Figure 1.
Clinical presentation of the spontaneous skin blistering phenotype in DEB rats.
A, Progression of skin blistering from birth to adulthood. At birth, blistering is mainly restricted to the footpads. With time, it spreads to the belly and the chest, and large erosions occur on the limbs. With the beginning of fur growth – which leads to skin stabilization – blistering decreases. Poorly healing paws lead to mutilating deformities. B, Soon after birth (4–10 days), small blisters develop on the back skin; these heal with scarring (arrows). C, In adult rats the phenotype is mainly restricted to the paws; note the dystrophic claws or toes with lost claws (yellow circle). E, Histological analysis of the skin from newborn mutant rats shows separation of the epidermis from the dermis. These findings are compatible with DEB. Left panel: H&E staining. Right panel: C7 antibody staining reveals a signal at the blister roof. Scale bar = 100 µm.
Figure 2.
Electron microscopy demonstrates anchoring fibril abnormalities in mutant rat skin.
A, Transmission electron microscopy images of the skin from four-day-old wild-type and mutant rats. Electron dense anchoring fibrils (arrowheads) are clearly visible under the lamina densa in wild-type rats, whereas mutant rat skin contains fewer and thinner anchoring fibrils (arrowheads). In blistered skin abnormal and collapsed anchoring fibrils are attached to the blister roof (arrowheads). Scale bar = 250 nm B, Quantification of the electron micrographs confirm the notion of fewer and thinner anchoring fibrils in mutant rat skin.
Figure 3.
The mutant rats carry a Col7a1 missense mutation resulting in substitution of a glycine by aspartic acid.
A, DNA sequencing chromatograms of exon 69 in Col7a1 of wild-type (left panel) and homozygous mutant (right panel) rats. The arrows indicate the G to A mutation (c.G5600A). B, The mutation leads to a codon change resulting in a glycine to aspartic acid substitution (p.G1867D). C, The mutated exon 69 codes for a stretch of the collagenous domain, N-terminally from the non-collagenous hinge region of C7. D, Both the mutated base and the amino acid are conserved in humans (ENST00000328333), suggesting that a similar mutation could result in human DEB.
Figure 4.
Dominant inheritance and gene-dosage effect of the mutation.
A, Two-day-old rat pups, wild-type, heterozygous or homozygous carriers of the p.G1867D mutation. The heterozygous pups were generated from breeding wild-type males with homozygous females. Heterozygous rats display blistering of paws and back skin (arrows); the phenotype is milder than that of the homozygous pups (gene-dosage effect). B, H&E and immunofluorescence staining of the skin of newborn wild-type, heterozygous or homozygous rats. Note the limited dermal-epidermal separation in heterozygous skin, but extensive separation in homozygous skin. The mutation leads to a reduction in the C7 content at the DEJZ, as seen by C7 immunostaining. The reduction is more pronounced in homozygous animals. Scale bar = 100 µm. C, Semiquantification of C7 staining in B.
Figure 5.
The p.G1867D mutation impacts protein stability.
A, C7 expression in cultured fibroblasts from wild-type and p.G1867D homozygous rats. qPCR analysis (left panel) and Western blot (right panel) demonstrate no notable change in mRNA or protein expression. B, Limited trypsin digestion reveals greatly reduced thermal stability of mutant C7, as shown by Western blot detecting the intact collagenous domain of C7. Mutant C7 containing the p.G1867D substitution was degraded at lower temperature than wild-type C7, indicating a reduction in thermal stability due to faulty triple helix folding of the collagenous domain. WT = wild-type; Ho = homozygous for p.G1867D. C, Limited trypsin digestion of C7 extracted from wild-type, heterozygous and homozygous keratinocytes. The lysates were heated to 30°C and allowed to cool down to room temperature before limited trypsin digestion. Western blot detecting the intact collagenous domain of C7 (left panel) shows reduced stability of heterozygous molecules and loss of stability of homozygous molecules. The right panel shows densitometric quantification of the collagenous domain of C7 and normalization to collagen XVII, which was used as a loading control. The value of wild-type C7 was set to 100% and the percent stability, as indicated by resistance to trypsin digestion, of heterozygous and homozygous C7 was calculated in relation to this value. WT = wild-type; Het = heterozygous for p.G1867D; Ho = homozygous for p.G1867D.