Methods of determining resilience

Abstract

The present invention relates to methods of predicting, measuring and improving levels of psychological resilience in a subject in response to stress, based on identifying the DNA methylation status of one or more genomic regions and using the methylation status to predict or measure psychological resilience. The invention also relates to methods of identifying one or more genomic regions associated with psychological resilience and arrays for predicting or measuring psychological resilience.

Description

[0001] Methods of determining resilience

[0002] Field of invention

[0003] The present invention relates to methods of predicting, measuring and improving levels of psychological resilience in a subject based on identifying the DNA methylation status of one or more genomic regions and using the methylation status to predict or measure psychological resilience, alone or in response to a therapeutic regime. The level of psychological resilience can be altered using therapeutic agents that target DNA methylation.

[0004] Background

[0005] Psychological resilience is a defining factor in the capacity of individuals to successfully cope with adversities and traumas that threaten their function, as well as their ability to restore function (1). Psychological resilience is a highly individual quality and people will respond differently when exposed to a life trauma, such as being diagnosed with cancer. As a result, some individuals will develop serious psychopathological conditions, thus are more in need of psychosocial support, while others maintain relatively stable physical and psychological functions overtime (2). Consequently, there has been an increasing interest in the concept of resilience within the field of cancer. In breast cancer, for example, with 5-year survival rates approaching 90% (3), survival is increasingly becoming a matter of quality of life. Thus, there is a need to define strategies promoting health-related quality of life during and beyond the course of the disease. Several studies have also shown that highly resilient cancer patients are not only less likely to suffer from depression and anxiety but also manage to promote their health-related quality of life and are experiencing improved treatment outcomes (4-6).

[0006] Despite years of research evaluating the psychosocial factors involved, the molecular background of psychological resilience is not yet defined (7). However, to foster an individual's psychological resilience and unleash its full potential, there is a need for a comprehensive understanding of the factors involved, including any somatic background.

[0007] In addition to established environmental and psychological components, like socioeconomic factors, research now recognizes the influence of biological aspects, such as neurochemical and (epi-) genetic factors (7). Epigenomics refer to changes in gene expression that are not encoded in the DNA sequence itself but are instead mediated by e.g. DNA methylation, histone modifications or RNA-mediated silencing. Among these, DNA methylation is one of the best characterized epigenetic mechanisms and involved in regulation of many cellular functions, such as development, transcription, or chromosomestability (8). In mammals, the addition of a methyl group to DNA is mainly restricted to cytosines that are followed by a guanine base, termed a CpG site. DNA methylation and other epigenetic modifications are, in contrast to the genetic code, plastic and can change throughout life in response to environmental factors (8). In studies on stress and anxiety, which are closely related to psychological resilience, associations to DNA methylation have been established in e.g. childhood trauma where changes in DNA methylation patterns have been identified as a result of exposure to stress (9). Similarly, adaptive changes in DNA methylation have been observed in the monoamine oxidase A gene, involved in neurotransmitter regulation, following therapeutic intervention in patients suffering from acrophobia, a type of anxiety disorder (10). On the other hand, findings of DNA methylation patterns associated to psychiatric conditions, are often inconsistent between studies and can rarely be replicated (11).

[0008] The inventors herein identified that epigenetic markers of psychological resilience could prove valuable in predicting and / or monitoring the impact of traumatic events and the success of therapeutic interventions.

[0009] Detailed description of invention

[0010] The inventors set out to define a molecular portrait of psychological resilience by analysing the blood epigenome of over 500 patients with known psychological resilience status at the time of breast cancer diagnosis.

[0011] The inventors have herein succeeded in identifying relevant genomic regions and CpG sites for which methylation patterns differed between subjects with high and low psychological resilience. Furthermore, the inventors have demonstrated that this difference in methylation signal displayed a dose-response-like configuration and that CpG sites located in the LY6G5C genomic region are particularly useful for discriminating between high and low resilience. These findings inform the use of therapeutic agents to manipulate the DNA methylation status, that can be used to treat low resilience, and improve patients' quality of life. They can also inform treatment strategies for patients who have been diagnosed with a disease, as they can be used to predict how a patient may react psychologically to a particular regimen.

[0012] In a first aspect, the invention provides a method of predicting or measuring psychological resilience of a subject, the method comprising:

[0013] (i) providing a sample from the subject to be tested;(ii) measuring the DNA methylation status in one or more of the genomic regions defined in Table 1,

[0014] wherein the DNA methylation status of the one or more genomic regions in Table 1 is indicative of the psychological resilience state in the subject.

[0015] In a second aspect, the invention provides a method of improving psychological resilience of a subject, the method comprising:

[0016] (i) providing a sample from the subject to be tested;

[0017] (ii) measuring the DNA methylation status in one or more of the genomic regions in Table 1,

[0018] wherein the DNA methylation status of the one or more genomic regions in Table 1 is indicative of the psychological resilience state in the subject, and

[0019] (iii) administering one or more therapeutic agents to alter the methylation status of one or more genomic regions in Table 1, thereby improving the psychological resilience of the subject.

[0020] In a third aspect, the invention provides a therapeutic agent for use in improving psychological resilience of a subject, wherein the use comprises administering a therapeutically effective amount of the therapeutic agent to the subject to alter the methylation status of one or more genomic regions in Table 1, which are associated with psychological resilience, wherein the therapeutic agent alters the methylation status to a status associated with improved psychological resilience.

[0021] "Psychological resilience" is defined herein as the ability of a subject to successfully adapt to or overcome negative conditions, experiences, or adversities. Resilience also includes the ability of an individual to return to their baseline state quickly, i.e. to bounce back. In other words, resilience refers to a person's positive adaptation when faced with adversities. Highly resilient persons are more likely to regain stability and are less likely to develop conditions such as depression, anxiety and PTSD. Such negative conditions or experiences or adversities could include, but are not limited to: physical health conditions and illnesses and the treatment thereof (for example cancer diagnosis and treatment), mental health conditions and illnesses and the treatment thereof (for example post-traumatic stress disorder), injury, trauma, stress, death of relatives or family, divources and burn out. Insome embodiments, these negative conditions or experiences may be referred to as traumatic events.

[0022] In some embodiments of the above aspects, the invention provides a method or a therapeutic agent for use in predicting, measuring or improving psychological resilience in a subject in relation to or in response to a traumatic event. By "traumatic event" we include, but are not limited to, all of the negative conditions and experience listed above.

[0023] In some embodiments, "traumatic event" excludes diagnosis and treatment of some chronic health conditions, for example fibromyalgia.

[0024] Psychological resilience can be measured using a variety of well-established tools. The skilled person will be well aware of how to utilise these tools to measure psychological resilience in a subject. For example, resilience can be measured using the Connor- Davidson Resilience Scale (CD-RISC). This scale is validated and widely used. It has three variations depending on the number of items measured - CD-RISC 2, CD-RISC 10, and CD-RISC 25. Each of the variations measures resilience as a function of five components: personal competence, acceptance of change and secure relationships, trust / tolerance / strengthening effects of stress, control, and spiritual influences. The CD-RISC 25 scale is widely used, and uses a scale of 0-100 (where a lower score is lower resilience). For example, a score of below about 30 is generally considered to relate to particularly low resilience, whereas a score of above about 70 is considered to relate to particularly high resilience.

