Filaggrin genotype does not determine the skin's threshold to UV-induced erythema.

To the Editor:

Profilaggrin and filaggrin play multiple roles in the formation and function of the epidermal barrier, contributing to protection against dehydration, mechanical stress, infection, and, it has been proposed, photodamage. Loss-of-function mutations in the gene encoding filaggrin (FLG) represent the strongest and most significant genetic risk factor for atopic dermatitis (AD) identified to date. Proteolysis of filaggrin releases histidine and other amino acids into the stratum corneum. Histidine is converted by the enzyme histidase (histidine ammonia-lyase) to trans-urocanic acid (trans-UCA), which can then undergo photoisomerization on absorption of UVB to produce cis-UCA (see Fig E1 in this article's Online Repository at www.jacionline.org). There is experimental evidence to suggest that cis-UCA has immunomodulatory and photoprotective effects. The local and systemic immunosuppressive effects of cis-UCA were initially demonstrated in murine models, and more recently, histidinemic mice deficient in cutaneous UCA because of a mutation in Hal, the gene encoding histidase, have been reported to show increased propensity to UVB-induced DNA damage. Mice deficient in caspase-14 (an enzyme in the profilaggrin-filaggrin proteolytic pathway) show accumulation of cyclobutane pyrimidine dimers in response to UVB radiation and increased apoptosis in the epidermis, indicating a role for caspase-14 in UVB scavenging within the stratum corneum. The immunosuppressive effects of cis-UCA have been demonstrated in human keratinocytes and leukocytes in vitro; knockdown of FLG in organotypic culture results in increased susceptibility of keratinocytes to UV-induced apoptosis. Loss-of-function mutations and copy number variation in FLG are known to result in lower levels of filaggrin breakdown products, including UCA, in human stratum corneum. Therefore it has been postulated that FLG genotype might in part determine the photoprotective capacity of human skin (see Fig E1), but experimental evidence in vivo is lacking.

Fig E1

Diagrammatic summary of factors affecting the profilaggrin–cis-UCA pathway

Previous studies have demonstrated the effects of variation within FLGE6, E7, E8 and levels of TH2 cytokinesE10, E9 on filaggrin expression. The role of caspase-14 in profilaggrin processing has been illustrated in mice. Filaggrin is degraded to release a pool of amino acids rich in histidine in the stratum corneum, contributing to barrier function through hydration and acidification. The conversion of histidine to trans-UCA is catalyzed by histidase, and the trans-isomer is converted to cis-UCA by UVB. UVB absorption might contribute to cutaneous photoprotection, and cis-UCA might have additional immunomodulatory effects.E16, E17, E18, E19, E20

We aimed to test the hypothesis that filaggrin deficiency resulting from loss-of-function mutations in FLG is associated with increased erythemal sensitivity to UV radiation. Cutaneous response to UV radiation was assessed by using the minimal erythema dose (MED; the lowest dose of UV causing just perceptible skin redness) as a quantifiable surrogate end point for cutaneous damage. We used detailed monochromator phototesting of 71 adult volunteers of white European ethnicity with clinically normal skin; the demographic characteristics are summarized in Table I. A calculation performed before this study commenced indicated that 7 or 8 FLG mutation carriers within a total study size of 70 to 80 subjects would provide sufficient statistical power to detect a 1.8-fold difference in MED. This sample size estimation was based on known variability in MEDs from previous studies and assuming comparisons of arithmetic means of log-transformed data (therefore able to back–transform differences into fold differences). Details of the power calculation are shown in the Methods section in this article's Online Repository at www.jacionline.org.

Table I

Demographic data and FLG genotype results for 71 volunteers with clinically normal skin

Sex	43 male/28 female
Age (y), range (median)	22-70 (41)
FLG wild-type subjects (no.)	61
FLG heterozygotes (no.)	10
Total (no.)	71

Volunteers were screened for the 6 most prevalent FLG loss-of-function mutations in the population. Five subjects were heterozygous for R501X, 3 were heterozygous for 2282del4, 1 was heterozygous for R2447X, and 1 was heterozygous for S3247X. No 3673delC or 3702delG mutations were detected, and there were no homozygotes or compound heterozygotes. Fitzpatrick skin phototype was recorded for 45 of 71 subjects, and there was no significant difference (P = .14, χ2 test) in skin phototypes between the genotype subgroups.

This work was approved by the East of Scotland Research Ethics Committee (reference 14/ES/0030), and the study was conducted in accordance with the Declaration of Helsinki.

Participants were screened for the 6 most prevalent loss-of-function mutations in FLG in the white European population (R501X, 2282del4, R2447X, S3247X, 3673delC, and 3702delG) by using published methodology. Ten (14%) of 71 were found to be heterozygous for a loss-of-function mutation in FLG (Table I). Fitzpatrick sun-reactive skin phototype was recorded for 45 of 71 subjects, and no difference was detected (P = .14, χ2 test) in skin phototypes between the genotype subgroups.

