Patent Application
20220033902
The present invention relates to an epigenetic method for obtaining information useful to determine the effect of the use of sun protection products on the skin of an individual.
DNA methylation is an epigenetic determinant of gene expression. Patterns of CpG methylation are heritable, tissue specific, and correlate with gene expression. The consequence of methylation, particularly if located in a gene promoter, is usually gene silencing. DNA methylation also correlates with other cellular processes including embryonic development, chromatin structure, genomic imprinting, somatic X-chromosome inactivation in females, inhibition of transcription and transposition of foreign DNA and timing of DNA replication. When a gene is highly methylated it is less likely to be expressed. Thus, the identification of sites in the genome containing 5-meC is important in understanding cell-type specific programs of gene expression and how gene expression profiles are altered during both normal development, ageing and diseases such as cancer. Mapping of DNA methylation patterns is important for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumour suppressor gene silencing in human cancers.
Ultraviolet (UV) irradiation present in sunlight is an environmental human carcinogen. The toxic effects of UV from natural sunlight and therapeutic artificial lamps are a major concern for human health. The major acute effects of UV irradiation on normal human skin comprise sunburn inflammation erythema, DNA damage, tanning, and local or systemic immunosuppression.
Sun protection products are topically applied skin treatments or oral supplements or foods capable of preventing or reducing UV rays reaching the skin and are therefore used to prevent or reduce the effect of exposure of the skin to sun.
It would be a highly valuable progression to be able to assess the epigenetic impact of the use of sun protection products in order to assess damage or health and to use that information to provide sun protection advice and interventions based on the epigenetic response of an individual to such products.
Various studies have investigated epigenetic changes in skin (i.e. DNA methylation) associated with sun exposure. Raddatz et al (Epigenetics & Chromatin 2013, 6:36) found that the aging epidermis methylome is characterized by considerable stability and does not show any global methylation loss. However, local age-related methylation changes could be observed, and were enriched at weak or poised promoters and at enhancer regions, thus suggesting a functional relevance of epigenetic mechanisms for skin aging. Gronniger et al. (PLoS Genetics 6(5): e1000971) found that aging and sun exposure are associated with comparably small, but significant changes in the DNA methylation patterns of human epidermis and dermis samples.
However, neither study investigated the epigenetic effect of the use of sun protection products. There therefore remains the need to provide an epigenetic method to assess the use of sun protection products, assess the sun protection requirements of the individual and their response to such products, and potentially use that information to provide advice and interventions personalised to the epigenetic sun protection profile of an individual.
The inventors have surprisingly found a specific set of CpG sites that have enhanced efficacy in determining the effect of use of sun protection products on an individual and that can be used in an epigenetic method to assess the impact of the use of sun protection products.
Therefore, the present invention provides a method for obtaining information useful to determine the effect of the use of sun protection products on the skin of an individual, the method comprising the steps of:
Preferably >5, >10, >15, >20, >30, >50, >75, >100, >125, >150, >175 most preferably all 185 loci are observed.
Preferably step (b) comprises observing the cytosine methylation of at least two CpG loci in the genomic DNA selected from the group consisting of:
Preferably step (b) comprises observing the cytosine methylation of at least two CpG loci in the genomic DNA selected from the group consisting of:
Preferably step (b) comprises observing the cytosine methylation of at least two CpG loci in the genomic DNA selected from the group consisting of:
Preferably step (b) comprises observing the cytosine methylation of at least two CpG loci in the genomic DNA selected from the group consisting of:
The inventors have further found that specific subsets of the above CpG sites are particularly effective in determining the effect of the use of sun protection products on chronic sun exposure.
Therefore, the method preferably provides a method for obtaining information useful to determine the effect of the use of sun protection products on chronic sun exposure on the skin of an individual, the method comprising the steps of:
Preferably >5, >10, >15, >20, >30, >50, >75, >100, most preferably all 103 sites of this subset are observed.