[0025] In some embodiments, a CD-RISC 25 score of below about 50 is considered to relate to low resilience and a CD-RISC 25 score of above 50 is considered to relate to high resilience.

[0026] In some embodiments, a CD-RISC 25 score of below 30 is considered to relate to low resilience and a CD-RISC 25 score of above 70 is considered to relate to high resilience.

[0027] In some embodiments, the methods of the present invention are capable of predicting or determining the psychological resilience of a subject when expressed as a CD-RISC 25 score.

[0028] As described above, the invention also provides for the use of therapeutic agents in improving psychological resilience. These therapeutic agents are useful in the context of improving psychological resilience of subjects defined as having low psychological resilience, but are also useful in the context of improving psychological resilience of subjectwho have "intermediate" levels of resilience. Therefore, an aim of the present invention is to improve the resilience of patients to high levels of resilience.

[0029] In some embodiments, the methods of the present invention are validated by comparing the level of resilience determined by the methods described herein with a CD-RISC 25 score calculated according to psychometric scoring of the subject.

[0030] Other measures of resilience also exist in the art. For example, these include but are not limited to: the Resilience Scale for Adults (RSA), Predictive 6-Factor Resilience Scale, Scale of Protective Factors (SPF), Resilience Scale, Brief Resilience Scale, Ego Resilience Scale, Response to Stressful Experiences Scale, and Academic Resilience Scale (ARS).

[0031] All of the measures described herein utilise patient questionnaires to establish scores on a numerical scale. The skilled person will know how to determine whether the scores obtained relate to lower or higher psychological resilience.

[0032] In the embodiments of the present invention that are used to predict or measure psychological resilience, the level of resilience determined can be expressed using any suitable scale. For example, it may be expressed on a scale of 0-100 (similarly to CD- RISC 25) to allow for easy comparison with current scales based on patient questionnaires. In some other embodiments, resilience may be expressed as a categorisation. For example, subjects may be categorised as having low, medium or high resilience depending on the level of methylation measured in the one or more genomic regions listed in Table 1.

[0033] In some cases, in the embodiments of the present invention that are used to predict or measure psychological resilience, the level of resilience determined does not need to be expressed in terms of a scale. For instance, if one of more of the genomic regions in Table 1 are found to be in the methylation status that is known to be associated with lower resilience, then the subject can be categorised as having low resilience. Conversely, if one of more of the genomic regions in Table 1 are found to be in the methylation status that is known to be associated with higher resilience, then the subject can be categorised as having high resilience.

[0034] In some embodiments, the the present invention is particularly useful in determining and improviding the psychological resilience of subjects who have experienced negative conditions or experiences. Such negative conditions or experiences could include, but are not limited to: physical health conditions and illnesses, mental health conditions andillnesses, injury, trauma, stress, death of relatives or family, divources and burn out, and serious events such as earthquake, war, storms, or other natural disasters.

[0035] In some embodiments, the present invention is particularly useful in determining and improving the psychological resilience of subjects who have been diagnosed with serious health problems that require treatments with high levels of intervention. For example, such health problems can include, but are not limited to: cancers, cardiac issues (for example myocardial infarction, stroke, heart failure), dementia, Alzheimer's disease, diabetes, neurodegenerative conditions, polygenic diseases and serious mental health problems.

[0036] In some preferred embodiments, the subject has been diagnosed with cancer. For example, in some embodiments, the subject has been diagnosed with breast cancer. The skilled person will appreciate that the invention is potentially useful in relation to other types of cancer, as the treatment regimens employed in the treatment of cancer are broadly similar. The invention is also useful in relation to patients undergoing invasive treatments for any condition: for example surgery, chemotherapy, radiotherapy, transplants and the like.

[0037] The present invention may be useful in determining and improving the psychological resilience of subjects who have recently or newly been diagnosed with a health problem, or whether they have been suffering with a particular health problem for a period of time already.

[0038] In some embodiments, the invention involves ongoing monitoring of psychological resilience of the subject, for example, during a treatment regimen. The methods may involve sampling and monitoring of the subject at multiple time points, for example, every week, every month, or every three months, as appropriate.

[0039] In some embodiments, the subject is a mammalian subject, for example a human, cat, dog, or horse. In some preferred embodiments, the subject is a human subject.

[0040] The sample to be tested is any sample that contains the DNA of the subject. For example, this may be a blood sample, tissue sample, skin sample, or saliva sample. In some preferred embodiments, the sample obtained from the subject is a whole blood sample. In some embodiments, the whole blood sample may be processed to obtain a serum or plasma sample for further testing according to the invention. By "providing a sample from the subject to be tested", in some embodiments we mean that the sample has alreadybeen obtained from the subject to be tested, i.e. the step does not involve a surgical step of obtaining the blood sample from the subject. Therefore, in some embodiments, step (i) of the methods described herein may involve providing a sample that has been obtained from the subject to be tested.

[0041] The skilled person will appreciate how to measure the methylation status of the one or more genomic regions in Table 1. DNA methylation is the process by which methyl groups are added to the DNA molecule without changing the DNA sequence. Methylation of the genome mainly occurs at cytosine residues at position 5 on the pyrimidine ring. The vast majority of DNA methylation in eukaryotes occurs at cytosine residues that are followed by a guanine reside (at so called "CpG sites"). This may occur at CpG islands, which are regions of sequence with high levels of GC content and high levels of CG repeats. DNA methylation is a type of epigenetic modification that influences the expression activity of a DNA segment without changing the base sequence. For example, when a promoter region is methylated, this typically acts to repress gene transcription. DNA methylation is carried out by DNA methyltransferase enzymes.

[0042] As used herein the term "DNA methylation status" means determining or measuring the degree of methylation of a particular genomic region. This includes determining the level of methylation of a particular genomic region and / or determining whether specific sites within the genomic region are methylated or not. By "methylated" we particularly include methylation at cytosine residues that are immediately followed by a guanine residue, i.e. so-called CpG sites or CpG dinucleotides.

[0043] In some embodiments of the methods disclosed herein, the step of determining the methylation status of the one or more genomic regions in Table 1 involves determining the methylation status of one or more CpG dinucleotides within each genomic region. For example, this may include determining the methylation status of two, three, four, five, six, seven, eight, nine, or ten or more CpG dinucleotides within each genomic region. In some preferred embodiments, this includes determining the methylation status of eight CpG dinucleotides within each genomic region.

[0044] In some embodiments, the DNA methylation status can be expressed as a level of the CpG dinucleotides within a particular genomic region that are methylated. For example, this may be expressed as a percentage of the dinucleotides that are methylated. For example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% of the CpG dinucleotides within a particular region may be methylated or unmethylated. In some embodiments, at least half of the CpG dinucleotides are methylated in a highly resilient individual.In some preferred embodiments, this includes determining the methylation status of eight CpG dinucleotides within the LY6G5C genomic region.

[0045] The skilled person will be aware of the various techniques available to determine the DNA methylation status. For example, the methylation status of specific sequences can be analysed using one or more of the following techniques: methylation-specific PCR, Methylation Sensitive Single Nucleotide Primer Extension Assay (msSNuPE), nanopore sequence, and the Illumina Methylation Assay.

