Jonathan L. Rees

Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease

Darier disease (DD) is an autosomal-dominant skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization. Recently we constructed a 2.4-Mb, P1-derived artificial chromosome contig spanning the DD candidate region on chromosome 12q23-24.1. After screening several genes that mapped to this region, we identified mutations in the ATP2A2 gene, which encodes the sarco/endoplasmic reticulum Ca2+ -ATPase type 2 isoform (SERCA2) and is highly expressed in keratinocytes. Thirteen mutations were identified, including frameshift deletions, in-frame deletions or insertions, splice-site mutations and non-conservative missense mutations in functional domains. Our results demonstrate that mutations in ATP2A2 cause DD and disclose a role for this pump in a Ca2+-signalling pathway regulating cell-to-cell adhesion and differentiation of the epidermis.

Evidence for variable selective pressures at MC1R.

It is widely assumed that genes that influence variation in skin and hair pigmentation are under selection. To date, the melanocortin 1 receptor (MC1R) is the only gene identified that explains substantial phenotypic variance in human pigmentation. Here we investigate MC1R polymorphism in several populations, for evidence of selection. We conclude that MC1R is under strong functional constraint in Africa, where any diversion from eumelanin production (black pigmentation) appears to be evolutionarily deleterious. Although many of the MC1R amino acid variants observed in non-African populations do affect MC1R function and contribute to high levels of MC1R diversity in Europeans, we found no evidence, in either the magnitude or the patterns of diversity, for its enhancement by selection; rather, our analyses show that levels of MC1R polymorphism simply reflect neutral expectations under relaxation of strong functional constraint outside Africa.

The Genetics of Sun Sensitivity in Humans

Humans vary >100-fold in their sensitivity to the harmful effects of ultraviolet radiation. The main determinants of sensitivity are melanin pigmentation and less-well-characterized differences in skin inflammation and repair processes. Pigmentation has a high heritability, but susceptibility to cancers of the skin, a key marker of sun sensitivity, is less heritable. Despite a large number of murine coat-color mutations, only one gene in humans, the melanocortin 1 receptor (MC1R), is known to account for substantial variation in skin and hair color and in skin cancer incidence. MC1R encodes a 317-amino acid G-coupled receptor that controls the relative amounts of the two major melanin classes, eumelanin and pheomelanin. Most persons with red hair are homozygous for alleles of the MC1R gene that show varying degrees of diminished function. More than 65 human MC1R alleles with nonsynonymous changes have been identified, and current evidence suggests that many of them vary in their physiological activity, such that a graded series of responses can be achieved on the basis of (i) dosage effects (of one or two alleles) and (ii) individual differences in the pharmacological profile in response to ligand. Thus, a single locus, identified within a Mendelian framework, can contribute significantly to human pigmentary variation.

A retinoic acid response element is present in the mouse cellular retinol binding protein I (mCRBPI) promoter.

Genomic and cDNA sequences for the mouse cellular retinol binding protein I (mCRBPI) are presented. A specific cis‐acting element responsible for retinoic acid (RA) inducibility of the mCRBPI promoter was identified and characterized. Deletion mapping of a CRBPI promoter‐‐chloramphenicol acetyltransferase reporter gene construct localized this element to a 259 bp restriction fragment located approximately 1 kb upstream from the transcription start‐site. A sequence closely resembling the previously characterized RA response element (RARE) of the RA receptor beta 2 (RAR‐beta 2) promoter, and consisting of a direct repeat of the motif 5′‐GGTCA‐3′ separated by three nucleotides, was found within this restriction fragment. Mutation of these 5′‐GGTCA‐3′ motifs to GGAGC and GGGGC abolished RA‐inducible transcription whereas a mutation to a direct repeat of the GTTCA motif found in the RARE of the RAR‐beta 2 promoter resulted in enhanced inducibility. Oligonucleotides containing the direct repeat of the GGTCA motif were able to confer RA‐dependent transcriptional enhancement to the herpes simplex thymidine kinase promoter, as well as to bind directly all three retinoic acid receptors (RARs) alpha, beta and gamma, as determined by gel retardation/shift assays. The control of CRBPI gene transcription by RA‐RAR complexes interacting with the RARE characterized here may correspond to a feedback mechanism important in regulating retinoid metabolism and action.

p53 protein expression in benign and malignant skin tumours

The skiu affords an excellent model of human carcinogenesis because a variety of lesions from benign tumours to invasive malignancy, with or without metastatic potential, are commonly found, and are accessible to biopsy. To date, few genetic alterations have been observed in skin neoplasia. In this study we have used a recently developed monoclonal antibody (DO7) to examine p53 protein expression in a wide variety of benign and malignant skin lesions. Benign skin lesions were negative, but a significant number of malignant epithelial lesions showed detectable p53: 56% of squamous carcinomas and 42% of basal cell carcinomas were positive. A smaller proportion of dysplastic epithelial lesions were positive (27%). and only 3.6% of malignant melanomas were positive. Thus, although detectable p53 protein is a common occurrence in malignant epithelial lesions, it does not correlate with the malignant phenotype or with metastatic potential. The finding of a lower proportion of positivity in dysplastic lesions. and absence of staining in benign tumours, suggests that p53 mutation may be involved in the progression towards invasive malignancy in human squamous skin lesions.

