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Previous Volume 331:1408-1415 November 24, 1994 Number 21 Next

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ABSTRACT

Background Many skin disorders are characterized by a mosaic pattern, often with alternating stripes of affected and unaffected skin that follow the lines of Blaschko. These nonrandom patterns may be caused by a postzygotic mutation during embryogenesis. We studied the genetic basis of one such disorder, epidermal nevus of the epidermolytic hyperkeratotic type. Epidermolytic hyperkeratosis is an autosomal dominant blistering skin disease arising from mutations in the genes for keratin (K) 1 and 10. The offspring of patients with epidermal nevi may have generalized epidermolytic hyperkeratosis.

Methods We studied the K1 and K10 genes in blood and in the keratinocytes and fibroblasts of lesional and nonlesional skin from three patients with epidermal nevi and four of their offspring with epidermolytic hyperkeratosis.

Results In the patients with epidermal nevi, point mutations in 50 percent of the K10 alleles of epidermal cells were found in keratinocytes from lesional skin; no mutations were detected in normal skin. This mutation was absent or underrepresented in blood and skin fibroblasts. In the offspring with epidermolytic hyperkeratosis, the same mutations as those in the parents were found in 50 percent of the K10 alleles from all cell types examined.

Conclusions Epidermal nevus of the epidermolytic hyperkeratotic type is a mosaic genetic disorder of suprabasal keratin. The correlation of mutations in the K10 gene with lesional skin and the correlation of the normal gene with normal skin provide evidence that genetic mosaicism can cause clinical mosaicism.

View larger version (112K): [in this window] [in a new window]   Figure 1. Clinical Features of Cutaneous Mosaicism along the Lines of Blaschko. Panel A shows the surface lines of Blaschko1,2,3. Several skin diseases display epidermal patterns of clinical mosaicism that follow these lines. The lines do not follow any known underlying vascular, neural, or dermal structures but instead are thought to arise from genetic mosaicism as a consequence of postzygotic mutation. Panel B shows epidermolytic hyperkeratosis along the lines of Blaschko on the lower left side of the trunk of Patient A-1. Immediately adjacent areas of skin are clinically normal. Panel C shows generalized epidermolytic hyperkeratosis on the back of the neck of Patient A-2.

 
Epidermal nevi affect about 1 in 1000 people. Epidermal nevi appear at or shortly after birth as localized lines of epidermal thickening. The extent of skin involvement varies markedly. Epidermal nevi tend to follow the lines of Blaschko. The disease may be a mosaic disorder resulting from a postzygotic mutation.

A rare subgroup of epidermal nevi is clinically indistinguishable from other epidermal nevi but displays histopathological features typical of epidermolytic hyperkeratosis. Patients with this type of epidermal nevi sometimes have offspring with generalized epidermolytic hyperkeratosis,^8,9,10,11,12 which is characterized by mechanical stress-associated skin blistering arising from fragile cells and rupturing within the suprabasal layers of epidermis (Figure 2). Although basal cells have normal morphologic features in this disease, suprabasal cells show clumping of keratin filaments, which make up the structural framework of the epidermal keratinocyte¹³. Recently, mutations in the genes for keratin (K) 1 and 10 have been found in family members with epidermolytic hyperkeratosis^{14,15,16,17,18,19,20,21,22,23}. These keratins are expressed after a cell has started to differentiate terminally and move toward the skin surface. In contrast to K5 and K14, which form the keratin filaments of the basal layer, K1 and K10 are restricted to suprabasal, differentiating layers (Figure 2). Because keratins form obligatory heteropolymers, mutations in one member of a pair can impair the ability of the other member to assemble into filaments¹⁴.

View larger version (167K): [in this window] [in a new window]   Figure 2. Terminal Differentiation in Epidermis. The photograph (left) shows a 0.75-microm section of normal skin stained with toluidine blue. The diagram of the epidermis (right) shows the four stages of terminal differentiation. All cells above the innermost (i.e., basal) layer of the epidermis are considered to be suprabasal. Keratin (K) 1 and 10 are present in the spinous layer, and K5 and K14 in the basal layer.