[0046] For example, the Illumina Methylation Assay works by treating the DNA with bisulfite to convert methylated cytosine to unmethylated cytosine. Any cytosine at originally unmethylated loci is converted to uracil by this step. The whole genome is then amplified and applied to a chip containing two bead types for each CpG site being analysed. Each bead is attached to a single stranded DNA oligonucleotide - with one bead for the methylated locus and one for the unmethylated. The amplified DNA products hybridise to the chip via allele-specific annealing to either the methylation specific or non-methylation specific probe. Hybridisation can be detected by fluorescent staining and scanning of the chip.

[0047] In the context of the present invention, the genomic regions of interest defined herein were determined using the Infinium MethylationEPIC vl.O BeadChip array. However, as the regions of interest have been identified herein, it could be that an array using only probes for the specific genomic regions definer herein is used in the methods defined herein.

[0048] As disclosed herein, the DNA methylation status of one or more of the genomic regions defined in Table 1 are determined:

[0049] Table 1: Annotations and Locations in GRCh37.pl3 gene assembly of differentially methylated regions

[0050]

[0051]

[0052] In some embodiments, the locations of the genomic regions defined in Table 1 are identified using the Genome Reference Consortium (GRC) assembly. For example, the genomic regions may be as defined above, and either defined using the GRCh37.pl3 or GRCh38.pl4 assemblies. In some preferred embodiments, the genomic regions are defined according to the locations in the GRCh38.pl3 GRC assembly.

[0053] In some embodiments, the genomic regions are one or more of the following: LY6G5C, ZFP57, PF4, RP11-16E12.1 / RP11-16E12.2, CDH9, ZNF727, and / or C8orf31.

[0054] In some embodiments, the genomic regions as defined herein are as shown in SEQ ID NOs 1-7.

[0055] In some embodiments, the genomic regions are one or more of the following: LY6G5C, ZFP57, CDH9, ZNF727, and / or C8orf31.

[0056] In some preferred embodiments, the genomic regions are LY6G5C or ZFP57.

[0057] In some embodiments, the genomic regions are one or more of the following: LY6G5C, PF4, RP11-16E12.1 / RP11-16E12.2, CDH9, ZNF727, and / or C8orf31. In some embodiments, the genomic region is not ZFP57.

