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Epidermolysis Bullosa Simplex with mottled pigmentation: mutation analysis in the first reported Hispanic pedigree with the largest single generation of affected individuals to date

European Journal of Dermatology. Volume 16, Number 2, 132-5, March-April 2006, Genes and skin

Summary

Author(s) : Daniel Shurman, Jacqueline Losi-Sasaki, Ronald Grimwood, Sirpa Kivirikko, Elizabeth Tichy, Jouni Uitto, Gabriele Richard , Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA; USA, Division of Dermatology, University of Texas Health Science Center at San Antonio, San Antonio, TX. USA, Department of Clinical Genetics, Helsinki University Central Hospital, Helsinki, Finland, Genedx, Gaithersburg, MD. USA.

Summary : Epidermolysis bullosa simplex with mottled pigmentation (EBS-MP), characterized by trauma-induced blisters, distinct pigmentary changes of the trunk and extremities, and acral hyperkeratotic papules, is almost exclusively caused by a common KRT5 missense mutation affecting the V1 region of keratin 5. We studied the first Hispanic family, the largest single generation of affected family members in which 5 out of 10 siblings inherited EBS-MP from their affected father, as well a second large pedigree, the first reported of Finnish ancestry. In both families, the heterozygous transition mutation 74C→T of the keratin 5 gene, which results in amino acid substitution P25L, completely co-segregated with the EBS-MP phenotype.

Keywords : blistering skin diseases, epidermolysis bullosa simplex, genodermatoses, keratin mutations, mechanobullous skin diseases

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ARTICLE

Auteur(s) : Daniel Shurman¹, Jacqueline Losi-Sasaki², Ronald Grimwood², Sirpa Kivirikko³, Elizabeth Tichy², Jouni Uitto^1,*, Gabriele Richard^1,4

¹Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA; USA
²Division of Dermatology, University of Texas Health Science Center at San Antonio, San Antonio, TX. USA
³Department of Clinical Genetics, Helsinki University Central Hospital, Helsinki, Finland
⁴Genedx, Gaithersburg, MD. USA

accepté le 20 Decembre 2005

Epidermolysis bullosa simplex (EBS) with mottled pigmentation (MP) is a distinct variant of EBS first described by Fisher and Gedde-Dahl in 1979 [1]. The disorder shows three major characteristics. First, relatively mild, trauma-induced blistering of the palms, soles and the extremities is present since infancy or early childhood. They heal without scarring, milia or hyperpigmentation, and decrease in frequency with age. Secondly, also at a young age, the patients develop small, brown, hyperpigmented macules ranging from 2-5 mm in size. These lesions tend to coalesce on the trunk and extremities and give the skin a characteristic mottled pigmentation. Thirdly, acral hyperkeratotic papules typically develop in adolescence or early adulthood. Numerous sporadic cases and multi-generation families have been reported, all being consistent with autosomal dominant inheritance [1-9].Molecular studies have revealed a single KRT5 missense mutation in all but one EBS-MP family analyzed to date. The mutation is a C→T transition at position 74 of KRT5 and results in replacement of a conserved proline residue with leucine at position 25 (P25L) [10]. The mutation lies in the non-helical variable head domain of KRT5, thus residing outside the mutation hot spot regions, namely the helix initiation and termination motifs and linker regions, found in other more severe forms of EBS. The P25L mutation does not interfere with alignment and assembly of keratin intermediate filaments (KIF), thus explaining the mild blistering phenotype of EBS-MP [10].

Methods

Clinical features of the families

Family 1

We studied a large Mexican family with EBS-MP, which is the first reported family with this EBS subtype of Hispanic origin. Clinical evaluation of 6 affected individuals, including an affected father and 5 of his affected children, revealed small blisters on the palmoplantar surfaces (Family 1 in ( figure 1 )). All affected individuals showed characteristic mottled pigmentation of the skin, which consistently spared the face and was most apparent in the younger children (( figure 2 )). Consistent with previously reported cases, the pigmentary changes in the 34-year-old affected father were subtle and had nearly resolved [10]. In contrast, acral keratotic papules were much more striking in the older (13- and 14-year-old) children as well as the father. While only a few discrete acral papules were observed in the younger children, 2 and 10 years of age, the 11- and 12-year-old children had experienced a rapid progression, expansion and confluence of papules along the dorsolateral palms and soles within the last year. In all cases, the oral mucosa and other mucosal surfaces were spared and there was no evidence of photosensitivity in this family.

Family 2

We also studied a 47-year-old female patient of Finnish ancestry with a family history of EBS-MP (Family 2 in ( figure 1 )). The disorder manifested in early childhood with hyperpigmentation of the face, trunk, and legs as well as trauma-induced skin blistering without evidence for scarring or milia. The patient also had severe, diffuse palmoplantar keratoderma, papular hyperkeratotic lesions on the dorsal hands and mild nail dystrophy.

