDS)

Abstract

Classical Ehlers-Danlos syndrome (EDS) is a heritable disorder characterized by joint hypermobility, skin hyperextensibility, and abnormal wound healing. The majority of affected individuals have alterations in 1 of the 2 type V collagen genes, COL5A1 and COL5A2. The most common mechanism is COL5A1 haploinsufficiency due to instability of the transcript of one allele. In dermal fibroblasts from our population of 76 individuals with clinical features of classical EDS, there were 21 (29.5%) with decreased expression of one COL5A1 allele, consistent with published estimates of the frequency of null alleles. We identified the causative mutation in nine of these cell strains (mutations for seven others had been previously described), and found two nonsense mutations, five splice mutations, and two insertion/deletions. The same type of genomic change at splice sites can have different effects at the RNA level and the outcome could not be predicted from the primary genomic DNA alteration.

Figure 1

Amplification of COL5A1 overlapping large cDNA fragments made from whole cell RNA. A. P12; amplification of exons 14–37 has a normal fragment and one with faster than expected mobility, as well as a heteroduplex.. B. P11; amplification of exons 27–48 has a fragment with normal mobility and a heteroduplex, indicative of a size alteration in some of the fragments of apparently normal size. C. P1; amplification of exons 44–57 with a small amount of a smaller than expected product and a heteroduplex. D. P8; amplification of exon 60-3′UTR. The heteroduplex in the control results from an alternative splice variant. There is an additional heteroduplex in the subject sample. C= control individual; P= subject; HD= heteroduplex

Figure 2

Distribution of abnormal allele RNA. A. TspRI digestion of PCR product from P7 (COL5A1 exon 46 c.3680_3681insT). Virtually all the uncut product (from the mutant allele) is in the nuclear fraction. B. Amplified products from P8 (COL5A1 c.4916_4931del16). The cDNA was amplified with primers in exons 60 and 63. The expected 391 bp product of exons 60–63 is indicated. The product from the abnormal allele with the 16 basepair deletion is seen only in the nuclear compartment. C. P6: (COL5A1 c.1654C>T; p.Gln552X). The mutation introduces a new BsrI site in the amplified cDNA. The majority of the abnormal product is in the nucleus with virtually none in the cytoplasmic fraction. wt= wildtype; mut= mutant; n= nuclear RNA; c= cytoplasmic RNA; T=TspRI site; B-BsrI site.

Figure 3

Effects of COL5A1 mutations on splicing. A. P9: COL5A1 c.1989+1G>A(IVS19+1G>A); Amplification primers in exons 17 and 21 produced the expected 219 bp product. The product from the abnormal allele results from use of an intronic splice site and inclusion of four nucleotides (atag) from intron 19. The heteroduplex is seen in both nuclear and cytoplasmic samples in low abundance. B. P11: COL5A1 c.2844+1G>A (IVS35+1G>A). Primers in exons 33 and 37 were used and the expected 268 bp product is indicated. The smallest band in lanes 1 and 2 results from skipping of exon 35. The other product from the abnormal allele results from use of an intronic splice site and inclusion of 118 nucleotides from intron 35. The heteroduplex derived from the normal product and the intron inclusion product is seen only with the nuclear material (HD3). C. P12: COL5A1 c.2845-2A>C (IVS35-2A>C). Primers in exons 33 and 37 were used and the expected 268 bp product is indicated. The smallest band in lanes 1and 2 results from skipping of exon 36. The other product from the abnormal allele results from use of a cryptic exonic splice site and deletion of nineteen nucleotides from exon 36. Heteroduplex 1 (HD1) results from interaction between the normal product and the exon skip product and is seen in the nuclear and cytoplasmic fractions. HD2, which results from interaction between the normal product and the partial exon deletion product, is poorly represented in the cytoplasmic fraction. D. P10: COL5A1 c.2386-1G>A (IVS27-1G>A). Primers in exons 25 and 30 were used and the expected 295 bp product is indicated. The product from the abnormal allele results from the use of a splice site generated by the mutation that is 1 nt downstream and results in deletion of one nucleotide from exon 28. It is seen almost exclusively in the nuclear fraction. n=nuclear RNA; c=cytoplasmic RNA; HD=heteroduplex; arrows indicate location of primers used for PCR-amplification.

Figure 4

Identification of abnormal splice products in cells from P1 (COL5A1 c.3874G>A; p.Glu1292Lys). A. Samples from cDNA were amplified with primers in exons 48 and 54 of COL5A1. The expected 447 bp product is indicated. The smaller band, seen only in the nuclear fraction, results from use of a cryptic splice donor site within exon 49 and deletion of all but 19 basepairs of the exon. B. Determination of amounts of non-deletion products expressed from each allele. Samples from cDNA synthesized from nuclear and cytoplasmic RNA were PCR-amplified with primers in exons 44 and in the segment of exon 49 deleted as a result of the use of the newly formed donor site. The exon 49 reverse primer differed from the wildtype sequence by 1 nucleotide so that a HinfI restriction enzyme site was created with the normal product. The amplification product is 373 bp; 21 bp are removed from the product of the normal allele after digestion. A little less than half the product in the cytoplasm represents the mutant sequence. n=nuclear RNA; c=cytoplasmic RNA; HD=heteroduplex; H=HinfI site; arrows indicate location of primers used for PCR-amplification.

Figure 5

SDS-PAGE of radiolabeled collagens from P4. Theα1(V) andα2(V) bands in the P4 cell layer are broader than expected (lane 4 vs. lane 3), indicating overmodification of the protein. C=control sample; P= patient sample.