This method more preferably involves observing cytosine methylation of at least two CpG loci in the genomic DNA selected from the group consisting of:
Preferably >5, >10, >15, most preferably all 19 sites of this subset are observed.
This method even more preferably involves observing cytosine methylation of at least two CpG loci in the genomic DNA selected from the group consisting of:
Preferably >5, >10, most preferably all 12 sites of this subset are observed.
This method most preferably involves observing cytosine methylation of at least two CpG loci in the genomic DNA selected from the group consisting of:
Preferably 2, most preferably all 3 sites of this subset are observed.
The inventors have further found that specific subsets of the above CpG sites are particularly effective in determining the effect of the use of sun protection products on acute sun exposure.
The invention therefore also preferably provides a method for obtaining information useful to determine the effect of the use of sun protection products on acute sun exposure on the skin of an individual, the method comprising the steps of:
Preferably >5, >10, >15, >20, >30, >50, >75, >100, >110, >120, >130, >140, most preferably all 149 sites of this subset are observed.
This method more preferably involves observing cytosine methylation of at least two CpG loci in the genomic DNA selected from the group consisting of:
Preferably >5, >10, >15, >20, >30, >50, >75, >100, >110, >120, most preferably all 121 sites of this subset are observed.
This method even more preferably involves observing cytosine methylation of at least two CpG loci in the genomic DNA selected from the group consisting of:
Preferably >5, >10, >15, >20, >30, >50, >75, >100, most preferably all 110 sites of this subset are observed.
This method yet more preferably involves observing cytosine methylation of at least two CpG loci in the genomic DNA selected from the group consisting of:
Preferably >5, >10, >15, >20, >30, >50, >75, >90, most preferably all 99 sites of this subset are observed.
This method yet more preferably still involves observing cytosine methylation of at least two CpG loci in the genomic DNA selected from the group consisting of:
Preferably >5, >10, >15, >20, >30, >50, >75, most preferably all 81 sites of this subset are observed.
This method yet most preferably involves observing cytosine methylation of at least two CpG loci in the genomic DNA selected from the group consisting of:
Preferably >5, >10, >15, >20, >30, >35, most preferably all 38 sites of this subset are observed.
Ultraviolet (UV) irradiation present in sunlight is an environmental human carcinogen. The toxic effects of UV from natural sunlight and therapeutic artificial lamps are a major concern for human health. The major acute effects of UV irradiation on normal human skin comprise sunburn inflammation erythema, tanning, and local or systemic immunosuppression. Malignant melanoma can be caused by indirect DNA damage from UVA radiation. In addition, other wavelengths of sunlight have also been linked to skin ageing (e.g. blue light and infra-red light). As used herein the terms “sun exposure”, “exposure”, “exposure to sun”, “UV exposure”, and the like are intended to refer to exposure to either natural sunlight or artificial exposure such as sun beds and sun lamps. The term therefore refers to exposure to the harmful radiation associated with such, including but not limited to UVA and UVB radiation.
Sun exposure can occur over varying time scales. As used herein the term chronic sun exposure means exposure over the period of from 1 to 24 months, in particular about 5 months. As used herein the term acute sun exposure means exposure over the period of from 1 to 30 days, in particular about a week.
In the context of the present invention, sun protection products means topically applied skin treatments capable of preventing or reducing UV rays reaching the skin. Sun protection products may be referred to by terms such as sunscreen, sunblock, sun cream, or suntan lotion. They may be in the form of a lotion, spray, gel or other topical product. They may act by absorbing or reflecting some or all of the ultraviolet radiation from the sun, in particular UVA and/or UVB and therefore protect against sun exposure. Sun protection products often have an SPF rating (sun protection factor) which is a measure of the fraction of sunburn-producing UV rays that reach the skin.
As used herein, CpG sites/loci refer to the unique identifiers found in the Illumina CpG loci database (as described in Technical Note: Epigenetics, CpG Loci Identification ILLUMINA Inc. 2010, https://www.illurnina.com/documents/products/technotes/technote_cpg_loci_identification.pdf). These CpG site identifiers therefore provide consistent and deterministic CpG loci database to ensure uniformity in the reporting of methylation data.