Up to 7 separate wavebands from 295 to 430 nm, representing a spectrum from UVB to UVA and visible light, were tested on the 71 subjects. A detailed description of phototesting methods is given in the Methods section in this article's Online Repository. Subjects were grouped according to FLG genotype, and MEDs were compared by using nonparametric rank-based methods (because some MED values were greater than or less than test dose ranges) with the Mann-Whitney U test (see the Methods section in this article's Online Repository) to derive CIs for differences in median MEDs (see Table E1 in this article's Online Repository at www.jacionline.org). We detected no significant differences in MEDs (defined as P ≤ .05) between the FLG wild-type and FLG heterozygous groups at any of the wavebands tested (Fig 1 and see Table E1). The CIs for differences were sufficiently narrow to make any large differences in MEDs between the genotype groups unlikely.

Table E1

Results of monochromator phototesting of 71 healthy volunteers stratified according to FLG genotype

Waveband	295 ± 5 nm	300 ± 5 nm	305 ± 5 nm	335 ± 30 nm	365 ± 30 nm	400 ± 30 nm	430 ± 30 nm
Median MED (mJ/cm2)
FLG wild-type subjects	10.0 (n = 53)	18.0 (n = 53)	47.0 (n = 61)	5,600 (n = 61)	24,000 (n = 61)	>82,000 (n = 61)	>82,000 (n = 61)
FLG heterozygous subjects	8.2 (n = 8)	15.0 (n = 8)	47.0 (n = 10)	4,950 (n = 10)	20,000 (n = 10)	82,000 (n = 10)	>82,000 (n = 10)
Difference	1.8 mJ/cm2 higher in FLG wild-type	3.0 mJ/cm2 higher in FLG wild-type	No difference	650 mJ/cm2 higher in FLG wild-type	4,000 mJ/cm2 higher in FLG wild-type	Not quantifiable	Not quantifiable
95% CI for difference	−2.6 to 5.0	−3.0 to 7.0	−9.0 to 14.0	−1,200 to 2,300	−7,000 to 9,000	Not quantifiable	Not quantifiable
P value (Mann-Whitney U test)	.41	.35	.79	.62	.74	.58	.70

Some cells are “not quantifiable” because results of greater than the highest test dose were recorded.

Fig 1

Box plots showing monochromator phototesting MED results in healthy volunteers of different FLG genotypes. The findings for 5 distinct wavebands are shown. Results for the 400 ± 30 and 430 ± 30 nm wavebands showed no difference between FLG wild-type and heterozygotes (see Table E1); these data are not displayed because the median MEDs and ranges are not quantifiable. Boxes indicate interquartile ranges, and the bar within each box marks the median result. The difference in median MEDs (and 95% CIs) are shown above each plot. All values are in millijoules per square centimeter. Median MEDs were compared by using the Mann-Whitney U test. There were 61 FLG wild-type subjects and 10 FLG heterozygous subjects tested in each group, with the exception of the 295 nm and 300 nm wavebands, in which data were obtained on 53 FLG wild-type subjects and 8 FLG heterozygous subjects.

It has previously been reported that AD might be associated with photosensitivity, a lower threshold to UVB-induced erythema, or both. Some epidemiologic data also suggest a higher incidence of multiple nonmelanoma skin cancers in subjects with a history of AD. Loss-of-function mutations in FLG are strongly associated with AD, and there is widespread downregulation of filaggrin expression in the skin of patients with atopic eczema, which has been demonstrated at the transcriptome level by means of direct RNA sequencing, and in the breakdown products of filaggrin in the stratum corneum, which was quantified by means of HPLC. A partial reduction in expression of filaggrin might result from the effect of circulating inflammatory cytokines, whereas a more profound deficiency results from loss-of-function mutations in FLG leading to near-complete absence of profilaggrin in the homozygous or compound heterozygous state. Therefore it can be hypothesized that filaggrin deficiency contributes to the observed photosensitivity and/or reduced threshold to UVB-induced erythema in patients with AD. We have performed a detailed analysis of cutaneous photoresponse in clinically normal skin to avoid the confounding effects of atopic inflammation. Our findings have excluded a large effect of FLG genotype on photosensitivity (≥1.8-fold difference in MED) at any of the wavebands tested. In addition, the results of our monochromator phototesting did not indicate a differential erythemal sensitivity within the wavelengths representing UVB, as would be predicted from the known absorption spectrum of UCA.

One limitation of our study is that the healthy volunteers did not include any subjects with ichthyosis vulgaris, and therefore we have not excluded the possibility that FLG homozygous (or compound heterozygous) subjects might show greater erythemal sensitivity than wild-type subjects. However, FLG-null heterozygosity has a significant effect on filaggrin expression in vivo,9, 10 and therefore we would expect an effect to be observed in FLG heterozygotes if this was substantial.

The fact that observations of UVB-induced damage in murine and in vitro models have not been supported by clinical data suggest that different mechanisms lead to cutaneous erythema in vivo than the markers of UV damage studied in vitro and in mice. For example, apoptosis is known to occur within areas of skin damaged by UV exposure, and this is associated with cutaneous erythema, but the relationship is nonlinear. Furthermore, the photoprotective effect of the FLG wild-type genotype might be attributable to a mechanical filtering of UV radiation by the stratum corneum rather than by chemical photoimmunosuppression.

In conclusion, our FLG genotype–stratified analysis of responses to UV and visible radiation in clinically normal skin does not support the hypothesis that the breakdown products of filaggrin play a major role in the sensitivity of human skin to UV-induced erythema. This has relevance to the ongoing search for predictors of patient response in phototherapy for AD and for the development of personalized medicine.