[0058] In some preferred embodiments, the genomic region is LY6G5C. As shown in Table 1, by LY6G5C we mean the region of sequence in chromosome 6, positions 31,644,461-31,651,817 (as determined by the GRCh37.pl3 assembly). The forward strand of this sequence is shown in SEQ ID NO. 1 below:AACAGTTTACACACAAATTTATTTGGGAGAAACATCCAGGGACTAGGGGACAAGAGAGGAAACCTGGTGGGC AGTAGGGCTGGGGGTACAGAGTAGCAGTAAGTGTGCTGAAGGGCGTCAACCAAGAGGAAGAGCCAAGGCT GGGGTCCAGTGGCTGGAGGGAGGCAAGGAGGGCTGGTATGAGGGACTAGAAGTCCTGGCCAAGCCCAGAT AGAAGTCAGGAAGGTGGCTGGAAACTGGTGGAATTTTACACCAAAGTTTGCTGCAGTCACACTAAGGAGTAT AGAGCCCTCTGTTTTGAGGGTCATTGCAGAAATCCAGGAAGCAGTATTGAGAGAATATCCAGAAGCCAGACAC CGGAGAAGTTCGGGTATTTGAACAATCACTCATCTGCTCCTTACTTCGGCAGTCACTCACCATGACGTCAGAA CCGCTGCCTGGGGAGGGACAGTGGGCACCAGTGATACGGAAGTCCCCAGGAAGAGCCCCAAATCCTCTCAT CCCCACACTCATAAGTCAAAAAAAAAAGAAAAAGAAAAGATTCCTGTAGTTAGGCATGGGTGGACATGCCCAG TGTTCACCAGCCATGGAACTCCACTGAAGTTCCCATGCAAGGCTGGAGGAAAAGAGCCATATGAAATGTAATG GTTGGAGGGGGAGTTGGGAGTTACTGAGCCAAGTGAGGAGAACTAGCACCATAGGACCATGTGAGAAAAAG CTGGGAAATGTTTTGGAGATTGGGTGGCAGGAAGGAGGTGTATTGTTATTTATTTTTCAGACCAAAAGAGAATA AGATGATGTCTGCTGCTGTTATACATAATAGAGAAAAATCTTTGTGCCTGCATCCCAAGAAGTCATGTTCAGGG ATGTTTGCTGCTGCCCTGCTTGAGAGAAATGACCAAAATGCCCATCAATAGTGGGATGGGGAAATCAGCTGTG ATATGCGCATGCTATGGAGTAGTATACAGCAGGTCAATAAAACAAGGAAGCTGTTTACAAACTGATATCGGAAC ATTCAGTTCCCCTAACTTAAATGTGGAATAATGTTTACAGTGGGATGCTACTATCTTGGGTTGGGGCGGGGGAA GAGGTGAAAAAATAGTAAACAGCATATTTGTGCAGGGTGGAATGTGCATAAAAGATTGCAGGAGGGATCATCC AGAAAGTAAAAAAAGTGGTCACATGTGCAGGGGAGCCAGGTGGGTTAGGGTAGTAGCGGGAGACTTTGGTT TGATGGTATTGTATACTCTGATATTTGACCCACATCTGTGCATCGGCTATGTTAAAAGGGTAGTAAGAGGACTT GAACACAGGCAGCTGCATGCAGTGGTTGTTGAGAGCACCATCTCTGGAGCCATCACAAATTCTGGCTCAGCA TCTGTGAGACTCAGGCAAGGTTATGACCTTTCTGCACCTGTTTCCTCATCTGTAAAATGCACATAGTAATAATAC CTGCCTCAGCGGATTGCAAGTGTTTAGAACAGTGCCTAGCACATATTATGTGTTACGTTTTTGCTAACTTAAGAA AGGTGGGGGGTCGGTGGAAGAGCAGGCATCGGGAAGGAGTCAATTTTCAGCGAGGGAGATGTCCAGTGGT CAACGGGATATGAGGAGAGCGGTTTGACATAACATTCAGATTCAGAAGGAAGTGGTATGTGGCTGCTGGTTG AAGCCAGCAAAGCAGATAAAATCCTCTGCTTTTGAGTATATGAAGTGGGAAGACAGCTAAGGACCAAACCTTG GTGAACATGAACCACTAAGGGTCAGAGAGAAAACGCTCCATGAAGGAGACTGAAGAAGCCGTGGAGGATGC AGGAGAAGAGCAACACCAGCAGTAACTGCAGACAGATGCGGAAGCAGACAGCTTGAGGACAGGCAAGGGC ACCTGGAGATCTGGAGGGTCCCCGTCAAAGCTGCGCACCTTGATAGGGTAGAAGCTATTCAGCTACAGATTG AGGAGAGAAGGTTAGTGGAAGTGGAGACAGAGTGTGGCTCTGAAGAAAAGGGAAGAGAGGCTGGGCACGG TGGCTCACGCCTGTAATCCCAGCACTCTGGGAAGCTAAGGTGGGTGGATCACCTGAGGTCAGGAGTTCGAG ACCAGCCTGGCCAACATGGTGAATCCCCATCTCTACTAAAAATACAAAAAATTAGCTGGGCGTGGTGGCGTGC ACCTTTAATCCCAGCTGCTTGGGAGACTGAGGCACAAGAATTGCTTGAACTGGAGAGGTGGAGGTTGCAGTG AGCCAAGATTGCGCCACTGCACTCCAGCCTGGGTGACAGAGCAGCAAAAAAAAAAAGACAGGATTGGAGCA ATGTCTTATGGGATTATGGGAACAAGACTTGGGGTGCAGCTTAGGAGGCTGAGAGAGTTTCCGTTTGGGAGA GTGCTGGGCCCATGACAGGAGAAGGCCACTTACTGTTCTTTTTGTGGAGAGTGATGCAGCTGCTGCCAGCTG GGGTGAGGCAGATGTCAGATCCCAGAAGGCACCCTAACTCCTTGGTCTCCAAGAGGCATCGGTAGCAGCGC AGGTATTTGGGGAATGGAAGTGGTTGAGGGGGTTCCCAATTGACAGGAACAAACTTACCTAGAACACAGAGA AGTGCTGACCCCACTCACACCCCATTCTACCTCACACCCTACCACTGCCTGATTCCAGGCCACTCAGCCCCAC TCCTCCCTCCCTTCCTGTCTCAGAAAACCATCAAAGCCCCAATTCTCTGCTTCCTTCCCCAACTGCATACACATA CATCCCCCTTTTCCTCTGGTCCTAAGGCCAGACCACATGTTAACAAATCCCCAGACCCAGCAGAGCACTTGGT GTTAGGCAGAGGAAAGTGCTAAACCAACACTTTGAATCCTGTGTCTCTGTGGCTGGTGCTTTGCAGCCAAGTG GGGAGCCCAGCAGGCTGGACTCAGTCTTGTTCTATCCTGTGGATTCTGGTTTTCTCATCCAGCACACTCCCTA ACCCTCCCTATTCTATGTTGCCCTCAGATCCAGAGAGGATTCCTTCAGTATCTCTATTCAGGTCACTGCTGTGAA GTGAGACAGCCCTGGGGTGGTCACTAGAAATCTCCTTCAGAGGCTGGGTGCGGTGGCTCACGCCTGTAATCC CAGCACTTTGGGAGGCCAAGGCGGGCAGGTACCTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGT GAAACCCCGTCTCTACTAAATATACAAAAATTAGCTGGGCTTGGTGGCTTATGCCTGTAATCCCAGTTATTCGG GAGGCTGAGGCATGAGAATCGCTTGAACCCGGGAGGTGGAGGTTGCAGTGAGCCGAGATCTCGCCACTGCA CTCCGGCCTGGGATACAGAGCGAGACTCCATCTCAAAAATAATAATAATAATAAATTTTTAAAAATCTTCAGATT GCACATCAGTCCATGAGCAGGCATTCCCTACCAAACCCATCTGTCCCATCTCTCCTCCTGCATGGGTTTACCTG AGCATCCTGGACAGGTGTACCCAGACACTTGGTGTCTGTGGGTTTCTCCATCCAGGCCAGGAGACCCTTCTG