 
In previous studies of epidermolytic hyperkeratosis, we identified a family (referred to here as Family C) whose affected members had a substitution of A for G at nucleotide 467 of one K10 allele, resulting in a substitution of His for Arg at codon 156¹⁵. Analysis of blood genomic DNA from the grandmother of this family showed that this mutation was underrepresented. Subsequent clinical and histopathological evaluation of this patient identified extensive epidermal nevi. From these findings, we reasoned that the epidermolytic hyperkeratotic form of epidermal nevus arises from a postzygotic K1 or K10 mutation in a cell destined to become an epidermal keratinocyte.

Methods

Clinical Characteristics of Patients

The clinical features of the three families have been described elsewhere^12,15. Family A included a man with epidermal nevi following the lines of Blaschko (Patient A-1) (Figure 1B) and his son, who had generalized epidermolytic hyperkeratosis (Patient A-2) (Figure 1C). Family B included a woman with epidermal nevi (Patient B-1) and her daughter, who had epidermolytic hyperkeratosis (Patient B-2). Family C (referred to as Family EH6 in a prior report¹⁵) included a woman with epidermal nevi (Patient C-1) and her daughter and granddaughter, both of whom had generalized epidermolytic hyperkeratosis. All the patients with epidermolytic hyperkeratosis had generalized scaling, erythroderma, and superficial blistering. Patients A-2 and B-2 also had marked palmoplantar keratoderma.

The study protocol was reviewed and approved by the institutional review committee of the University of Chicago. All participants were informed of the purpose of the study and gave written consent.

Skin-Biopsy Specimens

Keratinocytes and fibroblasts were cultured from biopsy specimens of normal and affected skin from the patients with epidermal nevi, as described elsewhere²⁴. Additional specimens were prepared for ultrastructural and immunoelectron microscopy. After embedding, immunogold labeling was performed with the use of rabbit anti-human K1 antiserum,²⁵ followed by 30 nm of colloidal gold-conjugated goat anti-rabbit antibody (Amersham, Arlington Heights, Ill.). Grids were then counterstained briefly with lead citrate.

Isolation of Genomic DNA and RNA and Mutational Analyses

Genomic DNA was isolated from blood and from fibroblasts of normal and lesional skin. Keratinocyte RNA was purified,²⁶ primed with random hexamers (Pharmacia, Piscataway, N.J.), reverse-transcribed into complementary DNA,²⁷ and then used directly for polymerase chain reaction (PCR) to amplify fragments encompassing K1 and K10 coding sequences. PCR primers were designed from K1 (GenBank numbers M11215, M11845, and M11846) and K10 (GenBank number X14487) sequences. PCR was repeated in duplicate to verify that mutations did not arise from polymerase artifacts, and DNA was sequenced with a CircumVent Thermal Cycle sequencing kit (New England Biolabs, Beverly, Mass.). In some cases, products were subcloned into pCRII vectors (Invitrogen, San Diego, Calif.) before sequencing by the dideoxy method²⁸.

Results

Clinical and Ultrastructural Diagnosis of Epidermal Nevus

The affected parents had verrucous hyperkeratotic lesions arranged along the lines of Blaschko,¹² and their affected offspring had such lesions over the entire surface of their bodies. Histologic and ultrastructural examination of lesional skin showed the classic signs of epidermolytic hyperkeratosis: a normal basal layer, with cytolysis and hyperkeratosis in the suprabasal layers (Figure 2 and Figure 3A). In contrast, the epidermis was morphologically normal in uninvolved areas (Figure 3B). Spinous layers of affected skin showed clumping of amorphous material (Figure 3C), which was labeled with an antiserum specific for suprabasal keratin (Figure 3D).

View larger version (161K): [in this window] [in a new window]   Figure 3. Ultrastructural Features of Lesional and Nonlesional Skin from Patient A-1. Panel A shows lesional skin with the typical signs of epidermolytic hyperkeratosis: normal basal cells but cytolysis (*) and clumping of keratin (KC) in suprabasal spinous cells. Nu denotes nucleus. Panel B shows nonlesional skin, which is indistinguishable from normal skin. In Panel C a higher magnification of suprabasal cells from lesional skin shows aggregates of keratin surrounding the nuclei and clumping of keratin. In Panel D immunogold labeling with anti-K1 antibodies shows an abundance of gold particles over clumps of aberrant filaments composed of K1 and K10 heterodimers in a suprabasal cell from lesional skin. (Uranyl acetate and lead citrate stains.).