Identification of CpG Sites Potentially Associated with Use of Sun Protection Products
A study was carried out to identify CpG sites potentially associated with protection from sun exposure though the use of sun protection products.
Data from a single centre, cross-sectional biopsy study involving 24 Chinese and 24 European female participants in which 24 young and 24 old females had enrolled was used to identify CpG sites with potential value for the assessment of sun protection. Biopsy samples of skin were collected from two different areas of each subject: samples from a sun exposed area of the skin; and samples from a sun protected area of the skin. Areas of the skin designated as sun exposed were located on the lower outer arm and areas designated as sun protected were located on the upper inner arm, typically half way between the elbow and axilla area.
DNA methylation data from samples collected from exposed areas of skin in the young European subjects was compared with data from protected areas in the same young European subjects. After p-value filter and multiple testing correction (<0.05 FDR and 5% delta beta) 4450 CpG sites were found to be significantly different in the sun exposed samples relative to the sun protected samples and are therefore likely to be associated with sun protection.
Methylation data from samples collected from exposed skin areas in the young European subjects was compared with data from exposed skin areas in the old European subjects. After p-value filter and multiple testing correction (<0.05 False Discover Rate (FDR) and 5% delta beta) 6972 CpG sites were found to be significantly different in the old exposed samples relative to the young exposed samples and are therefore likely to be associated with prolonged sun exposure.
It was found that there was an overlap of 455 CpG sites between these two lists (i.e. between the 4450 and the 6972 CpG sites) and that of these 455 CpG sites, 399 were in the same direction—i.e.
These 399 CpG sites found from the above single centre, cross-sectional biopsy study were then reassessed in a publicly available dataset (Vandiver A. R. et al.: Age and sun exposure-related widespread genomic blocks of hypomethylation in nonmalignant skin. Genome Biology (2015) 16:80, Gene Expression Omnibus accession number: GSE51954). The Vandiver dataset contained epidermal samples (N=38) from 20 Caucasian subjects. Paired punch biopsy samples, 4 mm in diameter, had been collected under local anaesthesia from sun exposed areas (outer forearm or lateral epicanthus) and sun protected areas (upper inner arm).
Of the 399 CpG sites identified from this study, 198 CpG sites were found to be significantly different (FDR for 399 tests, p<0.05, delta beta greater than 5%) comparing young to old exposed sites, and also significant (FDR for 399 tests, p<0.05, delta beta greater than 5%) comparing exposed to protected (separately tested) in the young samples from the Vandiver dataset.
Moreover, all 198 of these CpG sites were found to change in the same direction in both the Vandiver dataset and the above single centre, cross-sectional biopsy study (135 being hypo-methylated and 63 being hyper-methylated in the older photo-exposed samples).
These 198 CpG sites were therefore taken forward and assessed for their ability to measure the effect of using sun protection products in general and specifically the effect of using sun protection products during chronic and acute sun exposure.
Identification of CpG Sites Associated with Use of Sun Protection Products on Chronic Sun Exposure
A clinical study was carried out to investigate the epigenetic effect of using sun protection products on chronic sun exposure.
One aim of the study was to assess of the ability of the 198 CpG sites identified above to measure the effect of the use of a sun protection product on chronic sun exposure.
In the study, skin biopsy samples were collected from both forearms at the start of the study. The study then ran for 5 summer months during which a sun protection product (Pond's Age Miracle Firm and Lift Day Cream, Sun Protection Factor 30) was applied to one of the forearms (“treated”) but not the other (“untreated”). At the end of the 5 months, skin biopsy samples were collected from both forearms to assess the effect of chronic sun exposure on the skin and to also assess the effect of using and not using the sun protection product for those 5 months.
The study was a single-centre, split forearm study with 42 subjects completing. Subjects were female, age 22-58, Fitzpatrick skin type II-III, in general good health, not regular users of sun protection (on the arms) and routinely went outside and were exposed to the sun (at least 10 hours per week).