AACCCTTGGAGCCACTTACCAAACACCAAGCTCATCATGACCAGCACTATTAAGAGGACCGTGTAGAGGGCTT GGGGGCTGCTGTGGAAGCACAGGGGACCCAGACTCTGGCTCCCTGCAGGGCCTGCCATAAAACGCATGACT GCCTGCTGGCCTCCAGTTTGGGCTTATATTGGTGGAAGAGAGGTTGGCCAAGAGGAAGGAGAGAGGCAACA CCAGCTCAGGGTGGAAATCAGTGCCAGACCAGCCAGAGGGGCAGAATGTTCGCACCCACAGCCACTCTGGG GCATAACATCCTGCTTGAGGGCAGGGGACCAGCAATAGGGGAATGAGAAAAGGAACTGTCTTTCCTATTAATT GGACAGATGTTTATTGAATCACTGCATCAGATGCTGGGGATACAACCCTGCACAAAGTCTCCACCCTCACAGG GCACAGTCTAGTAGGGGAGACAAGTCCACCAGCAATGATGTGGGGAGGGCAGAGTGCTGCCAGGAGCACCT CGACAGTTAAACCACTGACCAGAGGGATTTCGGCAGAGGAGTAACTTGATCGGATTTCTGTTTATAAAAGATT GCCATGGCTGCACATTGCATTTGGGTCAAGAGTGGAGGCCGCCGGGAAGTAGGACGCTATTCCCGAGTCCG GTCACAAGATGGCGGACTGGTCCGGCAGAAGACGAGCAGGGACGAGGAAGCGGGGCTAATGAACCTGAGA TACAGTTAGAAGACTGGACAGATTTGCTGTTGGACTGAACGAGGGGTGAGGGAACAGGGGTAGGCTTGCAC AAGGAAGTGGTACCATTTTCCAAGATAGGAAACATGTGGTCTGTCTCAAAAAAAAAAAAAAAAAAGCAAATAG GGGGTGCCCAGTCCCACTTCTCATACCCTGGGGACACCTGTCAGACATCCTAAAACAAGGACACCTGGATCC CAAGCGATACGTACTCAGCTCAGTGCTCCCTTGGGGTTCCAGGAACCCAGCGCCTTCCCTCACCTCATCCTTTTTCCTGCCCCGCCTGTGCTCAGCTGCGGCTCAGTGGGCCTGAACTCCGGAGCCCACAGAATCTGGCGCTGG GCGTCCGCTCTCCGCGCCTGACCGCACCTCAGAACTCCGGTAGGACGGGGGGGTGGCCCCCCGCTCAAGC TCTGTTCCCTGGGGAAGAAACCTGGAAAGTGCGAACCGCGCGTCGGGACCCAAGCGTCGGGCCCCAGCGG ACATCCGGAGCCCGAAGCGGCTCCCCAGGAAGGCGGCGCCGTAGCGCCACTCTCCCTCCCAGGCGAATTCT GGAGACCGCGGCCCCAGGCGTCTCACCCATTTTCTCCGCTGGGGACCCGCTGGGCTCCCCATCCACGCCTA CTCGGTCCCCACCCCACCAGCTCAGTCTTGACTCAGAAACTCAGGGTTTTTACTTTTAGGATCGTTGGGCTGT GCGTTAGGGGAGGAGGTGGTCCTCAGCGTCCTGGAACGACACCACCTGCTCCAATTTCCCGTCTGGAGGTTC TGGTCGAGGCTCCGAACTCGGGTTCCCTGCTACCTCCCAGACTATTCAAGAATTATCCAGTCCCAGGATGATA AGGGGGAAGATGGGAAGAAACAGACGGGAGACGCCCGCCCAGAAAGACTGCGGGAAGAAAGAAATTCGAG AGGAAACTGCACGCCACTGAGCGCCTCCCAAAAGCCTTGGAATGAATGAATTTAAAAACTATATTAGGGCCGG ACTGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGTGGACTACCTGAGGTCAGGA GTTCGCACCCAGCCTGGCTAACATGGTGAAACCCCGTTTCTACTACAAATACCAAAAATTAGCCGGGCGTGGC GGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGTGGGGGATCATTCGAGGTCAGGAGTTCGCAACC AGCCTGAGCAACATGGTGAAACCCCGTCTCTATCAAAAAATACAAAAACATTAGCCAGGTGTGGTGGCGCAC GCCTGTAGTCCTGGCTACTCGGGAGGCTGAGGCAGGAGAATCTCTTGAACCTGGGAGGCAGAGGTTGCAGT GAGCCGAGATCGCACCACTGCACTCCAGCCTGGGCGACAGAGTGAGACTCTGTCTTAAAGAAATAATAACAC AAAATAAATTGTATTAGAGAAAAGCCAGAGTAGTGGAGAACTGCAGAGGAACGCGGGGCACCTACATAAATG TCTTGAATGAATGAGTGCACAGAGTGATAGACAAAAAGAATCAGAGGGCCGGGCTCCGTGGCTCACGCCTGT AATCCCAGCACTTTGGGAGGCCGAGCTGGGCGGATCACAAGGTTAAGAGATCGAGACCATCCTGGACAATAT GGTGAAACCCCGTCTCTACTAAACATACAAAAATTAGCCAGGAGTGGTGGCGCCTGCCTGTAGTCCCAGCTAC TCAGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGACGGAGGTTGCAGTGAGCCGAGATCGCGCCA CTGCACTCCAGCTTGGCGACAGAGCAAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAGAGAGCCA GGGGCTCCTCTTGAAGCGAAGAGGGCAAAGGGCAAAGGGGAAGCACAGGGGAACTTCGCGGCGCCCTCTG AAGCTCCCTCTCGAATATAATCGCAACGAAAAGGCCAACGACTAGAGGCTTTGCGAGGCTGAGGCTGGGCTT CGGGAGGGGATTGCCCTGAGAGGTCCGGGAGGACTTGCTGTGGAATTCAAGCGACCGTGGGCCTTGAGGG AACCGGGGGGCAAGACACCCACCCAGCATTCGCGGAATATTTCCTCGAATTATTTCGGGGAGGGGTGAGGCC GGGGCAGGGTGGGGCCTTCTTCGGAGGGGGCGCGGCCTCCGAGTAATTAATCCCGTCTTTGTTGCGTTTTGC TCCTCTCCTGTCCACCCAGCAGGGCCAGCCCAGGGCGCGCTAAGAGTCCAGAGAGTTCGTTTCCATGGTGAC GGGTTCCGCGAAGGTTTTCCTGGGGTGAAGAGGCAGGGCGTTGAATAATCGCCATGGCGACAGCAGCAGAT GACGGTGTCCCTTCTGAGTGCTCCTACCTAGAGTTAAGGGATACCTGAGGGTAAGCAACCGAGTGACGAAAC AAAGAAGGCGGGGCCTGAGGACAGAACGCCAAGGTTAGGGGAATGGAGCCAGGCAAACGAGGGGCGGGG CTGTAGATGACCCGGTCGGGAGAGGGCCACGGTTTGTTGGGGGAGCGGCTCGAGATTGCGTTCTAGAGAGG AACCAGAGAGAGGGTCTTTAACCTAAATATAAATGAATGACTGGATTCCTGAAGAATCCGGAATGGCTTGTTGA TTGGATAGATGGATGGATGGATGGACGGACGGACGGACCGATGGATGGAAATCTGGCTATCACTGACGCCTG AGCTCCCCACCCTCTTGGGCCCTCCACCTCCGGAGCCCTCACTCGCTTGTGACAGCTGTACGAGAAATACAT GCCTCTCCTAGGAGCAAACCCTCAACCCAAACAGGCAGCACAGAGCCAGTCCAGCACCTCACACTGGAGGC ACTCAGGGTGGAGCCCAGGTCGATGAGACGGCGTAGGATGAGGCTTTTTGGCCCAGCTGGGAACCACTTCT TTCCAGATTTCCCGTCCAGAGTCTAACTTTCCTTTCTCCCAGCGCCATCTTTTCTGCTAGTTTGCCCAGCTCCTC AGGGTGCCTGGACTTTCAGGCCTCACCTTGTGTCCAGTATAGCAGGGTCCAGCGCCCCAGCAACTGGGAAG GTCTGCAT (SEQ ID NO: 1)

