We analyzed the role of genetic variation in the histidase gene (HAL) in altering the risk of NMSCs and found evidence of effects in response to environmental and innate exposures, such as UV radiation, glucocorticoids and estrogen. Specifically, a high number of lifetime sunburns, indicative of intense UV exposure, increased risk of NMSCs in the presence of the variant G allele. Pigmentation did not modify the effects of HAL genotype on risk of skin cancer. Additional interactions were found with estrogen and glucocorticoids, both transcriptional activators of histidase expression. Glucocorticoid users had an increased risk of SCC in the presence of the variant G allele, as did women and those with a history of OC usage.
To our knowledge, the functionality of this SNP has not been studied previously in either humans or animal models. While the change from an isoleucine to a valine residue is relatively conservative, it is plausible that the amino acid change alters substrate affinity in the catalytic binding pocket. Nearby amino acid 444 is a highly conserved phenylalanine group present in humans, rats and mice, as well as plant, yeast and bacterial strains such as Pseudomonas putida
). In experiments on histidase from P.putida
, mutation of this phenylalanine results in a 100 to 2500-fold decrease in activity, as well as a 4-fold decrease in affinity for histidine (25
). Based on the crystal structure, phenylalanine 444 is hypothesized to be involved in π-stacking interactions with a catalytic intermediate, resulting in stabilization of that intermediate to allow extraction of the appropriate β-proton (25
). There is also evidence that loss of this residue results in improper formation of the 4-methylidene-imidazol-5-one group (25
), the active site of this enzyme. In light of the importance of the positioning of this residue, an alteration in a neighboring amino acid such as I439V could potentiate a slight repositioning of F444, resulting in altered enzyme kinetics. The structure prediction software PMut (http://mmb2.pcb.ub.es:8080/PMut/
) was used to estimate the effects this amino acid change would have on protein structure, based on sequence information. The program predicted this coding change to be neutral, suggesting that I439V may not be the causal SNP for the associations found in this study with NMSC. However, these results must be interpreted with caution, as the PMut software only has a 66.5% rate of improvement over random with sequence data (28
). While this is one of the best programs currently available, the results are not definitive.
Whereas I439V is the only non-synonymous coding SNP, HapMap lists at least 40 synonymous coding or non-coding SNPs with minor allele frequencies
0.05 in the 32.2 kb region (nucleotides 94864874–94897073) encoding for and surrounding HAL
. It remains possible that I439V may not be directly causing the effect, but may instead be linked to another functional SNP in the untranslated region, promoter or intronic regions. The PMut prediction of a neutral substitution supports this theory. Also, the interactions we have found between genotype and two transcriptional inducers of HAL
could be due to a linked promoter SNP as the causal variant.
studies have shown that increased exposure to UV directly correlates with increased UCA isomerization rates (29
). As isomerization is a key on/off switch controlling production of cis
-UCA, intense sun exposure could create higher epidermal concentration of cis
-UCA and a consequent immunosuppressed state. Our results from the combined effects of HAL
genotype and lifetime number of severe sunburns support this. If a high lifetime number of severe sunburns is considered a marker of intense UV exposure, our data show a gene dosage effect with increasing numbers of the variant G allele for both BCC and SCC among those with intense exposure. Therefore, our results suggest a role for intense, intermittent sun exposures in the etiology of UV-induced immunosuppression and NMSC.
The lack of biologic interaction between pigmentation and genotype agrees with previous literature that has shown a lack of effect of pigmentation on UV-induced immunosuppression. Vermeer et al.
) found that UV-B susceptibility was a polymorphic trait that occurred at similar frequencies in Caucasian skin, heavily tanned skin and genetically determined black skin. This seems logical when considering the distribution of melanin among the layers of the epidermis. While melanin forms crescent-shaped vesicles over the nucleus of basal keratinocytes, they are degraded throughout the differentiation process, appearing as sparse particles in the uppermost stratum corneum (30
). This degradation process most probably impairs the ability of melanin to effectively absorb UV in this top layer of the skin, allowing UCA photoisomerization to occur regardless of pigmentation.