Subjects used their typical facial and body cleansing and care products and routines and did not change their products and routine until the study completed.
Applications of the sun protection product were conducted daily, at home, by the subjects. Subjects were instructed to apply the product at least twice daily (subjects could apply morning, prior to going outside, every 2 hours while outside, or evening) to a randomly selected (right or left) forearm (consistent throughout study). Subjects were allowed to apply as many times a day as desired and when they went out in the sun. Subjects kept a diary on their sun exposure and sun protection product usage for 5 months.
At baseline and at month 5, 3-mm punch biopsies (1 from each extensor forearm at each time point) were collected by a licensed certified dermatologist. Resulting in the following biopsies
Baseline and month 5 biopsy sites were randomized within the forearm, either at upper (close to elbow), or lower (close to wrist) location. The two biopsy sites were about 4-5 cm apart.
A total of 168 biopsies were collected (42 subjects×2 forearms per subject×2 time points per forearm). Biopsies were analysed for epigenetic modifications caused by sun exposure on Illumina Infinium MethylationEPIC BeadChip arrays.
One subject was missing one of the four sampling points (due to low DNA quantity), and as a result all observations for this subject were excluded from the analysis resulting in 41 individuals being available for further analysis (total 164 biopsies).
Of the 198 CpG sites identified above that were taken forward and assessed in the context of sun exposure and sun protection, 185 sites were present on the MethylationEPIC BeadChip arrays used.
Samples were filtered to remove probes that contained known polymorphisms (23,356 probes), were cross-hybridised (42,529 probes) and non-CpG probes (1,652 probes) identified by McCartney et al (Genomics Data, Volume 9, September 2016, Pages 22-24). Those probes on X and Y chromosomes (14,404 probes) and those probes where more than 25% of samples had a signal indistinguishable from negative control probes were also removed (7,094 probes). 83,801 probes were therefore excluded from the original 866,836, leaving 782,437 probes. None of the above 185 sites/probes were lost as a result of this filtering. The resultant dataset was corrected using ComBat to control for a batch effect in the data (related to the technical factor “Sentrix Barcode”).
The overall aim of the study was to identify differentially methylated sites between baseline and the end of the study for both treated and untreated arms. Following this, the differences in post vs pre M values (paired delta-M values) were calculated for the treated and untreated arms. Delta-M measures for each probe were then modelled as a function of baseline M-value (a covariate), and subject and treatment group (fixed effects) using ordinary least squares. Specific contrasts of interest were then assessed by determining least-square means (or estimated marginal means) for delta-M (being different from 0); these contrasts were for
An extensive analysis of the 185 CpG sites of interest was carried out. Random sampling and permutation testing was performed to determine the performance of these 185 sites.
For each probe and treatment group, the mean absolute change in M-value (from baseline) was calculated over samples. It was then determined whether this measure changed between untreated and treated samples over the set of probes.
To visualise changes for each of the 185 probes (averaged over samples within a treatment group), box plots (
To test changes in M-value measures between untreated and treated samples the point estimate of change in M-value measure for the 185 probes between groups was first calculated. The significance of this estimate was tested using permutation testing in which 10,000 permutations of the procedure were performed for randomly chosen sets of 185 probes to construct the null distribution of change in M-value measure. It was then tested whether our point estimate was significantly different to this null distribution (
Permutation testing therefore demonstrates an observed value of −0.0073, indicating that the mean difference in mean absolute change from baseline across 185 probes was 0.0073 higher in the untreated group relative to the treated group. This mean difference in mean absolute change from baseline was significantly different to the permuted (null) distribution (p<0.05).
Although individual probes showed mixed patterns of change in M-value measure between groups (with both negative and positive changes), when averaging over 185 probes, it was found that the observed mean change was significantly different to that observed for randomly chosen probes.