[0059] LY6G5C, as part of the Ly6 gene family and belonging to the Ly6 / uPAR superfamily, is characterized by a domain with a specific cysteine pattern that creates a three-fingered structural motif, the exact function of which is not yet identified in humans (28). The LY6G5C protein is glycosylated and while experimental analyses suggested it belongs to the secreted members of the Ly6 protein family, rather than being GPI-anchored, isoforms predicted to be membrane bound exist (29, 30). In blood, LY6G5C RNA is expressed across most immune cell compartments with low specificity for a certain cell type (30, 31). FACS analysis of different cell lines has suggested that ligands for LY6G5C exist on undifferentiated megakaryocytes, macrophages, and B-cells, although it could not be excluded that ligands were present elsewhere (29). Moreover, LY6G5C RNA expression is enhanced in brain tissue, particularly the cerebellum and basal ganglia (30, 32). The direct function of LY6G5C is not known, although it was shown in mice that hippocampal transcription of the LY6G5C homologue was downregulated in offspring affected bymaternal immune activation (MIA). MIA can have lasting negative impact on neurodevelopment of the off-spring, including hippocampal dysfunction (33). This finding is of relevance, since the hippocampus, as part of the limbic system, is involved in memory formation and learning, including appropriate responses and the resulting resilience to stress (34).

[0060] In some embodiments, by "determining the methylation status" of the genomic region in question, we mean determining (for example using the methods described herein and known in the art) whether the CpG dinucleotides in the region of interest are methylated or unmethylated. By "methylated" we mean that the 5 position of the pyrimidine ring is methylated. By "unmethylated" we mean that the 5 position of the pyrimidine ring is not methylated.

[0061] In some embodiments, the methods and uses of the present invention involve determining the methylation status of any of the CpG dinucleotides in each of the sequences within Table 1. In some preferred embodiments, there are particular CpG dinucleotides within each region that are particularly informative on resilience.

[0062] For example, in relation to the LY6G5C region, the following sequence (reverse complement) shows positions which are particularly useful in correlating with resilience, in some embodiments:

[0063] Legend:

[0064] CpG site in classifier (bold underline)

[0065]