 
Point Mutations of the K10 Gene in the Offspring

We analyzed genomic DNA from the offspring with epidermolytic hyperkeratosis for possible mutations of the K1 and K10 genes. PCR was used to amplify sequences encoding the large -helical rod segment of each keratin. The ends of these rod segments, which are critical for the elongation of keratin filaments, harbor the majority of mutations in severe cases of epidermolytic hyperkeratosis¹⁴. The son of Patient A-1 (Patient A-2) had a point mutation in one of the two K10 alleles (Figure 4A). This was a T-to-C mutation at nucleotide 449, resulting in a potential Met-to-Thr mutation at amino acid residue 150. The daughter of Patient B-1 (Patient B-2) had a C-to-T mutation at nucleotide 466, resulting in a potential Arg-to-Cys mutation at amino acid 156 in one of the two K10 alleles (Figure 4B). Patient C-1 and her daughter and granddaughter had a G-to-A mutation at nucleotide 467, resulting in a potential Arg-to-His mutation at amino acid 156 in one of the two K10 alleles¹⁵. PCR-amplified complementary DNA from control keratinocytes showed only ATG (Met) at codon 150 and CGC (Arg) at codon 156.

View larger version (100K): [in this window] [in a new window]   Figure 4. Sequence of K10 Alleles in Families A and B. PCR-amplified fragments of the K10 coding sequence were generated from keratinocyte complementary DNA or blood genomic DNA (gDNA) from the offspring with epidermolytic hyperkeratosis and the parents with epidermal nevi in Family A (Panel A) and Family B (Panel B). DNA sequencing was performed either directly or after subcloning. K10-coding-strand sequences are shown that encompass regions where point mutations were found. The sequences, from left to right, are from gDNA from blood obtained from the offspring with epidermolytic hyperkeratosis (Patient A-2 in Panel A and Patient B-2 in Panel B); gDNA from blood obtained from the parents with epidermal nevi (Patient A-1 in Panel A and Patient B-1 in Panel B); and complementary DNA from epidermal keratinocytes in nonlesional and lesional skin from the parents with epidermal nevi (Patient A-1 in Panel A and Patient B-1 in Panel B). The arrowheads indicate the sites of autosomal dominant mutations.

 
All these mutations were in the conserved amino end of the rod segment of K10. One of these mutations, a Met-to-Thr mutation at amino acid residue 150, was in the same methionine that was replaced by arginine in another patient with epidermolytic hyperkeratosis²³. The other K10 mutation, the substitution of Cys or His for Arg, has been found in at least 12 unrelated people with epidermolytic hyperkeratosis^{15,16,18,20,21,22,23}. Prior analyses of about 200 wild-type K10 alleles have shown that mutations at the Met and Arg sites are not polymorphic variations^15,23.

To examine these mutations further, we selected primers to amplify a 587-bp fragment and a 206-bp fragment, which encompass the Met-to-Thr mutation at codon 150 and the Arg-to-Cys or Arg-to-His mutation at codon 156, respectively. Restriction endonuclease NlaIII cuts the wild-type 587-bp fragment to generate a 443-bp fragment that can be seen on agarose gel (Figure 5A; the 106-bp and 38-bp fragments are not visible). The mutation in Patients A-1 and A-2 eliminates one of these cleavage sites, resulting in 549-bp and 38-bp fragments (Figure 5A). Restriction endonuclease AciI cuts the wild-type 206-bp fragment at one site, yielding 89-bp and 117-bp fragments. The mutation in Patients B-1 and B-2 destroys this site (Figure 5B). Enzymatic digestion of the PCR DNA from Patients A-2 and B-2 left approximately 50 percent (on a molar basis) of the relevant restriction-endonuclease site undigested. These findings are consistent with the presence of an autosomal dominant mutation that obliterates the cleavage site in one of the two alleles.

