John J. DiGiovanna

Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis

Atopic disease, including atopic dermatitis (eczema), allergy and asthma, has increased in frequency in recent decades and now affects ∼20% of the population in the developed world. Twin and family studies have shown that predisposition to atopic disease is highly heritable. Although most genetic studies have focused on immunological mechanisms, a primary epithelial barrier defect has been anticipated. Filaggrin is a key protein that facilitates terminal differentiation of the epidermis and formation of the skin barrier. Here we show that two independent loss-of-function genetic variants (R510X and 2282del4) in the gene encoding filaggrin (FLG) are very strong predisposing factors for atopic dermatitis. These variants are carried by ∼9% of people of European origin. These variants also show highly significant association with asthma occurring in the context of atopic dermatitis. This work establishes a key role for impaired skin barrier function in the development of atopic disease.

Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris.

Ichthyosis vulgaris (OMIM 146700) is the most common inherited disorder of keratinization and one of the most frequent single-gene disorders in humans. The most widely cited incidence figure is 1 in 250 based on a survey of 6,051 healthy English schoolchildren. We have identified homozygous or compound heterozygous mutations R501X and 2282del4 in the gene encoding filaggrin (FLG) as the cause of moderate or severe ichthyosis vulgaris in 15 kindreds. In addition, these mutations are semidominant; heterozygotes show a very mild phenotype with incomplete penetrance. The mutations show a combined allele frequency of ∼4% in populations of European ancestry, explaining the high incidence of ichthyosis vulgaris. Profilaggrin is the major protein of keratohyalin granules in the epidermis. During terminal differentiation, it is cleaved into multiple filaggrin peptides that aggregate keratin filaments. The resultant matrix is cross-linked to form a major component of the cornified cell envelope. We find that loss or reduction of this major structural protein leads to varying degrees of impaired keratinization.

Prevention of Skin Cancer in Xeroderma Pigmentosum with the Use of Oral Isotretinoin

To confirm reports that skin cancer can be prevented with retinoids, we conducted a three-year controlled prospective study of oral isotretinoin (also called 13-cis retinoic acid) in five patients with xeroderma pigmentosum who had a history of multiple cutaneous basal-cell or squamous-cell carcinomas. Patients were treated with isotretinoin at a dosage of 2 mg per kilogram of body weight per day for two years and then followed for an additional year, without the drug. Before, during, and after treatment, biopsies of all suspicious lesions were performed, and skin cancers were surgically removed. The patients had a total of 121 tumors (mean, 24; range, 8 to 43) in the two-year interval before treatment. During two years of treatment with isotretinoin, there were 25 tumors (mean, 5; range, 3 to 9), with an average reduction in skin cancers of 63 percent (P = 0.019). After the drug was discontinued, the tumor frequency increased a mean of 8.5-fold (range, 2- to 19-fold) over the frequency during treatment (P = 0.007). Although all patients experienced mucocutaneous toxic effects, and triglyceride, liver-function, or skeletal abnormalities developed in some, high-dose oral isotretinoin was effective in the chemoprophylaxis of skin cancers in patients with xeroderma pigmentosum.

Developmental defects in gorlin syndrome related to a putative tumor suppressor gene on chromosome 9

Gorlin syndrome is an autosomal dominant disorder that predisposes to basal cell carcinomas of the skin, ovarian fibromas, and medulloblastomas. Unlike other hereditary disorders associated with cancer, it features widespread developmental defects. To investigate the possibility that the syndrome is caused by mutation in a tumor suppressor gene, we searched for loss of heterozygosity in 16 sporadic basal cell carcinomas, 2 hereditary basal cell carcinomas, and 1 hereditary ovarian fibroma and performed genetic linkage studies in five Gorlin syndrome kindreds. Eleven sporadic basal cell carcinomas and all 3 hereditary tumors had allelic loss of chromosome 9q31, and all informative kindreds showed tight linkage between the Gorlin syndrome gene and a genetic marker in this region. Loss of heterozygosity at this chromosomal location, particularly in hereditary tumors, implies that the gene is homozygously inactivated and normally functions as a tumor suppressor. In contrast, hemizygous germline mutations lead to multiple congenital anomalies.

Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis

Erythrokeratodermia variabilis (EKV, OMIM 133200) is an autosomal dominant genodermatosis with considerable intra- and interfamilial variability. It has a disfiguring phenotype characterized by the independent occurrence of two morphologic features: transient figurate red patches and localized or generalized hyperkeratosis (Fig. 1). Both features can be triggered by external factors such as trauma to the skin. After initial linkage to the RH locus on 1p (Refs 2,3), EKV was mapped to an interval of 2.6 cM on 1p34-p35, and a candidate gene (GJA4) encoding the gap junction protein α-4 (connexin 31, Cx31) was excluded by sequence analysis. Evidence in mouse suggesting that the EKV region harbours a cluster of epidermally expressed connexin genes led us to characterize the human homologues of GJB3 (encoding Cx31) and GJB5 (encoding Cx31.1). GJB3, GJB5 and GJA4 were localized to a 1.1-Mb YAC in the candidate interval. We detected heterozygous missense mutations in GJB3 in four EKV families leading to substitution of a conserved glycine by charged residues (G12R and G12D), or change of a cysteine (C86S). These mutations are predicted to interfere with normal Cx31 structure and function, possibly due to a dominant inhibitory effect. Our results implicate Cx31 in the pathogenesis of EKV, and provide evidence that intercellular communication mediated by Cx31 is crucial for epidermal differentiation and response to external factors.

Missense mutations in GJB2 encoding connexin-26 cause the ectodermal dysplasia keratitis-ichthyosis-deafness syndrome.

