Field of the Invention

The present invention relates to an epigenetic method for indicating actual or potential ultraviolet radiation (UVR)-induced skin damage in an individual.

Background of the Invention

Historically, humans have been exposed to ultraviolet radiation (UVR) mainly through occupational exposure to sunlight. Recreational UVR exposure, however, has increased dramatically in recent years because of outdoor leisure activities and to purposely tan for cosmetic purposes.

Excessive exposure to UVR, either from natural sunlight or from artificial sources such as tanning beds, can cause a variety of harmful effects on human skin, including increased susceptibility to melanoma and non-melanoma skin cancers.

In addition to the risk of cancers, damage to the skin due to UVR overexposure may also lead to concerns from a cosmetic perspective, such as the visual and textural skin changes which are collectively termed photoaging. Freckles (ephelides), fine wrinkling, and dilation of the small blood vessels under the skin (telangiectasias) are often seen early in the photoaging process. In later years, photoaged skin may develop irregular pigmentation (lentigines) and become thick, wrinkled, and leathery. Since these changes occur gradually, often manifesting themselves many years after most of a person’s sun exposure, they are generally regarded as an unavoidable, normal part of growing older. However, with proper protection from UVR, much photoaging of the skin can be avoided.

Studies have suggested that the early years of sun exposure may be the most critical for skin damage and photoaging. However, adolescents and young adults tend to be the least compliant in practicing healthy sun-protective practices such as applying sunscreen, wearing protective clothing, and avoiding tanning beds.

One of the main difficulties in promoting healthy sun-protective practices in younger people is the temporal delay which exists between the cause (UVR) and effect (skin damage and photoaging) of unprotected sun exposure. There is a need for methods which can be used in younger people to assess the impact of sun exposure. Such methods may help to promote healthier sun-protective practices in younger people, by revealing changes which have already occurred at the molecular level but are not yet manifest as visible skin damage.

Efforts have been made in the prior art to provide epigenetic methods for obtaining information useful to determine the effect of sun exposure on the skin of an individual. WO 2019/238792 describes a specific set of CpG loci (aka CpG dinucleotides, CpG sites) that have enhanced efficacy in determining the effect of sun exposure on an individual and that can be used in an epigenetic method to assess the impact of sun exposure on the human skin. WO 2019/238793 describes a specific set of CpG loci 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.

The CpG loci disclosed in WO 2019/238792 and WO 2019/238793 are excellent markers of sun exposure in older subjects. However, they were less effective in younger subjects, where changes occurred to a lesser extent both in number and in magnitude.

The present invention addresses this problem.

Summary of the Invention

The present invention provides an epigenetic method for indicating actual or potential ultraviolet radiation (UVR)-induced skin damage in a human individual aged between 18 and 30 years; the method comprising:

- obtaining a test sample of genomic DNA from epidermal skin cells obtained from the individual;

- in the test sample, determining the cytosine methylation status of a set of CpG loci in the genomic DNA; wherein the cytosine methylation status so determined provides an indicator of actual or potential UVR-induced skin damage; and characterised in that the set of CpG loci is at least 2 of, preferably at least 10 of, more preferably at least 20 of, most preferably at least 30 of and ideally all the following 39 CpG loci: cg01079842, cg01665432, cg02078727, cg02737747, cg04812879, cg04857880, cg06090739, cg06636244, cg06823550, cg07198365, cg07482373, cg07869839, cg08464076, cg10488100, cg11148483, cg11607742, cg11920406, cg12092932, cg12299478, cg13027321 , cg13362436, cg13991350, cg14203437, cg15669600, cg15935526, cg17381727, cg21004104, cg21172464, cg22247002, cg22355498, cg22384883, cg23090732, cg23919385, cg25586364, cg25655489, cg26164878, cg26586287, cg26849382 and cg27579771.

The invention also provides a kit comprising reagents and instructions for carrying out the epigenetic method as defined above.

Detailed Description and Preferred Embodiments

The term “epigenetic” as used herein means relating to, being, or involving a modification in gene expression that is not primarily through alterations of DNA sequence.

The term “genomic DNA” as used herein refers to DNA derived from the genetic material in the chromosomes of a human individual.

Cytosine methylation is an epigenetic modification of DNA in which cytosine is converted to 5- methylcytosine in a reaction that involves flipping a target cytosine out of an intact double helix and transfer of a methyl group from S-adenosylmethionine by a methyltransferase enzyme. Cytosine methylation in vertebrates occurs predominantly at dinucleotide sequences where the cytosine is followed by guanine (CpG). Accordingly, the term “cytosine methylation status” as used herein refers to the presence or absence of a methylated cytosine (5-methylcytosine) at a particular CpG locus in the genomic DNA.

As used herein, the terms “CpG locus” or “CpG loci” denote respectively, one, or a plurality, of the unique identifiers found in the lllumina® CpG loci database (as described in Technical Note: Epigenetics, CpG Loci Identification © 2010 lllumina® Inc.,

(https://www.illumina.com/documents/products/technotes/te chnote_cpg_loci_identification.pd1).