View larger version (78K): [in this window] [in a new window]   Figure 5. Obliteration of a Restriction-Endonuclease Site in One of Two K10 Alleles in the Parents with Epidermal Nevi and Their Offspring with Epidermolytic Hyperkeratosis. Genomic DNA fragments generated by three different primer sets, two encompassing the Met-to-Thr K10 mutation at codon 150 and one encompassing the Arg-to-Cys K10 mutation at codon 156, were amplified and digested with restriction endonuclease NlaIII (Panel A and Panel C) or AciI (Panel B and Panel D). Lesion mutant denotes the cloned abnormal allele from lesional keratinocytes, and lesion normal denotes the cloned normal allele from lesional keratinocytes. Samples were resolved by electrophoresis through agarose gels, and bands were visualized by staining with ethidium bromide. The patient and tissue from which the DNA was obtained are indicated above each gel (the control is from a person without the mutation). The source of the keratinocytes and fibroblasts (from a lesion or normal skin) is also indicated. For Patient C-1, fibroblasts from lesions on the abdomen (lesion 1) and thigh (lesion 2) were studied. Bands characteristic of normal and mutant K10 alleles are indicated. Note that an NlaIII cleavage site is lost as a consequence of the Met-to-Thr mutation, and an AciI site is lost as a result of the Arg-to-Cys or Arg-to-His mutation.

 
K10 Mutations in Keratinocytes from the Parents

To test the hypothesis that epidermal nevus of the epidermolytic hyperkeratotic type arises from a postzygotic mutation in K1 or K10, we extracted RNA from keratinocytes cultured from specimens of lesional and nonlesional skin. After reverse transcription, PCR amplification, and sequencing, keratinocyte K10 complementary DNA from uninvolved skin showed only the wild-type pattern -- that is, ATG (Met) at codon 150 in the man with epidermal nevi (Patient A-1) and CGC (Arg) at codon 156 in the woman with epidermal nevi (Patient B-1) (Figure 4). Similarly, the amplified K10 DNA fragments from uninvolved keratinocytes showed the wild-type pattern of restriction-endonuclease sites. This finding confirmed the absence of the heterozygous K10 mutation found in the DNA from the offspring with epidermolytic hyperkeratosis (Figure 5A and Figure 5B).

In contrast, keratinocyte complementary DNA from lesional skin from Patient A-1 had a T-to-C mutation at nucleotide 449 of K10 (Figure 4A). Keratinocyte cDNA from lesional skin from Patient B-1 had a C-to-T mutation at nucleotide 466 of K10 (Figure 4B). Restriction-endonuclease analysis showed that amplified K10 DNA fragments from lesional keratinocytes had lost the appropriate restriction-endonuclease sites. These findings confirmed the presence of the heterozygous K10 mutation (Figure 5A and Figure 5B).

Sequence analysis of genomic DNA from our patients showed the wild-type K10 sequence at codons 150 and 156 (Figure 4); however, PCR analysis revealed a small amount of undigested, mutant DNA from the blood obtained from Patient B-1 under conditions in which wild-type controls were quantitatively digested (Figure 5B). In a prior study, we also detected an Arg-to-His K10 mutation at codon 156 at much less than 50 percent of allelic levels in the DNA extracted from leukocytes of Patient C-1,¹⁵ who was later found to have epidermal nevi.

We also examined fibroblast DNA from lesional and nonlesional skin specimens from the three patients with epidermal nevi (Figure 5C and Figure 5D). For Patient A-1, we selected new primers to detect the loss of the NlaIII site. The primers generated a 273-bp PCR K10 fragment, which is cleaved into fragments of 29, 106, and 138 bp in wild-type DNA and 244 and 29 bp in the allele harboring the Met-to-Thr K10 mutation at codon 150 (the 29-bp fragment is not shown). This mutation was detected in fibroblast DNA from Patient A-1, irrespective of whether the fibroblasts were from lesional or nonlesional skin (Figure 5C). For patients B-1 and C-1, digestion of PCR-amplified dermal fibroblast DNA from both lesional and nonlesional skin with the restriction endonuclease AciI showed the presence of a 206-bp DNA fragment that was resistant to digestion (Figure 5D). However, there was less of the undigested band than the cleaved fragments, indicating that the Arg-to-Cys or Arg-to-His mutation at codon 156 was present in only a minority of the fibroblasts.

