The molecular genetic analysis of the expanding pachyonychia congenita case collection.

BACKGROUND: Pachyonychia congenita (PC) is a rare autosomal dominant keratinizing disorder characterized by severe, painful, palmoplantar keratoderma and nail dystrophy, often accompanied by oral leucokeratosis, cysts and follicular keratosis. It is caused by mutations in one of five keratin genes: KRT6A, KRT6B, KRT6C, KRT16 or KRT17. OBJECTIVES: To identify mutations in 84 new families with a clinical diagnosis of PC, recruited by the International Pachyonychia Congenita Research Registry during the last few years. METHODS: Genomic DNA isolated from saliva or peripheral blood leucocytes was amplified using primers specific for the PC-associated keratin genes and polymerase chain reaction products were directly sequenced. RESULTS: Mutations were identified in 84 families in the PC-associated keratin genes, comprising 46 distinct keratin mutations. Fourteen were previously unreported mutations, bringing the total number of different keratin mutations associated with PC to 105. CONCLUSIONS: By identifying mutations in KRT6A, KRT6B, KRT6C, KRT16 or KRT17, this study has confirmed, at the molecular level, the clinical diagnosis of PC in these families.

Delayed-onset pachyonychia congenita associated with a novel mutation in the central 2B domain of keratin 16.

A young girl with clinical features of pachyonychia congenita type 1 was unusual in that the typical skin and nail changes were not noted until the age of 6 years. Direct sequencing of the KRT16A gene, encoding keratin K16, revealed a novel mutation K354N in the central 2B domain of the K16 polypeptide. The mutation created a new BsmI restriction site and therefore, the mutation was confirmed in the patient and excluded from both parents and 50 normal, unrelated individuals by BsmI digestion of KRT16A polymerase chain reaction products. This is the first time a mutation has been described in this location in a keratin other than K14, where similar mutations cause the milder Weber-Cockayne and/or Köbner types of epidermolysis bullosa simplex.

Photodegraded nifedipine stimulates uptake and retention of iron in human epidermal keratinocytes.

Photodegraded nifedipine has been shown to increase uptake of nontransferrin bound iron into erythroid cells. If iron could be loaded into keratinocytes, it might be possible to exploit epidermal desquamation for the purpose of eliminating potentially toxic amounts of iron from the body. We investigated the ability of photodegraded nifedipine to stimulate iron transport and accumulation in human epidermal keratinocytes. Nifedipine was degraded to its nitroso derivative by exposure to sunlight. 59Fe uptake was measured in keratinocyte monolayers, and total iron content was measured in stratified epidermal cultures. Photodegraded nifedipine increased iron uptake into keratinocytes 80-fold compared to controls. The effect of photodegraded nifedipine on iron uptake was rapid, was concentration dependent and occurred at physiologically relevant concentrations of nonprotein-bound iron. Stimulation of iron uptake by photodegraded nifedipine was independent of transferrin and worked equally well in the presence or absence of serum proteins. Iron content in keratinocytes was increased 3-fold by four daily treatments with photodegraded nifedipine. The increased iron content resulting from photodegraded nifedipine treatment was retained during a 4 d washout period. Photodegraded nifedipine may be a way delivering clinically significant amounts of iron to the epidermis.

Diagnosis of autosomal recessive lamellar ichthyosis with mutations in the TGM1 gene.

BACKGROUND: Autosomal recessive lamellar ichthyosis (ARLI) is a clinically and genetically heterogeneous disorder. In many cases, mutations in the transglutaminase 1 gene (TGM1) have been identified, however, other clinically indistinguishable cases have been linked to chromosomes 2, 3 and 19. Previous studies have failed to establish any correlation between clinical characteristics and genetic mutations. OBJECTIVES: To investigate the molecular basis of ARLI in 10 patients with the typical clinical presentation of the disorder. METHODS: We performed polymerase chain reaction and direct sequencing-based mutation screening in all of these patients, and TGM1 immunofluorescence microscopy and in vitro enzyme activity assays in selected patients. RESULTS: Mutation screening revealed 14 mutations, four of which have been previously described. While immunofluorescence microscopy was negative in patients with non-sense mutations or out-of-frame insertions or deletions, the results were variable in cases with mis-sense mutations and in cases with no mutations in the TGM1 gene. In vitro enzyme activity assays gave results consistent with the mutation data. CONCLUSIONS: Our findings support the importance of mutation screening in the evaluation of ARLI.

