Leonard M. Milstone

First-in-human Mutation-targeted siRNA Phase Ib Trial of an Inherited Skin Disorder

The rare skin disorder pachyonychia congenita (PC) is an autosomal dominant syndrome that includes a disabling plantar keratoderma for which no satisfactory treatment is currently available. We have completed a phase Ib clinical trial for treatment of PC utilizing the first short-interfering RNA (siRNA)-based therapeutic for skin. This siRNA, called TD101, specifically and potently targets the keratin 6a (K6a) N171K mutant mRNA without affecting wild-type K6a mRNA. The safety and efficacy of TD101 was tested in a single-patient 17-week, prospective, double-blind, split-body, vehicle-controlled, dose-escalation trial. Randomly assigned solutions of TD101 or vehicle control were injected in symmetric plantar calluses on opposite feet. No adverse events occurred during the trial or in the 3-month washout period. Subjective patient assessment and physician clinical efficacy measures revealed regression of callus on the siRNA-treated, but not on the vehicle-treated foot. This trial represents the first time that siRNA has been used in a clinical setting to target a mutant gene or a genetic disorder, and the first use of siRNA in human skin. The callus regression seen on the patients siRNA-treated foot appears sufficiently promising to warrant additional studies of siRNA in this and other dominant-negative skin diseases.

Mitotic Recombination in Patients with Ichthyosis Causes Reversion of Dominant Mutations in KRT10

Gaining from a Loss Mitotic recombination can cause a cell carrying heterozygous mutations in a tumor suppressor gene to lose the wild-type copy of the gene, setting the cell on the pathway to uncontrolled growth. But can mitotic recombination have beneficial effects in other settings—that is, lead to phenotypic correction of a diseased cell by facilitating loss of the disease-causing mutation? Choate et al. (p. 94, published online 26 August; see the Perspective by Davis and Candotti) now find evidence for this type of event in a rare skin disease called ichthyosis with confetti (IWC). Patients with IWC display severe scaling of the skin but have widespread patches of normal skin that reflect clonal expansion of revertant cells. The revertant cells showed loss of heterozygosity on chromosome 17q and, as a result of mitotic recombination, these cells selectively lost dominant disease-causing mutations in the keratin 10 gene (KRT10), but retained the wild-type copy of the gene. Patches of normal skin in patients with a rare skin disorder are due to cells that have lost the disease-causing mutation. Somatic loss of wild-type alleles can produce disease traits such as neoplasia. Conversely, somatic loss of disease-causing mutations can revert phenotypes; however, these events are infrequently observed. Here we show that ichthyosis with confetti, a severe, sporadic skin disease in humans, is associated with thousands of revertant clones of normal skin that arise from loss of heterozygosity on chromosome 17q via mitotic recombination. This allowed us to map and identify disease-causing mutations in the gene encoding keratin 10 (KRT10); all result in frameshifts into the same alternative reading frame, producing an arginine-rich C-terminal peptide that redirects keratin 10 from the cytokeratin filament network to the nucleolus. The high frequency of somatic reversion in ichthyosis with confetti suggests that revertant stem cell clones are under strong positive selection and/or that the rate of mitotic recombination is elevated in individuals with this disorder.

Premature epiphyseal closure in a child receiving oral 13-cis-retinoic acid

A boy with epidermolytic hyperkeratosis was treated systemically for 4 1/2 years with 13-cis-retinoic acid. At the age of 10 1/2 years, he developed pain in his right knee and radiographic evidence of partial closure of the proximal epiphysis of the right tibia. Similar radiographic changes have been described in individuals ingesting excessive amounts of vitamin A.

Native and a synthetic analogue of the malignancy-associated parathyroid hormone-like protein have in vitro transforming growth factor-like properties.

A human parathyroid-like protein (PLP) has recently been isolated and cloned from human tumors associated with the paraneoplastic syndrome, humoral hypercalcemia of malignancy. PLP shares NH2-terminal amino acid sequence similarity with PTH but has a unique primary structure thereafter. Studies reported to date have indicated that both native and synthetic amino-terminal PLP polypeptides display actions in vivo and in vitro that are similar to those of PTH. We report here that purified native PLP and synthetic 36Tyr(1-36)amide human PLP induce epidermal growth factor-dependent transformation of NRK 49F cells in soft agar. Further, the synthetic peptide induces a significant increase in the biosynthesis of fibronectin by human dermal fibroblasts. (1-34)PTH does not display either of these biological activities. These data indicate that there are qualitative differences between PTH and the recently identified PLP. The latter hormone appears to possess transforming growth factor-like properties that may be relevant to its physiological actions.

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.

A Large Mutational Study in Pachyonychia Congenita

Pachyonychia congenita (PC) is a rare autosomal dominant skin disorder characterized predominantly by nail dystrophy and painful palmoplantar keratoderma. Additional clinical features include oral leukokeratosis, follicular keratosis, and cysts (steatocysts and pilosebaceous cysts). PC is due to heterozygous mutations in one of four keratin genes, namely, KRT6A, KRT6B, KRT16, or KRT17. Here, we report genetic analysis of 90 new families with PC in which we identified mutations in KRT6A, KRT6B, KRT16, or KRT17, thereby confirming their clinical diagnosis. A total of 21 previously unreported and 22 known mutations were found. Approximately half of the kindreds had mutations in KRT6A (52%), 28% had mutations in KRT16, 17% in KRT17, and 3% of families had mutations in KRT6B. Most of the mutations were heterozygous missense or small in-frame insertion/deletion mutations occurring within one of the helix boundary motif regions of the keratin polypeptide. More unusual mutations included heterozygous splice site mutations, nonsense mutations, and a 1-bp insertion mutation, leading to a frameshift and premature termination codon. This study, together with previously reported mutations, identifies mutation hotspot codons that may be useful in the development of personalized medicine for PC.

Systemic retinoids in the management of ichthyoses and related skin types

The term retinoid includes both natural and synthetic derivatives of vitamin A. Retinoid‐containing treatments have been used since ∼1550BC by the early Egyptians. Treatment of ichthyosiform disorders with retinoids dates back at least to the 1930s. Early use of high‐dose vitamin A demonstrated efficacy, but because vitamin A is stored in the liver, toxicity limited usefulness. Interest turned to synthetic retinoids in an effort to enhance efficacy and limit toxicity. Acetretin, isotretinoin and, in the past etretinate, have provided the most effective therapy for ichthyosiform conditions. They have been used for a variety of ages, including in newborns with severe ichthyosis and for decades in some patients. Careful surveillance and management of mucous membrane, laboratory, skeletal, and teratogenic side effects has made systemic retinoids the mainstay of therapy for ichthyosis and related skin types.

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 cytoplasmic 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 tumors degree of differentiation.

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.

Growth and differentiation regulate CD44 expression on human keratinocytes.

SummarySeveral 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.