Discussion

Immunohistochemical, functional, and genetic studies show that defects in epidermal keratin genes can cause blistering skin disorders associated with the clumping of keratin filaments and cytolysis in cells expressing the mutated keratin gene^{14,15,16,17,29,30,31,32}. Transgenic mice expressing a truncated suprabasal K10 gene have clinical and ultrastructural features of epidermolytic hyperkeratosis,³³ and genetic mapping has linked the epidermolytic hyperkeratotic defect in one family to the keratin gene cluster on chromosome 12³⁴. Approximately 30 unrelated people with epidermolytic hyperkeratosis have been reported to have point mutations in their K1 or K10 genes^15,23. Studies of in vitro filament assembly, as well as keratinocyte transfections with mutant keratin genes, support the view that these mutations are responsible for the clinical manifestations of the disease^15,21,23.

Our studies provide in vivo evidence linking suprabasal keratin mutations with the clinical features of epidermolytic hyperkeratosis. Studies of three unrelated people with epidermal nevi demonstrated mutations in one of the two K10 alleles in keratinocytes cultured from lesional but not nonlesional epidermis, and these mutations were present in their offspring with epidermolytic hyperkeratosis. The correlation of clinical, ultrastructural, and genetic findings with the presence or absence of a K10 gene mutation in patients with epidermal nevi demonstrates that mutations in this gene cause epidermolytic hyperkeratosis.

Studies with mutant epidermal keratins have suggested that the highly conserved amino end of the -helical rod segment is critical for the assembly of keratin filaments^35,36,37. Within the amino end of the rod is a highly conserved arginine residue encoded by CGC, which appears to be a site of frequent C-to-T mutations³⁸. Our findings suggest that this codon may be a target for postzygotic mutations that give rise to epidermal nevi.

The embryologic alterations that lead to somatic mosaicism are poorly understood. Our ability to detect the K10 mutation in fibroblasts and blood leukocytes, as well as its transmission through the germ line, suggests that the mutations that cause epidermal nevus occur early in the development of the embryo. We found no concordance between either the presence or the amount of mutant K10 allele in fibroblasts and its presence or amount in keratinocytes from the same skin-biopsy specimen. This is consistent with the different embryologic development of the dermis and the epidermis and the presence of disease in the epidermis but not in the dermis.

Our findings suggest that the genetic diagnosis of epidermal nevus can be reliably made only from an examination of lesional epidermis, not from studies of other tissue or blood. Extensive skin involvement increases the risk of germ-line transmission. Because epidermolytic hyperkeratosis may cause devastating disfigurement and is resistant to therapy, prenatal genetic screening may be desirable. Rothnagel et al. have reported the prenatal diagnosis of epidermolytic hyperkeratosis by means of molecular analysis²². Our studies make similar approaches feasible for the offspring of patients with epidermal nevi.

In summary, the mosaic pattern of K10 mutations in patients with epidermal nevi of the epidermolytic hyperkeratotic type and the transmission of these mutations to offspring with epidermolytic hyperkeratosis provide evidence that genetic mosaicism can cause clinical mosaicism. Other skin diseases that are clinically heterogeneous and follow the lines of Blaschko should be studied to see whether a similar relation between genetic and clinical mosaicism is present.

Supported by grants from the National Institutes of Health (AR01811-ASP and R01-AR27883-EF) and the Howard Hughes Medical Institute.

Source Information

From the Departments of Pediatrics and Dermatology, Northwestern University Medical School, Chicago (A.S.P.); the Howard Hughes Medical Institute, Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago (A.J.S., Y.-M.C., Q.-C.Y., E.H., E.F.); and the Department of Dermatology, University of Milan, Milan, Italy (G.T.).

Address reprint requests to Dr. Paller at the Division of Dermatology, #107, Children's Memorial Hospital, 2300 Children's Plaza, Chicago, IL 60614.

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