Background Many skin disorders are characterized by a mosaicpattern, often with alternating stripes of affected and unaffectedskin that follow the lines of Blaschko. These nonrandom patternsmay be caused by a postzygotic mutation during embryogenesis.We studied the genetic basis of one such disorder, epidermalnevus of the epidermolytic hyperkeratotic type. Epidermolytichyperkeratosis is an autosomal dominant blistering skin diseasearising from mutations in the genes for keratin (K) 1 and 10.The offspring of patients with epidermal nevi may have generalizedepidermolytic hyperkeratosis.
Methods We studied the K1 and K10 genes in blood and in thekeratinocytes and fibroblasts of lesional and nonlesional skinfrom three patients with epidermal nevi and four of their offspringwith epidermolytic hyperkeratosis.
Results In the patients with epidermal nevi, point mutationsin 50 percent of the K10 alleles of epidermal cells were foundin keratinocytes from lesional skin; no mutations were detectedin normal skin. This mutation was absent or underrepresentedin blood and skin fibroblasts. In the offspring with epidermolytichyperkeratosis, the same mutations as those in the parents werefound in 50 percent of the K10 alleles from all cell types examined.
Conclusions Epidermal nevus of the epidermolytic hyperkeratotictype is a mosaic genetic disorder of suprabasal keratin. Thecorrelation of mutations in the K10 gene with lesional skinand the correlation of the normal gene with normal skin provideevidence that genetic mosaicism can cause clinical mosaicism.
Many skin disorders are characterized by a mosaic pattern, oftenwith alternating stripes of affected and unaffected skin. Thesestripes are referred to as lines of Blaschko1. They do not followthe vascular, neural, or lymphatic structures of the skin, nordo they correlate with dermatomes (Figure 1). This pattern hasbeen attributed to the clonal proliferation of two geneticallydistinct groups of cells that arise from a postzygotic mutationduring embryogenesis2. Skin diseases exhibiting such patternsare often linked to the X chromosome3. Examples are focal dermalhypoplasia, the Conradi-Hunermann syndrome, incontinentia pigmenti,and the carrier state for hypohidrotic ectodermal dysplasia.Clinical mosaicism may also be seen in disorders that are notlinked to the X chromosome. A mosaic disorder with an identifiedgene alteration is the McCune-Albright syndrome, a sporadicand rare disease that is probably lethal in utero in the nonmosaicstate4. Patients with this disease have cystic bone lesions,often with precocious puberty4. In addition, cafe au lait spotsare often distributed along the lines of Blaschko4. The defectiveallele in the McCune-Albright syndrome is underrepresented intissue that is clinically affected, although a correlation betweengenetic and clinical mosaicism has not been established5,6,7.
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 neviappear at or shortly after birth as localized lines of epidermalthickening. The extent of skin involvement varies markedly.Epidermal nevi tend to follow the lines of Blaschko. The diseasemay be a mosaic disorder resulting from a postzygotic mutation.
A rare subgroup of epidermal nevi is clinically indistinguishablefrom other epidermal nevi but displays histopathological featurestypical of epidermolytic hyperkeratosis. Patients with thistype of epidermal nevi sometimes have offspring with generalizedepidermolytic hyperkeratosis,8,9,10,11,12 which is characterizedby mechanical stress-associated skin blistering arising fromfragile cells and rupturing within the suprabasal layers ofepidermis (Figure 2). Although basal cells have normal morphologicfeatures in this disease, suprabasal cells show clumping ofkeratin filaments, which make up the structural framework ofthe epidermal keratinocyte13. Recently, mutations in the genesfor keratin (K) 1 and 10 have been found in family members withepidermolytic hyperkeratosis14,15,16,17,18,19,20,21,22,23. Thesekeratins are expressed after a cell has started to differentiateterminally and move toward the skin surface. In contrast toK5 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 ofthe other member to assemble into filaments14.
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 identifieda family (referred to here as Family C) whose affected membershad a substitution of A for G at nucleotide 467 of one K10 allele,resulting in a substitution of His for Arg at codon 15615. Analysisof blood genomic DNA from the grandmother of this family showedthat this mutation was underrepresented. Subsequent clinicaland histopathological evaluation of this patient identifiedextensive epidermal nevi. From these findings, we reasoned thatthe epidermolytic hyperkeratotic form of epidermal nevus arisesfrom a postzygotic K1 or K10 mutation in a cell destined tobecome an epidermal keratinocyte.
Methods
Clinical Characteristics of Patients
The clinical features of the three families have been describedelsewhere12,15. Family A included a man with epidermal nevifollowing the lines of Blaschko (Patient A-1) (Figure 1B) andhis son, who had generalized epidermolytic hyperkeratosis (PatientA-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 priorreport15) included a woman with epidermal nevi (Patient C-1)and her daughter and granddaughter, both of whom had generalizedepidermolytic hyperkeratosis. All the patients with epidermolytichyperkeratosis had generalized scaling, erythroderma, and superficialblistering. Patients A-2 and B-2 also had marked palmoplantarkeratoderma.