It is remarkable that methylation changes were observed in this specifically selected (pre-defined) probe set. Moreover, it is a novel insight that epigenetic changes were observed over just 5 months—typically such changes have been associated with many years of exposure.
It is therefore apparent that these 185 CpG sites, either alone or in combination, are capable of measuring changes associated with or caused by the use of sun protection products and are therefore used in the method of the first aspect of the invention in which a method for obtaining information useful to determine the effect of the use of sun protection products on the skin of an individual is provided, the method comprising the steps of:
These sites and the more preferred selections of the sites listed below may also be used in a kit for obtaining information useful to assess changes associated with or caused by the use of sun protection products.
The present invention may therefore also provide a kit comprising primers or probes specific for at least two genomic DNA sequences in a biological sample, wherein the genomic DNA sequences comprise CpG loci in the genomic DNA selected from a group consisting only of these 185 sites;
and
Preferably the kit comprises primers or probes specific for >5 of the genomic DNA sequences, more preferably >10, >15, >20, >30, >50, >75, >100, >125, >150, most preferably all 185.
The kit may comprise primers or probes specific for at least two genomic DNA sequences in a biological sample, wherein the genomic DNA sequences comprise CpG loci in the genomic DNA selected from a group consisting of any of the sites listed in the preferred subgroups below.
The 185 sites are shown in Table 1 in which annotations are based on Human Genome Build HG19 and the columns are:
Identification of CpG Sites Associated with Use of Sun Protection Products on Chronic Sun Exposure
Of the 151 sites from the initial 185 that changed in the expected direction it can be seen that 103 of these sites also changed less in the treated group than the untreated group. Therefore these 103 CpG sites are valuable in the epigenetic evaluation of the use of sun protection products and their effect against chronic exposure because they are not as affected as those from the untreated arm. These sites are provided in Table 2.
The invention therefore provides a method for obtaining information useful to determine the effect of the use of sun protection products on chronic sun exposure on the skin of an individual, the method comprising the steps of:
Preferably >5, >10, >15, >20, >30, >50, >75, >100, most preferably all 103 sites of this subset are observed.
The sites all have p-values calculated for the significance of the change in the untreated arm vs. the treated arm and therefore the sites can all be assessed for the significance of the probes in relation to the use of sun protection products.
Table 3 lists the 19 sites with a p-value of at most 0.2. These sites are a more preferable subset.
Preferably >5, >10, >15, most preferably all 19 sites of this subset are observed.
Table 4 lists the 12 sites with a p-value of at most 0.1. These sites are an even more preferable subset.
Preferably >5, >10, most preferably all 12 sites of this subset are observed.
Table 5 lists the 3 sites with a p-value of at most 0.05. These sites are the most preferable subset.
Preferably all 3 sites of this subset are observed.
Assessment of CpG Sites Associated with Use of Sun Protection Products on Acute Sun Exposure
A study was carried out to investigate the epigenetic effects of using sun protection products on acute sun exposure.
In the study, ex vivo human skin (discarded abdominal or breast skin from surgical procedures) was used.
Two aims of the study were:
Three different treatments were employed:
A Newport solar irradiator [Housing: 67005/PSU: 69911 (160-600 W)/Lamp: 6259 (300 W Xe)] with CGA-320 and UG-11 filters to block UVC and visible light was used to dose the ex vivo skin or cultured biopsies with ssUV dose of equivalent to 1MED (Minimal Erythema Dose). The 1MED dose was equivalent to 4 J/cm2 (as detected by a UVA+UVB sensor from the Solar Light Company) and took 5 minutes and 40 seconds to deliver to the ex vivo skin or biopsies at a distance of −2.5 cm.
Biopsies were removed from −80° C. freezer. Adipose tissue was removed from the biopsy using surgical scissors. The remaining skin biopsy was digested and genomic DNA extracted following the protocol from the QiaAMP DNA kit (Qiagen). The gDNA was resuspended in 1× Tris-EDTA (1×TE) and stored at −200 until use.