Prognostic significance of allelic losses in primary melanoma.

Loss of genetic material, including loss of loci on chromosome arms 6q, 9p, and 10q, occurs frequently in cutaneous melanoma but infrequently in benign melanocytic nevi or other melanocytic lesions, suggesting that these genetic alterations are important in the development and progression of melanoma. To examine whether allelic loss is of prognostic importance in melanoma, disease-free survival was related to loss of heterozygosity on 6q, 9p and 10q in 83 individuals with sporadic primary cutaneous melanoma. Loss of chromosome arms 6q and 10q were each significantly associated with a poorer clinical outcome (P=0.013 and P=0.001 respectively). In a subgroup of 41 subjects whose primary tumours were allelotyped, the fractional allelic loss (FAL) at 39 autosomal arms also significantly correlated with disease-free survival (P=0.013), with an increase in FAL associated with a poorer outcome; this association remained significant when controlled for tumour thickness (P=0.035). In addition, a greater proportion of cells were immunopositive for Ki67 antigen, p53 and p21WAF1 protein in the primary melanomas than in the benign melanocytic nevi, however, only p53 over-expression was significantly associated with improved survival (P=0.041).

The relation between p53 mutation and p53 immunostaining in non-melanoma skin cancer

Extensive study of the p53 gene has established its role as a tumour‐suppressor gene, and the involvement of mutant p53 in a wide spectrum of human malignancy. Many mutations of p53 result in a protein product that is abnormally stable, so that it becomes readily detectable by immunocytochemistry. In contrast, under normal conditions, it has been considered that levels of wild‐type p53 were too low to be detectable. Although positive immunocytochemistry has been used as a marker of mutation, recent evidence suggests that this assumption may not always be valid. We have carried out both PCR‐sequencing of exons 5‐8 of the p53 gene in 20 basal cell carcinomas (BCC), and immunocytochemistry of these tumours with the anti‐p53 antibody Do7. Twenty cases of Bowens disease, in which we had previously documented mutations, were also immunostained. We report a low rate of p53 mutation in the BCCs we examined (2/20), and a discrepancy between tumours with positive immunostaining and those with mutation in both Bowens disease and BCC. Of eight tumours in which we detected mutation, only four were immunopositive; of 19 immunopositive samples, only four showed detectable mutation. We discuss the implications of our results for the use of positive immunostaining in clinical diagnosis, and the involvement of p53 in skin carcinogenesis.

The relation between melanocortin 1 receptor (MC1R) variation and the generation of phenotypic diversity in the cutaneous response to ultraviolet radiation

The melanocortin 1 receptor (MC1R) is known to play an important role in determining physiological variation in human pigmentation, and consequently human susceptibility to ultraviolet radiation. A reason for wider interest is that the considerable phenotypic diversity has been in part generated by the effects of gene dosage, and the presence of a large number of mutations at this G-protein coupled receptor that are not functionally equivalent. Thus, a range of mutations at a single receptor locus can lead to a complex range of graded phenotypes.

Sex differences in susceptibility to development of contact hypersensitivity to dinitrochlorobenzene (DNCB)

We have investigated the differences between the sexes in the development of contact sensitivity induced by dinitrochlorobenzene (DNCB). Ten male and 12 female subjects were sensitized with DNCB (30 μg applied on a 1 cm patch test disc) and challenged 1 month later with doses of 8.8, 12.5, 17.7 and 25 μg. The responses were measured after 48 h as increase in skinfold thickness with Harpenden callipers. Females showed a larger response at all challenge doses studied, and the slope of the log‐dose response curve was significantly steeper in females. We conclude that there are significant differences in delayed type hypersensitivity between males and females.

Variation in Epidermal Morphology in Human Skin at Different Body Sites as Measured by Reflectance Confocal Microscopy

Two methods of estimating stratum corneum thickness using reflectance confocal microscopy were examined, and epidermal thickness measurements at multiple body sites were compared. Measurements of stratum corneum thickness were made using the derivative method, which is based on the rate of change of image intensity as a proxy for keratin concentration, and simple visual analysis of confocal images. To compare epidermal thickness we collected 1491 z-axis stacks of confocal images from 10 body sites in 39 subjects. An artefact associated with the imaging process interfered with the derivative method for stratum corneum thickness, and simple visual analysis is to be preferred. Although some epidermal properties varied by site, the most striking finding was the degree of within-site variation, which accounted for between 50% and 74% of the total variation observed. The majority of this variation was not due to measurement error, and represents genuine topographical irregularity. This fine-scale variation limits the ease of use of reflectance confocal microscopy for quantitative studies of the epidermis and stratum corneum.