Keratitis-ichthyosis-deafness syndrome (KID) is a rare ectodermal dysplasia characterized by vascularizing keratitis, profound sensorineural hearing loss (SNHL), and progressive erythrokeratoderma, a clinical triad that indicates a failure in development and differentiation of multiple stratifying epithelia. Here, we provide compelling evidence that KID is caused by heterozygous missense mutations in the connexin-26 gene, GJB2. In each of 10 patients with KID, we identified a point mutation leading to substitution of conserved residues in the cytoplasmic amino terminus or first extracellular domain of Cx26. One of these mutations was detected in six unrelated sporadic case subjects and also segregated in one family with vertical transmission of KID. These results indicate the presence of a common, recurrent mutation and establish its autosomal dominant nature. Cx26 and the closely related Cx30 showed differential expression in epidermal, adnexal, and corneal epithelia but were not significantly altered in lesional skin. However, mutant Cx26 was incapable of inducing intercellular coupling in vitro, which indicates its functional impairment. Our data reveal striking genotype-phenotype correlations and demonstrate that dominant GJB2 mutations can disturb the gap junction system of one or several ectodermal epithelia, thereby producing multiple phenotypes: nonsyndromic SNHL, syndromic SNHL with palmoplantar keratoderma, and KID. Decreased host defense and increased carcinogenic potential in KID illustrate that gap junction communication plays not only a crucial role in epithelial homeostasis and differentiation but also in immune response and epidermal carcinogenesis.

Xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome: a complex genotype-phenotype relationship.

Patients with the rare genetic disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS) have defects in DNA nucleotide excision repair (NER). The NER pathway involves at least 28 genes. Three NER genes are also part of the basal transcription factor, TFIIH. Mutations in 11 NER genes have been associated with clinical diseases with at least eight overlapping phenotypes. The clinical features of these patients have some similarities but also have marked differences. NER is involved in protection against sunlight-induced DNA damage. While XP patients have 1000-fold increase in susceptibility to skin cancer, TTD and CS patients have normal skin cancer risk. Several of the genes involved in NER also affect somatic growth and development. Some patients have short stature and immature sexual development. TTD patients have sulfur deficient brittle hair. Progressive sensorineural deafness is an early feature of XP and CS. Many of these clinical diseases are associated with developmental delay and progressive neurological degeneration. The main neuropathology of XP is a primary neuronal degeneration. In contrast, CS and TTD patients have reduced myelination of the brain. These complex neurological abnormalities are not related to sunlight exposure but may be caused by developmental defects as well as faulty repair of DNA damage to neuronal cells induced by oxidative metabolism or other endogenous processes.

Suppression of frequently recurring genital herpes: a placebo-controlled double-blind trial of oral acyclovir

We studied 35 otherwise healthy adults with frequently recurring genital herpes (greater than or equal to 1 episode per month), in a double-blind trial comparing oral acyclovir with placebo capsules for suppression of recurrent infection. The patients were treated for 125 days unless herpes recurred. Among 32 evaluable patients, there were significantly fewer recurrences during acyclovir treatment (4 of 16) than during placebo treatment (16 of 16, P less than 0.001). The mean duration of therapy was significantly longer for patients receiving acyclovir than for those receiving placebo (114.9 vs. 24.8 days, P less than 0.001). Of 19 patients who had recurrences in the blind trial, only 2 had recurrences when given acyclovir in a second, open-study phase. All patients had recurrences after completing acyclovir treatment. The therapy was well tolerated, with minimal gastrointestinal upset and one hypersensitivity reaction. Studies of the viral isolates demonstrated that lesions developing in patients receiving acyclovir contained drug-resistant virus. Later recurrences in these patients were associated with drug-sensitive virus. We conclude that oral acyclovir suppresses genital herpes in patients with frequent recurrences, but the potential for problems with drug resistance and the long-term safety need to be more fully explored.

A leucine→proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis

Epidermolytic hyperkeratosis is an autosomal dominant disorder affecting the structural integrity of the suprabasal layers of human epidermis. We have recently documented in one family linkage of the disease phenotype to the cluster of type II keratins. We have now identified a leucine----proline amino acid substitution in the conserved H1 subdomain of keratin 1 that is present only in affected family members. Using a quantitative assay and electron microscopy with synthetic peptides, we show that, whereas the wild-type H1 peptide rapidly disassembles preformed keratin filaments in vitro, the mutant peptide does this far less efficiently. Therefore the mutation in keratin 1 is likely to cause defective keratin filaments and hence a defective cytoskeleton in the epidermal cells in vivo.

Shining a light on xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is a rare, autosomal recessive disorder of DNA repair characterized by sun sensitivity and ultraviolet (UV) induced skin and mucous membrane cancers. Described in 1874 by Moriz Kaposi in Vienna, nearly 100 years later James Cleaver in San Francisco reported defective DNA repair in XP cells. This eventually provided the basis for a mechanistic link between sun exposure, DNA damage, somatic mutations and skin cancer. XP cells were found to have defects in 7 of the proteins of the nucleotide excision repair pathway and in DNA polymerase eta. XP cells are hypersensitive to killing by UV and XP cancers have characteristic “UV signature” mutations. Clinical studies at NIH found a nearly 10,000-fold increase in skin cancer in XP patients under age 20 years demonstrating the substantial importance of DNA repair in cancer prevention in the general population. About 25 % of XP patients have progressive neurological degeneration with progressive loss of neurons, probably from DNA damage induced by oxidative metabolism which kills non-dividing cells in the nervous system. Interestingly, patients with another disorder, trichothiodystrophy have defects in some of the same genes as XP but they have primary developmental abnormalities without an increase in skin cancer.