The study protocol was reviewed and approved by the institutionalreview committee of the University of Chicago. All participantswere informed of the purpose of the study and gave written consent.
Skin-Biopsy Specimens
Keratinocytes and fibroblasts were cultured from biopsy specimensof normal and affected skin from the patients with epidermalnevi, as described elsewhere24. Additional specimens were preparedfor ultrastructural and immunoelectron microscopy. After embedding,immunogold labeling was performed with the use of rabbit anti-humanK1 antiserum,25 followed by 30 nm of colloidal gold-conjugatedgoat 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 ofnormal and lesional skin. Keratinocyte RNA was purified,26 primedwith random hexamers (Pharmacia, Piscataway, N.J.), reverse-transcribedinto complementary DNA,27 and then used directly for polymerasechain reaction (PCR) to amplify fragments encompassing K1 andK10 coding sequences. PCR primers were designed from K1 (GenBanknumbers M11215, M11845, and M11846) and K10 (GenBank numberX14487) sequences. PCR was repeated in duplicate to verify thatmutations did not arise from polymerase artifacts, and DNA wassequenced with a CircumVent Thermal Cycle sequencing kit (NewEngland Biolabs, Beverly, Mass.). In some cases, products weresubcloned 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 arrangedalong the lines of Blaschko,12 and their affected offspringhad such lesions over the entire surface of their bodies. Histologicand ultrastructural examination of lesional skin showed theclassic signs of epidermolytic hyperkeratosis: a normal basallayer, with cytolysis and hyperkeratosis in the suprabasal layers(Figure 2 and Figure 3A). In contrast, the epidermis was morphologicallynormal in uninvolved areas (Figure 3B). Spinous layers of affectedskin showed clumping of amorphous material (Figure 3C), whichwas labeled with an antiserum specific for suprabasal keratin(Figure 3D).
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 epidermolytichyperkeratosis for possible mutations of the K1 and K10 genes.PCR was used to amplify sequences encoding the large -helicalrod 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 epidermolytichyperkeratosis14. The son of Patient A-1 (Patient A-2) had apoint mutation in one of the two K10 alleles (Figure 4A). Thiswas a T-to-C mutation at nucleotide 449, resulting in a potentialMet-to-Thr mutation at amino acid residue 150. The daughterof Patient B-1 (Patient B-2) had a C-to-T mutation at nucleotide466, resulting in a potential Arg-to-Cys mutation at amino acid156 in one of the two K10 alleles (Figure 4B). Patient C-1 andher daughter and granddaughter had a G-to-A mutation at nucleotide467, resulting in a potential Arg-to-His mutation at amino acid156 in one of the two K10 alleles15. PCR-amplified complementaryDNA from control keratinocytes showed only ATG (Met) at codon150 and CGC (Arg) at codon 156.
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 rodsegment of K10. One of these mutations, a Met-to-Thr mutationat amino acid residue 150, was in the same methionine that wasreplaced 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 epidermolytichyperkeratosis15,16,18,20,21,22,23. Prior analyses of about200 wild-type K10 alleles have shown that mutations at the Metand Arg sites are not polymorphic variations15,23.
To examine these mutations further, we selected primers to amplifya 587-bp fragment and a 206-bp fragment, which encompass theMet-to-Thr mutation at codon 150 and the Arg-to-Cys or Arg-to-Hismutation at codon 156, respectively. Restriction endonucleaseNlaIII cuts the wild-type 587-bp fragment to generate a 443-bpfragment that can be seen on agarose gel (Figure 5A; the 106-bpand 38-bp fragments are not visible). The mutation in PatientsA-1 and A-2 eliminates one of these cleavage sites, resultingin 549-bp and 38-bp fragments (Figure 5A). Restriction endonucleaseAciI cuts the wild-type 206-bp fragment at one site, yielding89-bp and 117-bp fragments. The mutation in Patients B-1 andB-2 destroys this site (Figure 5B). Enzymatic digestion of thePCR DNA from Patients A-2 and B-2 left approximately 50 percent(on a molar basis) of the relevant restriction-endonucleasesite undigested. These findings are consistent with the presenceof an autosomal dominant mutation that obliterates the cleavagesite in one of the two alleles.
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 epidermolytichyperkeratotic type arises from a postzygotic mutation in K1or K10, we extracted RNA from keratinocytes cultured from specimensof lesional and nonlesional skin. After reverse transcription,PCR amplification, and sequencing, keratinocyte K10 complementaryDNA 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 epidermalnevi (Patient B-1) (Figure 4). Similarly, the amplified K10DNA fragments from uninvolved keratinocytes showed the wild-typepattern of restriction-endonuclease sites. This finding confirmedthe absence of the heterozygous K10 mutation found in the DNAfrom the offspring with epidermolytic hyperkeratosis (Figure 5Aand Figure 5B).