[0066] >NC_000006.11:31644461-31651817 Homo sapiens chromosome 6, GRCh37.pl3 Primary Assembly, forward strand AACAGTTTACACACAAATTTATTTGGGAGAAACATCCAGGGACTAGGGGACAAGAGAGGAAACCTGGTGGG CAGTAGGGCTGGGGGTACAGAGTAGCAGTAAGTGTGCTGAAGGGCGTCAACCAAGAGGAAGAGCCAAGGC TGGGGTCCAGTGGCTGGAGGGAGGCAAGGAGGGCTGGTATGAGGGACTAGAAGTCCTGGCCAAGCCCAG ATAGAAGTCAGGAAGGTGGCTGGAAACTGGTGGAATTTTACACCAAAGTTTGCTGCAGTCACACTAAGGAG TATAGAGCCCTCTGTTTTGAGGGTCATTGCAGAAATCCAGGAAGCAGTATTGAGAGAATATCCAGAAGCCAG ACACCGGAGAAGTTCGGGTATTTGAACAATCACTCATCTGCTCCTTACTTCGGCAGTCACTCACCATGACGT CAGAACCGCTGCCTGGGGAGGGACAGTGGGCACCAGTGATACGGAAGTCCCCAGGAAGAGCCCCAAATCC TCTCATCCCCACACTCATAAGTCAAAAAAAAAAGAAAAAGAAAAGATTCCTGTAGTTAGGCATGGGTGGACA TGCCCAGTGTTCACCAGCCATGGAACTCCACTGAAGTTCCCATGCAAGGCTGGAGGAAAAGAGCCATATGA AATGTAATGGTTGGAGGGGGAGTTGGGAGTTACTGAGCCAAGTGAGGAGAACTAGCACCATAGGACCATGT GAGAAAAAGCTGGGAAATGTTTTGGAGATTGGGTGGCAGGAAGGAGGTGTATTGTTATTTATTTTTCAGACC AAAAGAGAATAAGATGATGTCTGCTGCTGTTATACATAATAGAGAAAAATCTTTGTGCCTGCATCCCAAGAA GTCATGTTCAGGGATGTTTGCTGCTGCCCTGCTTGAGAGAAATGACCAAAATGCCCATCAATAGTGGGATGG GGAAATCAGCTGTGATATGCGCATGCTATGGAGTAGTATACAGCAGGTCAATAAAACAAGGAAGCTGTTTAC AAACTGATATCGGAACATTCAGTTCCCCTAACTTAAATGTGGAATAATGTTTACAGTGGGATGCTACTATCTT GGGTTGGGGCGGGGGAAGAGGTGAAAAAATAGTAAACAGCATATTTGTGCAGGGTGGAATGTGCATAAAA GATTGCAGGAGGGATCATCCAGAAAGTAAAAAAAGTGGTCACATGTGCAGGGGAGCCAGGTGGGTTAGGG TAGTAGCGGGAGACTTTGGTTTGATGGTATTGTATACTCTGATATTTGACCCACATCTGTGCATCGGCTATGT TAAAAGGGTAGTAAGAGGACTTGAACACAGGCAGCTGCATGCAGTGGTTGTTGAGAGCACCATCTCTGGAG CCATCACAAATTCTGGCTCAGCATCTGTGAGACTCAGGCAAGGTTATGACCTTTCTGCACCTGTTTCCTCATC TGTAAAATGCACATAGTAATAATACCTGCCTCAGCGGATTGCAAGTGTTTAGAACAGTGCCTAGCACATATTA TGTGTTACGTTTTTGCTAACTTAAGAAAGGTGGGGGGTCGGTGGAAGAGCAGGCATCGGGAAGGAGTCAATTTTCAGCGAGGGAGATGTCCAGTGGTCAACGGGATATGAGGAGAGCGGTTTGACATAACATTCAGATTCAG AAGGAAGTGGTATGTGGCTGCTGGTTGAAGCCAGCAAAGCAGATAAAATCCTCTGCTTTTGAGTATATGAAG TGGGAAGACAGCTAAGGACCAAACCTTGGTGAACATGAACCACTAAGGGTCAGAGAGAAAACGCTCCATGA AGGAGACTGAAGAAGCCGTGGAGGATGCAGGAGAAGAGCAACACCAGCAGTAACTGCAGACAGATGCGGA AGCAGACAGCTTGAGGACAGGCAAGGGCACCTGGAGATCTGGAGGGTCCCCGTCAAAGCTGCGCACCTTG ATAGGGTAGAAGCTATTCAGCTACAGATTGAGGAGAGAAGGTTAGTGGAAGTGGAGACAGAGTGTGGCTCT GAAGAAAAGGGAAGAGAGGCTGGGCACGGTGGCTCACGCCTGTAATCCCAGCACTCTGGGAAGCTAAGGT GGGTGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAATCCCCATCTCTACTAAAA ATACAAAAAATTAGCTGGGCGTGGTGGCGTGCACCTTTAATCCCAGCTGCTTGGGAGACTGAGGCACAAGA ATTGCTTGAACTGGAGAGGTGGAGGTTGCAGTGAGCCAAGATTGCGCCACTGCACTCCAGCCTGGGTGACA GAGCAGCAAAAAAAAAAAGACAGGATTGGAGCAATGTCTTATGGGATTATGGGAACAAGACTTGGGGTGCA GCTTAGGAGGCTGAGAGAGTTTCCGTTTGGGAGAGTGCTGGGCCCATGACAGGAGAAGGCCACTTACTGTT CTTTTTGTGGAGAGTGATGCAGCTGCTGCCAGCTGGGGTGAGGCAGATGTCAGATCCCAGAAGGCACCCTA ACTCCTTGGTCTCCAAGAGGCATCGGTAGCAGCGCAGGTATTTGGGGAATGGAAGTGGTTGAGGGGGTTCC CAATTGACAGGAACAAACTTACCTAGAACACAGAGAAGTGCTGACCCCACTCACACCCCATTCTACCTCACA CCCTACCACTGCCTGATTCCAGGCCACTCAGCCCCACTCCTCCCTCCCTTCCTGTCTCAGAAAACCATCAAAG CCCCAATTCTCTGCTTCCTTCCCCAACTGCATACACATACATCCCCCTTTTCCTCTGGTCCTAAGGCCAGACC ACATGTTAACAAATCCCCAGACCCAGCAGAGCACTTGGTGTTAGGCAGAGGAAAGTGCTAAACCAACACTTT GAATCCTGTGTCTCTGTGGCTGGTGCTTTGCAGCCAAGTGGGGAGCCCAGCAGGCTGGACTCAGTCTTGTT CTATCCTGTGGATTCTGGTTTTCTCATCCAGCACACTCCCTAACCCTCCCTATTCTATGTTGCCCTCAGATCCA GAGAGGATTCCTTCAGTATCTCTATTCAGGTCACTGCTGTGAAGTGAGACAGCCCTGGGGTGGTCACTAGAA ATCTCCTTCAGAGGCTGGGTGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGCA GGTACCTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAATATACAAA AATTAGCTGGGCTTGGTGGCTTATGCCTGTAATCCCAGTTATTCGGGAGGCTGAGGCATGAGAATCGCTTGA ACCCGGGAGGTGGAGGTTGCAGTGAGCCGAGATCTCGCCACTGCACTCCGGCCTGGGATACAGAGCGAGA CTCCATCTCAAAAATAATAATAATAATAAATTTTTAAAAATCTTCAGATTGCACATCAGTCCATGAGCAGGCAT TCCCTACCAAACCCATCTGTCCCATCTCTCCTCCTGCATGGGTTTACCTGAGCATCCTGGACAGGTGTACCCA GACACTTGGTGTCTGTGGGTTTCTCCATCCAGGCCAGGAGACCCTTCTGAACCCTTGGAGCCACTTACCAAA CACCAAGCTCATCATGACCAGCACTATTAAGAGGACCGTGTAGAGGGCTTGGGGGCTGCTGTGGAAGCACA GGGGACCCAGACTCTGGCTCCCTGCAGGGCCTGCCATAAAACGCATGACTGCCTGCTGGCCTCCAGTTTGG GCTTATATTGGTGGAAGAGAGGTTGGCCAAGAGGAAGGAGAGAGGCAACACCAGCTCAGGGTGGAAATCA GTGCCAGACCAGCCAGAGGGGCAGAATGTTCGCACCCACAGCCACTCTGGGGCATAACATCCTGCTTGAGG GCAGGGGACCAGCAATAGGGGAATGAGAAAAGGAACTGTCTTTCCTATTAATTGGACAGATGTTTATTGAAT CACTGCATCAGATGCTGGGGATACAACCCTGCACAAAGTCTCCACCCTCACAGGGCACAGTCTAGTAGGGG AGACAAGTCCACCAGCAATGATGTGGGGAGGGCAGAGTGCTGCCAGGAGCACCTCGACAGTTAAACCACT GACCAGAGGGATTTCGGCAGAGGAGTAACTTGATCGGATTTCTGTTTATAAAAGATTGCCATGGCTGCACAT TGCATTTGGGTCAAGAGTGGAGGCCGCCGGGAAGTAGGACGCTATTCCCGAGTCCGGTCACAAGATGGCG GACTGGTCCGGCAGAAGACGAGCAGGGACGAGGAAGCGGGGCTAATGAACCTGAGATACAGTTAGAAGAC TGGACAGATTTGCTGTTGGACTGAACGAGGGGTGAGGGAACAGGGGTAGGCTTGCACAAGGAAGTGGTAC CATTTTCCAAGATAGGAAACATGTGGTCTGTCTCAAAAAAAAAAAAAAAAAAGCAAATAGGGGGTGCCCAGT CCCACTTCTCATACCCTGGGGACACCTGTCAGACATCCTAAAACAAGGACACCTGGATCCCAAGCGATACGT ACTCAGCTCAGTGCTCCCTTGGGGTTCCAGGAACCCAGCGCCTTCCCTCACCTCATCCTTTTTCCTGCCCCG CCTGTGCTCAGCTGCGGCTCAGTGGGCCTGAACTCCGGAGCCCACAGAATCTGGCGCTGGGCGTCCGCTCT CCGCGCCTGACCGCACCTCAGAACTCCGGTAGGACGGGGGGGTGGCCCCCCGCTCAAGCTCTGTTCCCTG GGGAAGAAACCTGGAAAGTGCGAACCGCGCGTCGGGACCCAAGCGTCGGGCCCCAGCGGACATCCGGAG CCCGAAGCGGCTCCCCAGGAAGGCGGCGCCGTAGCGCCACTCTCCCTCCCAGGCGAATTCTGGAGACCGC GGCCCCAGGCGTCTCACCCATTTTCTCCGCTGGGGACCCGCTGGGCTCCCCATCCACGCCTACTCGGTCCC CACCCCACCAGCTCAGTCTTGACTCAGAAACTCAGGGTTTTTACTTTTAGGATCGTTGGGCTGTGCGTTAGG GGAGGAGGTGGTCCTCAGCGTCCTGGAACGACACCACCTGCTCCAATTTCCCGTCTGGAGGTTCTGGTCGA GGCTCCGAACTCGGGTTCCCTGCTACCTCCCAGACTATTCAAGAATTATCCAGTCCCAGGATGATAAGGGGG AAGATGGGAAGAAACAGACGGGAGACGCCCGCCCAGAAAGACTGCGGGAAGAAAGAAATTCGAGAGGAAA CTGCACGCCACTGAGCGCCTCCCAAAAGCCTTGGAATGAATGAATTTAAAAACTATATTAGGGCCGGACTGC GGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGTGGACTACCTGAGGTCAGGAGTTC GCACCCAGCCTGGCTAACATGGTGAAACCCCGTTTCTACTACAAATACCAAAAATTAGCCGGGCGTGGCGG CTCATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGTGGGGGATCATTCGAGGTCAGGAGTTCGCAACCA GCCTGAGCAACATGGTGAAACCCCGTCTCTATCAAAAAATACAAAAACATTAGCCAGGTGTGGTGGCGCAC GCCTGTAGTCCTGGCTACTCGGGAGGCTGAGGCAGGAGAATCTCTTGAACCTGGGAGGCAGAGGTTGCAG TGAGCCGAGATCGCACCACTGCACTCCAGCCTGGGCGACAGAGTGAGACTCTGTCTTAAAGAAATAATAAC ACAAAATAAATTGTATTAGAGAAAAGCCAGAGTAGTGGAGAACTGCAGAGGAACGCGGGGCACCTACATAA ATGTCTTGAATGAATGAGTGCACAGAGTGATAGACAAAAAGAATCAGAGGGCCGGGCTCCGTGGCTCACGC CTGTAATCCCAGCACTTTGGGAGGCCGAGCTGGGCGGATCACAAGGTTAAGAGATCGAGACCATCCTGGAC AATATGGTGAAACCCCGTCTCTACTAAACATACAAAAATTAGCCAGGAGTGGTGGCGCCTGCCTGTAGTCCC AGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGACGGAGGTTGCAGTGAGCCGAGATCGCGCCACTGCACTCCAGCTTGGCGACAGAGCAAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAGA GAGCCAGGGGCTCCTCTTGAAGCGAAGAGGGCAAAGGGCAAAGGGGAAGCACAGGGGAACTTCGCGGCG CCCTCTGAAGCTCCCTCTCGAATATAATCGCAACGAAAAGGCCAACGACTAGAGGCTTTGCGAGGCTGAGG CTGGGCTTCGGGAGGGGATTGCCCTGAGAGGTCCGGGAGGACTTGCTGTGGAATTCAAGCGACCGTGGGC CTTGAGGGAACCGGGGGGCAAGACACCCACCCAGCATTCGCGGAATATTTCCTCGAATTATTTCGGGGAGGG GTGAGGCCGGGGCAGGGTGGGGCCTTCTTCGGAGGGGGCGCGGCCTCCGAGTAATTAATCCCGTCTTTGTT GCGTTTTGCTCCTCTCCTGTCCACCCAGCAGGGCCAGCCCAGGGCGCGCTAAGAGTCCAGAGAGTTCGTTTC CATGGTGACGGGTTCCGCGAAGGTTTTCCTGGGGTGAAGAGGCAGGGCGTTGAATAATCGCCATGGCGACA GCAGCAGATGACGGTGTCCCTTCTGAGTGCTCCTACCTAGAGTTAAGGGATACCTGAGGGTAAGCAACCGAG TGACGAAACAAAGAAGGCGGGGCCTGAGGACAGAACGCCAAGGTTAGGGGAATGGAGCCAGGCAAACGAG GGGCGGGGCTGTAGATGACCCGGTCGGGAGAGGGCCACGGTTTGTTGGGGGAGCGGCTCGAGATTGCGTT CTAGAGAGGAACCAGAGAGAGGGTCTTTAACCTAAATATAAATGAATGACTGGATTCCTGAAGAATCCGGAAT GGCTTGTTGATTGGATAGATGGATGGATGGATGGACGGACGGACGGACCGATGGATGGAAATCTGGCTATC ACTGACGCCTGAGCTCCCCACCCTCTTGGGCCCTCCACCTCCGGAGCCCTCACTCGCTTGTGACAGCTGTAC GAGAAATACATGCCTCTCCTAGGAGCAAACCCTCAACCCAAACAGGCAGCACAGAGCCAGTCCAGCACCTC ACACTGGAGGCACTCAGGGTGGAGCCCAGGTCGATGAGACGGCGTAGGATGAGGCTTTTTGGCCCAGCTG GGAACCACTTCTTTCCAGATTTCCCGTCCAGAGTCTAACTTTCCTTTCTCCCAGCGCCATCTTTTCTGCTAGTT TGCCCAGCTCCTCAGGGTGCCTGGACTTTCAGGCCTCACCTTGTGTCCAGTATAGCAGGGTCCAGCGCCCC AGCAACTGGGAAGGTCTGCAT (SEQ ID NO: 1)