Isolated genomic DNA was sent to Q Squared Solutions Expression Analysis LLC to perform their methylation analysis using their Illumina Infinium DNA Methylation Service and the Illumina MethylationEPIC BeadChips. Methylation analysis included:
Of the 198 CpG sites identified above that were therefore taken forward and assessed in the context of acute sun exposure and sun protection, 185 sites were present on the MethylationEPIC BeadChip arrays used.
Samples were filtered to remove probes that contained known polymorphisms (23,399 probes), were cross-hybridised (42,557 probes) and non-CpG probes (1,651 probes) identified by McCartney et al, 2016. Those probes on X and Y chromosomes and those probes where more than 25% of samples had a signal indistinguishable from negative control probes were also removed (3,682 probes). From a total of 866,836, 80,683 probes were excluded from the raw data, leaving 786,153 probes. None of the above 185 sites/probes were lost as a result of this filtering.
The aim of the epigenetic analysis was to:
For each probe and sample type, the mean absolute change in M-value (from baseline) was calculated over samples. It was then determined whether this change was different in “UV vs. Control” compared to “UV+SPF vs. Control” over the set of probes.
To visualise changes for each of the 185 probes (averaged over samples within a treatment group), box plots (
It is therefore apparent that these 185 CpG sites, either alone or in combination, are capable of measuring epigenetic changes associated with or caused by acute sun exposure. In addition, these CpG sites are capable of measuring the beneficial effects of using sun protection products to prevent the epigenetic effects of acute sun exposure.
It is remarkable that methylation changes were observed in this specifically selected (pre-defined) probe set. Moreover, it is a novel insight that epigenetic changes were observed over just 1 week—typically such changes have been associated with many years of exposure.
The 185 sites are shown in Table 6 in which annotations are based on Human Genome Build HG19 and the columns are:
It can be seen from Table 6 that there were 161 sites from the initial 185 that changed in the expected direction (hyper- or hypo-methylated). Of these 161 sites from the initial 185 that changed in the expected direction, 149 sites also change less in the treated group than the untreated group. Therefore these 149 are indicative of protection against acute exposure because they are not as affected as those from the untreated sample. These sites are provided in Table 7.
These 149 sites therefore have greater performance and are a preferable subset of the 185 CpG sites for the epigenetic assessment of use of sun protection products on acute sun exposure.
The invention therefore provides a method for obtaining information useful to determine the effect of the use of sun protection products on acute sun exposure on the skin of an individual, the method comprising the steps of:
Preferably >5, >10, >15, >20, >30, >50, >75, >100, >110, >120, >130, >140, most preferably all 149 sites of this subset are observed.
The sites all have p-values calculated for the significance of the change in the untreated sample vs. the treated sample and therefore the sites can all be assessed for the significance of the probes in relation to the use of sun protection.
Table 8 lists the 121 sites with a p-value of at most 0.2. These sites are a more preferable subset.
Preferably >5, >10, >15, >20, >30, >50, >75, >100, >110, >120, most preferably all 121 sites of this subset are observed.
Table 9 lists the 110 sites with a p-value of at most 0.1. These sites are an even more preferable subset.
Preferably >5, >10, >15, >20, >30, >50, >75, >100, most preferably all 110 sites of this subset are observed.
Table 10 lists the 99 sites with a p-value of at most 0.05. These sites are a yet more preferable subset.
Preferably >5, >10, >15, >20, >30, >50, >75, >90, most preferably all 99 sites of this subset are observed.
Table 11 lists the 81 sites with a p-value of at most 0.01. These sites are a still more preferable subset.
Preferably >5, >10, >15, >20, >30, >50, >75, most preferably all 81 sites of this subset are observed.
Table 12 lists the 38 sites with a p-value of at most 0.001. These sites are the most preferable subset.
Preferably >5, >10, >15, >20, >30, >35, most preferably all 38 sites of this subset are observed.
| Number | Date | Country | Kind |
|---|---|---|---|
| 18177979.4 | Jun 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/065416 | 6/12/2019 | WO | 00 |