There are more than 28 million CpG loci (aka CpG dinucleotides, CpG sites) in the human genome. CpG sequences are symmetric on forward and reverse strands of any double- stranded DNA. When identifying the methylation status of a CpG locus, it is necessary to unambiguously refer to the cytosine on the forward strand, reverse strand, or both. The lllumina® method consistently designates CpG loci based on the actual or contextual sequence of each individual CpG locus, taking advantage of sequences flanking a CpG locus to generate a unique CpG locus cluster ID. This method will consistently designate the same CpG locus identifier and orientation calls even if public databases and genome assemblies change. These CpG site identifiers thus provide a consistent and deterministic CpG loci database to ensure uniformity in the reporting of methylation data.

As used herein, "ultraviolet radiation (UVR)-induced skin damage" refers to skin damage resulting from exposure to UVR in the A (320-400 nm), B (290-320 nm), or C ranges (200-290 nm) or combinations thereof. Examples of UVR-induced skin damage include cosmetic damage (such as the visual and textural skin changes which are collectively termed photoaging) as well as skin pathologies such as melanoma and non-melanoma skin cancers. Sources of UVR in the context of this invention include natural sources (such as the sun), and/or artificial sources (such as black lights, welding equipment, lasers, and tanning equipment).

Epidermal skin cells for use in the epigenetic method of the invention may be obtained by a variety of techniques such as punch biopsy, surgical excision, and non-invasive or minimally invasive skin sampling methods such as wet swabbing, tape lift, cotton tip swabbing, skin scraping or employing a small gauge needle (for example, 28 gauge) to collect micro-cores of skin tissue.

Advantageously, the set of CpG loci (as defined above) shows improved sensitivity in younger subjects, and so can be used to provide an indicator of actual or potential UVR-induced skin damage in younger subjects. The term “younger subjects” in the context of this invention denotes human individuals aged from 18 to 30 years of age. The information provided by the epigenetic method of the invention can thus be used to provide personalised advice and interventions which enable effective prevention of future skin damage and photoaging in younger subjects.

The inventors have also found that specific subsets within the set of CpG loci (as defined above) are particularly sensitive indicators of actual or potential UVR-induced skin damage in younger subjects. Accordingly, in a preferred epigenetic method according to the invention, the set of CpG loci is at least 2 of, preferably at least 5 of, more preferably at least 10 of, most preferably at least 15 of and ideally all the following 19 CpG loci: cg01079842, cg01665432, cg02737747, cg04812879, cg06823550, cg07869839, cg11920406, cg12299478, cg13362436, cg13991350, cg14203437, cg15669600 cg15935526, cg21172464, cg22355498, cg25586364, cg25655489, cg26586287 and cg27579771.

In a more preferred epigenetic method according to the invention, the set of CpG loci is at least 2 of, preferably at least 5 of, more preferably at least 10 of and most preferably all the following 13 CpG loci: cg02078727, cg06636244, cg07198365, cg07482373, cg10488100, cg11148483, cg11607742, cg17381727, cg22247002, cg22384883, cg23090732, cg26164878 and cg26849382.

In the method of the invention, the cytosine methylation status of the set of CpG loci in the test sample provides an indicator of actual or potential UVR-induced skin damage.

Typically, the cytosine methylation status of the set of CpG loci in the test sample is compared to that of the same set of CpG loci in a control sample; and differences in the cytosine methylation status between the test sample and the control sample provides an indicator of actual or potential UVR-induced skin damage;

A “control sample” in the context of this invention may include a skin sample obtained from a subject that has not been exposed to UVR, or whose exposure to UVR has been blocked or attenuated; or a skin sample obtained from the test subject at a body site that has not been exposed to UVR, or whose exposure to UVR has been blocked or attenuated.

A “control sample” in the context of this invention may also include a standard reference value or range of values which may be determined empirically or historically from single or multiple skin samples from the same test subject, or from a reference subject or subjects, or a previously established range of values that represent baseline or normal values. The epigenetic method of the invention as described above may also be used to measure the effectiveness of a test agent in reducing actual or potential ultraviolet radiation (UVR)- induced skin damage in a human individual. Such a method suitably comprises:

- treating a test skin site of the individual by topically applying the test agent to the test skin site;

- irradiating the treated test skin site by exposing it, or allowing it to be exposed to, a source of UVR;

- obtaining a test sample of genomic DNA from epidermal skin cells obtained from the treated, irradiated test skin site of the individual;

- in the test sample, determining the cytosine methylation status of the set of CpG loci (as defined above);

- comparing the cytosine methylation status so obtained to (i) that of the same set of CpG loci in a first reference sample obtained from epidermal skin cells obtained from a skin site of the individual which has been irradiated in the absence of the test agent, and (ii) that of the same set of CpG loci in a second reference sample obtained from epidermal skin cells obtained from a skin site of the individual which has not been irradiated; wherein if the cytosine methylation status of the test sample is the same or substantially similar to the cytosine methylation status of the second reference sample, then the test agent is effective at reducing actual or potential UVR-induced skin damage; whereas if the cytosine methylation status of the test sample is the same or substantially similar to the cytosine methylation status of the first reference sample, then the test agent is not effective at reducing actual or potential UVR- induced damage.

