Pachyonychia congenita (PC) is a group of autosomal dominant disorders characterized by dystrophic nails and other ectodermal aberrations. A gene for Jackson-Lawler PC was recently mapped to the type I keratin cluster on 17q. Here, we show that a heterozygous missense mutation in the helix initiation motif of K17 (Asn92Asp) co-segregates with the disease in this kindred. We also show that Jadassohn-Lewandowsky PC is caused by a heterozygous missense mutation in the helix initiation peptide of K16 (Leu130Pro). The known expression patterns of these keratins in epidermal structures correlates with the specific abnormalities observed in each form of PC.
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 patient's siRNA-treated foot appears sufficiently promising to warrant additional studies of siRNA in this and other dominant-negative skin diseases.
Keratins K6 and K16 are expressed in suprabasal interfollicular epidermis in wound healing and other pathological conditions associated with hyperproliferation, such as psoriasis and are induced when keratinocytes are cultured in vitro. However, these keratins are also constitutively expressed in normal suprabasal mucosal and palmoplantar keratinocytes. Mutations in keratins have been reported in the basal keratin pair K5 and K14 in epidermolysis bullosa simplex and in suprabasal epidermal keratins K1, K2 and K10 in epidermolytic ichthyoses. Two families with autosomal dominant disorder of focal non epidermolytic palmoplantar keratoderma, have oral mucosal and follicular lesions in addition to the palmoplantar hyperkeratosis. Previous studies have shown linkage in these families to the type I keratin gene cluster at 17q12-q21 and this report shows that the cDNA of affected members of both families have novel heterozygous mutations in the expressed keratin 16 gene. These mutations (R10C and N8S) lie in the helix initiation motif of the 1A domain. These mutations do not appear to cause epidermolysis on light or electron microscopy, which may reflect differences in function, assembly or interaction of the 'hyperproliferative' or 'mucoregenerative' keratins from other major types of keratins. The mutations reported here are the first to describe the molecular pathology of focal non epidermolytic palmoplantar keratoderma.
CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.
White sponge nevus (WSN) is a benign autosomal dominant disorder which affects non-cornifying stratified squamous epithelia (MIM 193900) (ref. 1). Phenotypically it presents as white 'spongy' plaques (oral leukokeratoses), most commonly in the mouth but also reported in the esophagus and anogenital mucosa. Histologically, the plaques show evidence of hyperproliferation, acanthosis and tonofilament aggregation. These types of pathogenic changes are characteristic of many of the epidermal keratin disorders. Keratins are expressed in pairs by epithelial cells in a tissue and cell specific manner. The major differentiation specific keratins of the buccal mucosa, nasal, esophageal and anogenital epithelia are K4 and K13 (ref. 7). The tissue distribution and nature of the lesions in patients affected by WSN suggested that mutations in K4 and/or K13 might be responsible for this disorder. We have now confirmed this hypothesis and report here a three base-pair (bp) deletion in the helix initiation peptide of K4 in affected members from two families with this condition.
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