Mutation analysis

With written informed consent of all participants, genomic DNA was obtained from peripheral blood samples of 11 individuals in Family 1 and the proband of Family 2 (( figure 1 )). The molecular genetic studies were approved by the Institutional Review Board of Thomas Jefferson University and adhered to the Declaration of Helsinki principles. In the proband of each family, the complete coding sequence (exons 1-9) and exon-intron boundaries of the KRT5 gene were PCR amplified and subjected to bi-directional sequence analysis. Gene specific primers were created with Primer3 (http://cbr-rbc.nrc-cnrc.gc.ca/cgi-bin/primer3_www.cgi) and BLAST analysis and are available upon request from the authors. PCR was performed in 50 μl reactions using 200 ng of DNA, 50 ng of each primer, 1.0 IU of Taq Polymerase (Qiagen, Valencia, CA) and standard PCR conditions. Amplicons were separated by agarose gel electrophoresis, gel-purified (QIAquick gel extraction kit, Qiagen) and directly sequenced using the BigDye terminator sequencing system on an ABI Prism 377 sequencer (PE Applied Biosystems, Foster City, CA). The presence or absence of missense mutation P25L in other family members and a cohort of 47 unrelated, unaffected Hispanic control individuals was established by allele-specific PCR, as previously described [7]. The frequency of 2 sequence polymorphisms in exon 1 of KRT5, 591C→A (D197E) and 594C→A (T198T), was determined by sequence analysis.

Results

All affected members of Family 1 and the proband of Family 2 were found to be heterozygous for the C to T transition at position 74 in exon 1 of KRT5, replacing the proline codon (CCG) at position 25 with a leucine codon (CTG), a mutation denoted as P25L (( figure 1 )). In addition, 3 affected individuals of Family 1, all of whom had acral hyperkeratotic lesions, were heterozygous for two previously described polymorphisms 591C→A (D197E) and 594C→A (T198T) [10, 11] (( figure 1 )). Although the allelic frequency of both the coding polymorphism D197E and the silent polymorphism T198T in a Hispanic control cohort was relatively high (29%; 27 out of 94 alleles for both), it is conceivable that the presence of D197E might modulate the clinical phenotype of EBS-MP and increase the susceptibility for developing acral hyperkeratotic papules. This hypothesis, however, needs further investigation.

Discussion

To date, 23 patients with EBS-MP from 9 unrelated, non-consanguineous families have been reported to harbor the P25L mutation in KRT5, including families from diverse ethnic and geographic backgrounds from Japan, UK, Germany, Ireland, The Netherlands, and Scandinavia. As demonstrated here, the P25L mutation has also occurred in Hispanic and Finnish families, thus strongly refuting a founder effect in EBS-MP. Irvine et al. (2001) determined that P25L lies inside an 18 amino acid sequence that is highly conserved across several types of intermediate filaments in different species. In this region, the pattern of SXXSXXXPXXXR is likely to be involved in phosphorylation and/or O-linked glycosylation, and the presence of a CpG dinucleotide in codon 25 makes it vulnerable to mutation, reflecting the hypermutability of 5-methyl-cytosine [9].

In vitro studies, in the context of the P25L mutation, have demonstrated that K5 clones lacking the amino terminus were still able to form normal appearing 10 nm K14/K5 tetramers [12]. However, it has been demonstrated that transfection of kidney epithelial cells with K5 DNA harboring the P25L mutation resulted in the formation of slightly shortened K14/K5 intermediate filaments, and cells expressing the mutant K5 DNA showed signs of cytoskeletal collapse in comparison to wild-type K5 controls [10]. Furthermore, there is experimental evidence for the role of V1 in maintaining the dynamic nature of intermediate filaments and in protein-protein interactions [13]. This notion is supported by an earlier observation that the P25L mutation lies in a region of K5 that extends out from the keratin tetramers [14]. Improper keratin intermediate filament function provides a likely explanation for the cell fragility observed in EBS-MP as it has been well described for other forms of EBS. However, the pathomechanisms responsible for the unusual pigmentation and hyperkeratotic lesions in EBS-MP remain unexplained. It has been hypothesized that accumulation of mature melanosomes in those basal keratinocytes that demonstrate perinuclear vacuolization and cytolysis is responsible for both blisters and mottled pigmentation [10]. It has also been suggested that the V1 head region of K5 interacts with dynein, a cytoskeletal protein involved in melanosome transport [15]. Finally, it is conceivable that the P25L mutation might interfere with normal binding of the V1 domain of K5 with desmoplakin, which might alter cell-cell signaling and change the homeostatic proliferation and differentiation of the local epidermis [16].

The molecular findings in the Hispanic and Finnish EBS-MP families reported here confirm previous studies and underscore that the missense mutation P25L in KRT5 is consistently associated with an EBS-MP phenotype. Most likely, this mutation is responsible for all major clinical findings of EBS-MP, thus emphasizing the importance of keratin intermediate filament function for skin fragility, melanosome transport, and epidermal differentiation and proliferation.

Acknowledgments

We are grateful to the families that participated in our study. Carol Kelly assisted in preparation of this manuscript. This work was supported in part by the NIH/NIAMS grants K08-AR02141 and P01-AR38923.

References

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