Expression and localization of thymidine phosphorylase/platelet-derived endothelial cell growth factor in skin and cutaneous tumors.

Thymidine phosphorylase/platelet-derived endothelial cell growth factor (TPase/PD-ECGF) is a catabolic enzyme that has been shown to be chemotactic for endothelial cells in vitro and angiogenic in vivo. TPase/PD-ECGF expression is increased in a variety of tumors. In the skin, TPase is active in normal keratinocytes in vitro and in vivo. Our objective was to study the expression and localization of TPase/PD-ECGF by immunohistochemical analysis in normal skin and cutaneous tumors and to correlate this information with enzymatic activity of TPase. TPase/PD-ECGF expression was observed in keratinocytes with intense staining of the infundibulum of hair follicles but no staining of hair bulbs. Expression localized primarily to the nucleus of keratinocytes in the basal layer but was more intense and cytoplasrmic in suprabasal keratinocytes. Increased expression of TPase/PD-ECGF in differentiated cells was confirmed by in vitro studies of TPase activity. In cutaneous tumors, there was positive staining for TPase/ PD-ECGF in squamous cell carcinomas (10/10), eccrine poromas (3/4), eccrine syringomas (4/4), trichoepitheliomas (1/3), and tumors of the follicular infundibulum (2/3) and melanomas (5/8). There was no staining of any intradermal nevi (0/2), basal cell carcinomas (0/10) or Merkel cell carcinoma (0/1). We conclude TPase/PD-ECGF is found throughout the epidermis and its expression increases with differentiation of keratinocytes. In cutaneous tumors, expression of TPase/PD-ECGF may be linked to the cell of origin of the tumor as well as the tumor's degree of differentiation.

Growth and differentiation regulate CD44 expression on human keratinocytes.

Several members of the CD44 family of hyaluronan receptors are expressed on keratinocytes. To identify factors that might be important in regulating CD44 expression, we studied CD44 expression on keratinocytes growing in vitro under a variety of conditions and on cells isolated directly from epidermis. Using Western immunoblots and metabolic labeling, we showed that the pattern of CD44 proteins expressed by keratinocytes was strongly influenced by growth and differentiation. Many protein forms of CD44 are expressed on proliferating keratinocytes in preconfluent cultures, whereas only a few forms are expressed on differentiated cells and in confluent cultures. In preconfluent monolayers, at least four splice variants were identified, including epican, CD44H, CD44E, and a 180-kDa variant. In differentiated cells or in confluent cultures, by contrast, only epican and the 180-kDa protein variant were found. Synthesis of all variants is strongly downregulated when keratinocytes become confluent or when they differentiate. Epican is the predominant form of CD44 on keratinocytes under all conditions and is expressed as a heparan, chondroitin, or keratan sulfate proteoglycan. Preconfluent basal keratinocytes, but not confluent or differentiated keratinocytes, also express chondroitin sulfate proteoglycan forms of CD44E and of the 180-kDa core protein. The modal size of the epican expressed on differentiated keratinocytes is smaller than the size of the epican expressed on basal keratinocytes. Thus, cell confluence and differentiation regulate several aspects of CD44 expression on keratinocytes, suggesting nuances in function for the different protein forms.

Bone densities in patients receiving isotretinoin for cystic acne.

BACKGROUND: Few studies have been done of bone densities in humans receiving retinoids, despite a substantial amount of literature concerning retinoid-induced osteoporosis in animals. We prospectively measured bone density and calcium metabolism in young men (aged 17-25 years) receiving oral isotretinoin for cystic acne and in a group of healthy volunteers (aged 19-26 years). OBSERVATIONS: Compared with that in healthy control subjects, mean bone density was lower at all sites (spine, femoral neck, and Ward triangle) and was considerably more variable at the spine in young men with cystic acne even before treatment. Bone density at the Ward triangle decreased a mean of 4.4% (P = .03) after 6 months of isotretinoin use (1 mg/kg of body weight). Four patients showed decreased density of more than 9% at the Ward triangle. The difference between the mean change in bone density in the patient group and in the control group was significant at the Ward triangle (P = .04) but not at the other sites. Measurements of calcium metabolism did not change over time in either group. CONCLUSIONS: A loss of bone density occurring in the absence of measurable alterations of calcium metabolism is likely to be a direct effect of retinoids on bone. Further study of retinoid-induced osteoporosis in humans and of bone density in patients with cystic acne is needed.