The source of UVR in the context of the above method may include natural sources (such as the sun), and/or artificial sources (such as a simulated solar radiation (SSR) source).

A kit comprising reagents and instructions for carrying out the epigenetic methods as described above may suitably comprise primers or probes which specifically bind to the set of CpG loci as defined above; and a reagent used in: a genomic DNA polymerization process; a genomic DNA hybridization process; a genomic DNA direct sequencing process; a genomic DNA bisulfite conversion process; or a genomic DNA pyrosequencing process.

The term “primer” as used herein refers to a single-stranded oligonucleotide capable of acting as a point of initiation for template-directed DNA synthesis under suitable conditions for example, buffer and temperature, in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, for example, DNA or RNA polymerase or reverse transcriptase. The length of the primer, in any given case, depends on, for example, the intended use of the primer, and generally ranges from 15 to 30 nucleotides.

The term “probe” as used herein refers to a surface-immobilized molecule that can be recognized by a particular target.

The term "specifically bind" as used herein denotes a binding reaction between two molecules that is at least two times the background and more typically more than 10 to 100 times the background molecular association under physiological conditions.

Alternatively, a kit comprising reagents and instructions for carrying out the epigenetic methods as described above may comprise reagents used in direct sequencing methods which measure the methylation status of the CpG loci as defined above. Direct sequencing methods measure the nucleotide sequence of DNA, including the presence of DNA methylation, directly without the need of primers or probes (targeted or untargeted). An example of a direct sequencing method is nanopore sequencing, which works by monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore.

The invention will be further illustrated by the following, non-limiting Examples. EXAMPLES

An in-vivo study was carried out with the objective of assessing epigenetic outcomes following exposure of human skin to suberythemogenic doses of solar-simulating radiation (SSR).

Two groups of test subjects were recruited to an in-vivo study: a younger group (n=17, age range 18-30 years, average age 23 years) and an older group (n=12, age over 65 years, average age 70 years). No differences in gender composition between the groups were present (Fisher’s test; p = 0.73).

The minimal erythemal dose (MED), i.e. , the amount of SSR needed to produce reddening of the skin, was determined for each test subject.

For each test subject, a test skin site was irradiated with a total of 5 suberythemogenic doses of SSR (80% of the subject’s MED per dose), with each dose administered to the test skin site on separate days over a week.

For each test subject, irradiated skin from the test skin site and non-irradiated skin from a control skin site was biopsied and quadrisected to allow DNA methylation analysis. Epidermis was disassociated with dispase, and the methylation of the extracted DNA was interrogated using the lllumina® Infinium MethylationEPIC BeadChip. Differential methylation was determined by the limma package (Nucleic Acids Research, 2015, Vol. 43, No. 1 e47).

The results showed that although the older group had a much greater DNA methylation response to SSR, there were 39 CpG sites that were significantly (false discovery rate adjusted p-value of p<0.2) changing in the younger group. These sites are shown in Table 1 : Table 1 Ex-vivo studies were also carried out on human skin explants collected from mammoplasty and abdominoplasty surgeries in 3 separate experiments as follows: (i) Experiment A - Females, Caucasian, 43 years old and 50 years old, breast and abdominal skin: 6 control, 10 test samples

(ii) Experiment B - Female, Hispanic, 21 years old, breast skin: 2 control, 2 test samples

(iii) Experiment C - Female, Hispanic, 18 years old, breast skin: 2 control, 2 test samples Test samples were irradiated with SSR for 7 days.

The methylation of the extracted DNA was interrogated using the lllumina® MethylationEPIC (850k) microarray, to measure the differential methylation between irradiated test samples and non-irradiated control samples. The results showed that across the 3 separate ex-vivo experiments (i) to (iii), the methylation status of 39 CpG sites as shown in Table 1 also changed in the same expected direction in response to SSR.

The results also showed that a subset of 19 CpG sites changed in the expected direction in 2 out of 3 of the ex-vivo experiments (i) to (iii). These sites are shown in Table 2:

Table 2 The results also showed that a subset of 13 CpG sites changed in the expected direction in all 3 of the ex-vivo experiments (i) to (iii). These sites are shown in Table 3: Table 3

The results show that the set of CpG loci in T able 1 , and especially those in T ables 2 and 3, show improved sensitivity in younger subjects, and so can be used to provide an indicator of actual or potential UVR-induced skin damage in younger subjects.