Numerous studies in rats have shown a strong enhancement of hepatic histidase expression in the presence of estrogen (16
), with postpubescent females possessing greatly increased levels of histidase throughout adulthood, compared with male counterparts. However, little research has been devoted to confirming estrogenic regulation in the skin. Estrogen receptors, specifically estrogen receptor-β, are expressed in human keratinocytes (31
), and estrogen is known to promote proliferation of keratinocytes, wound healing and vascularization of skin (reviewed in ref. 34
). However, the effects of estrogen on epidermal histidase have not been well characterized. Only one study has been performed on rat skin, showing decreased levels of histidase activity in adult females compared with adult males (35
). However, epidermal thickness and stage of the hair follicle cycle were not accounted for in this study. As these correlate to the pattern of histidase activity, it is possible that one or both could be confounding the observed gender differences. Since there is a lack of validation of these findings in humans, the regulation of histidase by estrogen in the skin remains controversial. However, our findings lend support to the theory that human epidermal transcription of HAL
may be regulated similarly to that of rat hepatic HAL
Similarly, studies in male rat livers show that glucocorticoids, specifically hydrocortisone acetate, are effective in elevating histidase levels (17
). No studies performed in any model system have examined epidermal regulation of histidase by glucocorticoids. Therefore, studies into regulation of histidase by both estrogen and glucocorticoids in human keratinocytes are necessary to confirm the results found in rats and in our study.
While the effects of sunburns in combination with I439V seem to be similar between the two types of NMSC, differential effects on SCC and BCC appear to occur in the presence of estrogen or glucocorticoids and HAL
genotype. This corroborates with other epidemiological data that transplant patients on immunosuppressive therapy are more likely to develop SCCs than BCCs (36
), the latter of which are more prevalent in the general population. Additionally, OC usage has been shown in this case–control population to increase risk of SCC but not BCC (38
). However, there is currently no known mechanism for these differences between the histologies.
A limitation of this study worth noting is that many variables used are self-reported. Therefore, the possibility of reporting bias, particularly with sun exposure variables, exists. One Australian study found little evidence of systematic changes in recall of severe sunburn history, when participants were surveyed repeatedly (39
). Also, intraclass correlation coefficients did not significantly differ between cases and controls in this study. Additionally, for self-reporting of OC usage, Nischan et al
) showed in a breast cancer case–control population that there was no differential reporting of OC usage by case status. Finally, a previous study of this population showed that equal numbers of cases and controls reported using inhaled glucocorticoid steroids, implying that recall of oral steroid usage is probably also not related to case status (41
NMSC has an estimated incidence of >1 million cases per year in the USA (42
), and it is also one of the top five most costly cancers to Medicare, with treatment costs of over half a billion dollars in 1994–1995 (43
). This cost was up 41% from 1992 to 1993 (43
), and that number can be expected to grow as the ‘Baby Boomer’ generation reaches the peak age of onset. Additionally, ~11 million women in the USA are currently using OCs (44
), and another 10 million new prescriptions for oral corticoid steroids for men and women are written each year to treat a variety of inflammatory diseases (reviewed in ref. 45
). With such widely prevalent exposures and such a high morbidity rate of this disease, understanding the underlying susceptibilities and targeting at-risk populations early may help alleviate some of this burden. This highlights the importance of genetic susceptibility studies, such as this one, to understanding the etiology of NMSC and UV-induced immunosuppression. This research may have impact beyond even skin cancer as UV-induced immunosuppression has been associated with decreased pathogen response (46
), interferes with vaccine efficacy (51
) and has even been hypothesized to accelerate the onset and progression of AIDS in HIV-infected individuals (52
). Given the potential scope of effects UV-induced immunosuppression has, further investigation of its genetic basis is crucial.