In contrast, keratinocyte complementary DNA from lesional skinfrom Patient A-1 had a T-to-C mutation at nucleotide 449 ofK10 (Figure 4A). Keratinocyte cDNA from lesional skin from PatientB-1 had a C-to-T mutation at nucleotide 466 of K10 (Figure 4B).Restriction-endonuclease analysis showed that amplified K10DNA fragments from lesional keratinocytes had lost the appropriaterestriction-endonuclease sites. These findings confirmed thepresence of the heterozygous K10 mutation (Figure 5A and Figure 5B).
Sequence analysis of genomic DNA from our patients showed thewild-type K10 sequence at codons 150 and 156 (Figure 4); however,PCR analysis revealed a small amount of undigested, mutant DNAfrom the blood obtained from Patient B-1 under conditions inwhich wild-type controls were quantitatively digested (Figure 5B).In a prior study, we also detected an Arg-to-His K10 mutationat codon 156 at much less than 50 percent of allelic levelsin the DNA extracted from leukocytes of Patient C-1,15 who waslater found to have epidermal nevi.
We also examined fibroblast DNA from lesional and nonlesionalskin specimens from the three patients with epidermal nevi (Figure 5Cand Figure 5D). For Patient A-1, we selected new primersto detect the loss of the NlaIII site. The primers generateda 273-bp PCR K10 fragment, which is cleaved into fragments of29, 106, and 138 bp in wild-type DNA and 244 and 29 bp in theallele harboring the Met-to-Thr K10 mutation at codon 150 (the29-bp fragment is not shown). This mutation was detected infibroblast DNA from Patient A-1, irrespective of whether thefibroblasts were from lesional or nonlesional skin (Figure 5C).For patients B-1 and C-1, digestion of PCR-amplified dermalfibroblast DNA from both lesional and nonlesional skin withthe restriction endonuclease AciI showed the presence of a 206-bpDNA 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 codon156 was present in only a minority of the fibroblasts.
Discussion
Immunohistochemical, functional, and genetic studies show thatdefects in epidermal keratin genes can cause blistering skindisorders associated with the clumping of keratin filamentsand cytolysis in cells expressing the mutated keratin gene14,15,16,17,29,30,31,32.Transgenic mice expressing a truncated suprabasal K10 gene haveclinical and ultrastructural features of epidermolytic hyperkeratosis,33and genetic mapping has linked the epidermolytic hyperkeratoticdefect in one family to the keratin gene cluster on chromosome1234. Approximately 30 unrelated people with epidermolytic hyperkeratosishave been reported to have point mutations in their K1 or K10genes15,23. Studies of in vitro filament assembly, as well askeratinocyte transfections with mutant keratin genes, supportthe view that these mutations are responsible for the clinicalmanifestations of the disease15,21,23.
Our studies provide in vivo evidence linking suprabasal keratinmutations with the clinical features of epidermolytic hyperkeratosis.Studies of three unrelated people with epidermal nevi demonstratedmutations in one of the two K10 alleles in keratinocytes culturedfrom lesional but not nonlesional epidermis, and these mutationswere present in their offspring with epidermolytic hyperkeratosis.The correlation of clinical, ultrastructural, and genetic findingswith the presence or absence of a K10 gene mutation in patientswith epidermal nevi demonstrates that mutations in this genecause epidermolytic hyperkeratosis.
Studies with mutant epidermal keratins have suggested that thehighly conserved amino end of the -helical rod segment is criticalfor the assembly of keratin filaments35,36,37. Within the aminoend of the rod is a highly conserved arginine residue encodedby CGC, which appears to be a site of frequent C-to-T mutations38.Our findings suggest that this codon may be a target for postzygoticmutations that give rise to epidermal nevi.
The embryologic alterations that lead to somatic mosaicism arepoorly understood. Our ability to detect the K10 mutation infibroblasts and blood leukocytes, as well as its transmissionthrough the germ line, suggests that the mutations that causeepidermal nevus occur early in the development of the embryo.We found no concordance between either the presence or the amountof mutant K10 allele in fibroblasts and its presence or amountin keratinocytes from the same skin-biopsy specimen. This isconsistent with the different embryologic development of thedermis and the epidermis and the presence of disease in theepidermis but not in the dermis.
Our findings suggest that the genetic diagnosis of epidermalnevus can be reliably made only from an examination of lesionalepidermis, not from studies of other tissue or blood. Extensiveskin involvement increases the risk of germ-line transmission.Because epidermolytic hyperkeratosis may cause devastating disfigurementand is resistant to therapy, prenatal genetic screening maybe desirable. Rothnagel et al. have reported the prenatal diagnosisof epidermolytic hyperkeratosis by means of molecular analysis22.Our studies make similar approaches feasible for the offspringof patients with epidermal nevi.
In summary, the mosaic pattern of K10 mutations in patientswith epidermal nevi of the epidermolytic hyperkeratotic typeand the transmission of these mutations to offspring with epidermolytichyperkeratosis provide evidence that genetic mosaicism can causeclinical mosaicism. Other skin diseases that are clinicallyheterogeneous and follow the lines of Blaschko should be studiedto see whether a similar relation between genetic and clinicalmosaicism is present.
Supported by grants from the National Institutes of Health (AR01811-ASPand 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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131: 1460-1462
[Abstract]
-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. 
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