CD44 expression on epidermal melanocytes.

We examined CD44 expression on melanocytes to begin to understand what role CD44 might have in the normal behavior of melanocytes and to provide a basis for comparing CD44 expression in melanoma cells. CD44 was expressed on the entire surface of melanocytes and accentuated at the tips of dendritic processes. Two predominant forms of CD44 are expressed on cultured human foreskin melanocytes. One form has the covalent addition of chondroitin sulfate, whereas the other form has no chondroitin sulfate. Both use the hematopoietic, or CD44H, core protein. Using polymerase chain reaction primers that span the site where alternative splicing of CD44 occurs, we found only the cDNA coding CD44H. 12-O-Tetradecanoylphorbol 13-acetate increases the size of the chondroitin sulfate chain(s) attached to CD44 but not the proportion of CD44 molecules that carry chondroitin sulfate. Ninety percent of proteoglycans on melanocytes are chondroitin sulfate proteoglycans, and the CD44 chondroitin sulfate proteoglycan represented 10% of that total. These data show that CD44H is expressed as a "part-time" chondroitin sulfate proteoglycan on normal cultured melanocytes.

Histologic findings in acute HIV exanthem.

We describe a patient with acute HIV exanthem with papulovesicular lesions and epidermal necrosis. We also review the literature regarding the histopathologic findings of acute HIV exanthem, which appears to be most commonly characterized by a perivascular lymphocytic infiltrate without epidermal change, but which may be associated with spongiosis, vacuolar alteration, or epidermal necrosis.

Keratinocytes differentiate in response to inhibitors of deoxyribonucleotide synthesis.

We previously reported that methotrexate (MTX) caused an irreversible inhibition of growth and induced terminal differentiation of human keratinocytes. These effects of methotrexate were prevented by thymidine and thus, were attributed to depletion of thymine deoxyribonucleotides. The purpose of the research reported in this paper was to determine whether differentiation was induced by the general class of agents which are known to interfere with synthesis or utilization of deoxyribonucleotides. Agents examined included 5-fluorouracil, 5-bromodeoxyuridine, hydroxyurea, high-dose thymidine, aphidicolin, and AG#85, a newly reported thymidylate synthase inhibitor. All these agents increased the expression of involucrin and increased the amount of cornified envelope protein at doses that inhibited proliferation by > 75%. We demonstrated, however, that in our cell culture system not all conditions producing inhibition of proliferation induced differentiation; withdrawal of growth factors and supplemental amino acids inhibited proliferation but did not increase involucrin expression or production of cornified envelope protein.

Epican, a heparan/chondroitin sulfate proteoglycan form of CD44, mediates cell-cell adhesion.

Epican is a heparan/chondroitin sulfate proteoglycan form of CD44 and is expressed on the surface of keratinocytes from the basal layer to the granular layer of the epidermis. To analyze the adhesive properties of epican apart from the influence of other adhesive molecules found on keratinocytes, mouse L cell fibroblasts were transfected with CD44Epican cDNA. The epican expressed on the surface of transfected L cells was predominantly a heparan or chondroitin sulfate proteoglycan. The CD44Epican-transfected L cells acquired: (a) a self-aggregating phenotype that required hyaluronan but was calcium-independent; and (b) a new capacity to adhere to keratinocytes, a property that was blocked by an anti-epican antibody. Both aggregation and adhesion of CD44Epican-transfected cells were completely prevented by pretreatment with hyaluronidase, but were totally restored by the addition of exogenous hyaluronan. Aggregation of transfected L cells was minimally influenced by other glycosaminoglycans, but adhesion of transfected L cells to keratinocytes was substantially inhibited by heparin.

