A homozygous missense mutation in TGM5 abolishes epidermal transglutaminase 5 activity and causes acral peeling skin syndrome

Abstract

Peeling skin syndrome is an autosomal recessive genodermatosis characterized by the shedding of the outer epidermis. In the acral form, the dorsa of the hands and feet are predominantly affected. Ultrastructural analysis has revealed tissue separation at the junction between the granular cells and the stratum corneum in the outer epidermis. Genomewide linkage analysis in a consanguineous Dutch kindred mapped the gene to 15q15.2 in the interval between markers D15S1040 and D15S1016. Two homozygous missense mutations, T109M and G113C, were found in TGM5, which encodes transglutaminase 5 (TG5), in all affected persons in two unrelated families. The mutation was present on the same haplotype in both kindreds, indicating a probable ancestral mutation. TG5 is strongly expressed in the epidermal granular cells, where it cross-links a variety of structural proteins in the terminal differentiation of the epidermis to form the cornified cell envelope. An established, in vitro, biochemical cross-linking assay revealed that, although T109M is not pathogenic, G113C completely abolishes TG5 activity. Three-dimensional modeling of TG5 showed that G113C lies close to the catalytic domain, and, furthermore, that this glycine residue is conserved in all known transglutaminases, which is consistent with pathogenicity. Other families with more-widespread peeling skin phenotypes lacked TGM5 mutations. This study identifies the first causative gene in this heterogeneous group of skin disorders and demonstrates that the protein cross-linking function performed by TG5 is vital for maintaining cell-cell adhesion between the outermost layers of the epidermis.

Figure 1

Clinicopathological features of APSS. a–d, Superficial peeling of the skin, leaving residual, painless erythema (best seen in a and d). d, Painless, manual skin removal is also possible, demonstrating that the peeling is limited to the stratum corneum. e, Light microscopy of a representative skin lesion on the dorsum of the foot (proband of family 2). The arrows point to the level of dehiscence, where cytolysis can be seen. No other abnormalities are evident. f, Electron microscopy of a similar lesion (proband of family 1, 20,000× magnification), where B denotes a blister. The level of separation is evidently between the uppermost granular layer cells (GC) and the acellular stratum corneum (SC). The asterisks mark tonofilaments that appear intact. Widened spaces (s) were observed in the stratum corneum, but these can be seen in skin from normal individuals (not shown) and are probably artefactual.

Figure 2

Pedigrees of APSS families 1 and 2 showing haplotype information in the vicinity of the TGM5 locus on 15q15. In family 1, of Dutch origin, two brothers married two sisters in generation II, and both resulting sibships produced affected children with APSS. At the outset, consanguinity could not be formally established, so compound heterozygous linkage was sought. This was identified for markers in the interval between D15S1040 (recombinant) and D15S1016 (recombinant), where all affected persons inherited the same two haplotypes (red and black boxes), whereas the unaffected individuals III-1 and III-5 inherited only one or the other of these haplotypes. Within the interval, homozygosity-by-descent was identified with markers flanking the TGM5 gene (bold type). Subsequently, a genealogical connection between the grandparents in generation I (dotted lines) was traced back to the 1780s, confirming the distant consanguinity implied by the genotype data. Interestingly, the proband in family 2 was homozygous for the same haplotype close to the TGM5 gene. This family was nonconsanguineous and of Scottish origin, with no history of Dutch ancestry.

Figure 5

Identification of homozygous missense mutations in TGM5 in persons affected by APSS. a, Normal coding-strand DNA sequence derived from exon 3 of the TGM5 gene, showing codons 108–114 inclusive. b, The same region of TGM5, derived from the proband in family 1 (individual III-3). Two homozygous missense mutations are shown: 326C→T (left arrow) and 337G→T (right arrow), predicting the amino acid changes T109M and G113C, respectively. c, The same region of TGM5, derived from a heterozygous carrier in family 1 (individual III-5). All affected persons in families 1 and 2 were homozygous for both changes.

Figure 6

Biochemical studies of TG5 mutant forms. NHEK (a) and HEK-293 (b) cells were transfected for 48 h with the use of wild-type TG5 and the three mutant forms indicated. Total TG5 activities were measured for insoluble and fractions and standardized for transfection efficiency (GFP). The control vector indicates the endogenous TG5 activity (cells transfected only with the GFP control vector). The data obtained are reported as percentages of TG activity, with 100% corresponding to the activity for cells transfected with the wild-type construct. The data presented are the averages of two independent experiments. c, View of the entire TG5 model with the four main structural domains. The mutations described here are located in the N-terminal domain at the interface between the N-terminal domain and the catalytic domain. d, Effects of the mutations in a local environment. The G113C mutation, close to the active site, would probably cause a local change in the main protein structure. The poorly conserved residue T109 is present on the surface and is, therefore, unlikely to be functionally critical. Green indicates the mutated residues, and red represents wild-type TG5 residues.

Figure 3

Summary of genome scan data obtained for APSS family 1, chromosomes 1–10, with microsatellite markers from Applied Biosystems Linkage Mapping Set version 2. Blue, locus excluded for both homozygous and compound heterozygous linkage. Red, locus showing homozygosity for all affected persons tested. Homozygosity with D1S2797 was found to be due to uninformativeness in the family. Region excluded by closely flanked markers D1S2713 (north) and D1S2652 (south).

Figure 4

Summary of genome scan data obtained for APSS family 1, chromosomes 11–22, with microsatellite markers from Applied Biosystems Linkage Mapping Set version 2. Blue, locus excluded for both homozygous and compound heterozygous linkage. Red, locus showing homozygosity for all affected persons tested. Green, locus showing compound heterozygous linkage. Homozygosity with D13S285 was found to be due to uninformativeness in the family. Region excluded by closely flanking markers D13S1315 (north) and D13S293 (south). Heterozygous linkage in the region D15S1012–D15S978 was confirmed with high-density markers in the region. An area of homozygosity was identified within this region, as detailed in figure 2 in this article.