Overexpression of parathyroid hormone-related protein in the skin of transgenic mice interferes with hair follicle development.

Parathyroid hormone-related peptide (PTHrP) was initially discovered as the cause of the syndrome of humoral hypercalcemia of malignancy. Subsequently, the PTHrP gene has been shown to be expressed in a wide variety of normal tissues, including skin. Because the biological function of PTHrP in skin remains unknown, we used the human keratin 14 promoter to target overexpression of PTHrP to the skin of transgenic mice. We achieved a 10-fold level of overexpression in skin, and human keratin 14 promoter-PTHrP transgenic mice displayed a disturbance in normal hair follicle development. These mice either failed to initiate follicle development or showed a delay in the initiation of follicles. These findings suggest that PTHrP normally plays a role in the early stages of hair follicle development and support previous speculation that the peptide may function in regulating cellular differentiation.

Response of stratified cultures of human keratinocytes to disruption of proteoglycan synthesis by p-nitrophenyl-beta-D-xylopyranoside.

Proteoglycans play a role in regulating proliferation and adhesion of cells to each other and to the basal lamina. Synthesis of proteoglycans is disrupted by beta-xylosides, which serve as alternate substrate sites for glycosaminoglycan chain attachment and therefore prevent glycosylation of the core protein. We have investigated the effects of p-nitrophenyl-beta-D-xylopyranoside (PNP-xyloside) on cultured human keratinocytes. Stratified cultures were incubated for 7 days with PNP-xyloside (0.05-2.0 mM). Concentrations as low as 0.05 mM increased the secretion of free chondroitin sulfate by 10-15-fold over untreated cultures. Cell-associated proteoglycan decreased as PNP-xyloside concentration increased. At 2 mM PNP-xyloside, heparan sulfate as well as chondroitin sulfate addition to core proteins was disrupted: the core protein of epican, a heparan sulfate form of CD44 found on keratinocytes, was detected immunologically but lacked heparan sulfate. 2.0 mM PNP-xyloside reduced the number of attached cells by 20-25% after 7 days, but had little effect on morphology or protein synthesis. These results indicate that intact proteoglycans are not critical for maintaining epidermal keratinocyte stratification, cell-cell adhesion, or growth.

Dermatosparaxis in children. A case report and review of the newly recognized phenotype.

BACKGROUND: Dermatosparaxis is an autosomal recessive connective tissue disorder in animals that is caused by abnormal processing of type I procollagen and results in skin laxity and fragility. Only three humans with characteristic biochemical and electronmicroscopic findings have been recognized to date. OBSERVATIONS: We describe the clinical and electronmicroscopic findings in an affected boy who presented at birth with large full-thickness groin fissures, micrognathia, large fontanelles, umbilical hernia, and dental laminal cysts. He subsequently exhibited marked skin fragility, blue sclerae, joint laxity, increased bruisability, and growth retardation. The diagnosis of dermatosparaxis was made by electron-microscopic findings consisting of characteristic small, irregular, and circular collagen fibers in the skin. His phenotype is strikingly similar to two other reported children with the disorder, which is now classified in humans as Ehlers-Danlos VII-C. CONCLUSIONS: The newly recognized phenotype of Ehlers-Danlos VII-C is a distinct connective tissue disorder characterized by marked skin fragility and laxity, blue sclerae, increased bruisability, micrognathia, umbilical hernia, and growth retardation. A suspected clinical diagnosis can be confirmed by electron-microscopic and biochemical studies of connective tissue.

The core protein of epican, a heparan sulfate proteoglycan on keratinocytes, is an alternative form of CD44.

Epican, a heparan sulfate proteoglycan, was recently identified on the surface of keratinocytes with the aid of a monoclonal antibody to its core protein. Using that antibody to screen a human keratinocyte cDNA library, a clone encoding the entire epican core protein was selected and sequenced. The core protein of epican is a form of CD44. The deduced protein sequence of 699 amino acids has a novel 339 amino acid domain inserted into the proximal extracellular domain of the standard, leukocyte form of CD44. The additional domain adds a number of potential N- and O-linked glycosylation sites and two proteolysis sites to this form of CD44.