[0067] In some embodiments, the CpG sites that are measured in the context of the present invention are located in promoter or 5'UTR regions. In some other embodiments, the CpG sites that are measured in the context of the present invention are located in intron or exon regions. In relation to the LY6G5C genomic region, the sites are preferably located in intron or exon regions.

[0068] In some embodiments, the genomic regions referred to herein and in Table 1 are referred to as "differentially methylated regions" or DMRs. DMRs are defined as genomic regions that have been found to be differentially methylated in subjects known to have high or low psychological resilience. The DMRs described herein contain various differentially methylated sites (i.e. individual CpG dinucleotides) within these regions. The number of differentially methylated sites within each region will vary. For example, each DMR may contain, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 differentially methylated sites. In some preferred embodiments, the DMR contains at least four differentially methylated sites. In some preferred embodiments, the DMR contains at least eight differentially methylated sites.

[0069] In the case of the LY6G5C region, sixteen differentially methylated sites were identified. The skilled person will understand that not all of the differentially methylated sites need to be identified in order to determine or measure the resilience status of a subject. As shown in the Examples herein, it is not necessary to measure or identify methylation in all of the eight sites that were identified for an individual to be classified as highly resilient. In the case of LY6G5C, the inventors have shown that methylation at four sites gives a good classifier of high resilience. In some preferred embodiments, eight of the CpG sites in the LY6G5C region are differentially methylated.In some embodiments, the DMRs described herein are described as being "hypermethylated" or "hypomethylated". By "hypermethylated" we mean there is an increased level of methylation compared to a baseline level. By "hypomethylated" we mean there is a decreased level of methylation compared to a baseline level.

[0070] In some embodiments, the subject herein is defined as having low psychological resilience if there is hypomethylation in the LY6G5C genomic region. In some embodiments, the subject herein is defined as having high psychological resilience if there is hypermethylation in the LY6G5C genomic region.

[0071] In some embodiments, increased methylation of CpG sites is associated with greater psychological resilience. For example, as shown herein, increased methylation of CpG sites in the LY6G5C genomic region has been shown to be associated with greater psychological resilience.

[0072] In some embodiments, the genomic region is C8orf31. As shown in Table 1, by C8orf31, we mean the region of sequence in chromosome 8, positions 144,120,626-144,141,359 (as determined by the GRCh37.pl3 assembly). The forward sequence is shown in SEQ ID NO. 2 below, which also includes a region lOOObp upstream of C8orf31. In some embodiments, the genomic region as defined herein is the sequence defined in SEQ ID NO. 2.

[0073] Legend:

[0074] CpG site in classifier (bold underline)

[0075]

[0076] >NC_000008.10: 144119626-144141359 Homo sapiens chromosome 8, GRCh37.pl3 Primary Assembly, forward sequence GATATGGCACTGGAATGTTTCTAGGCAGCCCACTCCTGTAAGAGGGCCTGGGGCTGGGCCCAGTAGTTCAC ACCTGTAATCGCAGCACTTTGGGAGGCTGAGGCAGGAGGATCGCTTGAGCCTCGGAGTTCGAGAGCAGCCT GGGCAACATAGAGACCCCACTTCTACTTGAGAGACACACAGAGAGAGAGAGAGGGAGGGAGGGAGAGAGG GAGGGAGGGAGAGAGAGAGGGAGGGAGGGAGAGAGAGAGGGAGAGAGGGAGGGAGGGAGAGAGAGAG AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG GGAGAGAGAGAGAGAGGGAGGGAGGGAGGGAGGGAGAGAGAGAGGCTTCTGGTAAATGCTCCTATTTTG CAAACTCTCTTCCTATTTTGTGATTAGGATGCTCCATCAGTTTCTGCCACCAGCTTGCTGGAGACGCTGCGT GTCCCTGACTCCTCTCAAAGGGTGAAAAGCTCAGTCGCACCCGAGACCTGCTCCTCAGGCAGAGGATGAAC TGAGTCCCCGCCCCACCGCCCGTCTGGCCTCTGTGAGAGTGGCGTGCAGGCTGACCCTGGAAGCAGAGGG ACATTCGAACGGGCCGGCGGCCTTCTCCCCGCTAGGCTGGGCTCCGGGGTGGGCGGAGAGTGGCCGCCAC AGGCCATCAGCGTCCGGGGTTCTGGAAGCAGGGAGGGCAGAGGCTGGCCCAGCCTATGGGAGACCCTGC AGAGGCGTGGCCTCCAGGCTAATCCTACCCCAGCTGCCCCAGGGCGGGATTTCTTTTC I I I I I I CCTTTAAT CTCTTTATCTACAGGCGCCGAGGCTTAATGAGCTCCAGATGTCTCAACATACCAGGAGAGGTTTAGGATCCC CTGCCAAATACAAGGGAGATAAGAACGCACCAGACTTGCACAAACCAGAGCTGGTGACCCCCAGTTCCCTTT AAATCAGCGACATGTCATTACAAGGCGGAAGTCCTGCCCGAGAGGACAGTCCCCAGGCCTTGCTAAGCATG CGTGGTATCAAGAGGTGCATTGAGGCGCTCCTGCAGCCTCCTCCCGGCACGGGACTCCTCACAACCCCCAG CCTCAGCTTCCCTGACAGGGCAGGGGCTCACTCTTCTCCACTCCGGGCCAGGAACGCAGCCTGCTTGCCTT TTGTCCAATTGGGTGCTCTTTCTTTGCGACTGATACAAAGTGGGAACAAACTCCGCTTCCGGTGATGCCCGG GCCCCAGGAGGCTGGGGGAGAAGTGCACGCTGCCCAGCCTTCAACCTGAACTGCCCCCAGCACTTCCGAG CCCCGACCCCTGCAGCTGTGAGGTGAGGTGGGGAGGGGGCGGCCTGCGGGGAGGGGGCTGCCTGCGGG GAGGGGGCTGCCTGCAGGGCGGGCCGTGTGGGTCCCCCGAGCGTGGGGGACCAGTGACGTCAGGCAACA TTTCTCCAAAGGCTCCCTCACCTTGTCCTTTGGCATCTGGGGTGCTCAGAGGAAGGGGGAAGCCGGGAGGGTGTGGAGAGGGTCACTGGAGAGGGGAAAGGCCAGAGTGATCCCCTTGGGGACGAGGCAGAGGAAAAGCT GTGGAGACCTAAAGAGAAGAGGTGGGGTGCGCCTGGGCCTCTCAAGTTTGTTTCCCAGAACCAGGGAAGG TTACAGTGGAAGGGAGGACCCTGTGGGGAAGAAGCTTCCAGAGACCTTCTGGAGAGGTGAGCCAAAGGCG AGCCATGGCCAGAAACCCCAGGCCTGCTGCAGTGGAGAGTCAGTGCTGCAGGAGGCTTGGGAATCAGGGC TGCCCTCCTTGGGCTGAACCCTCTATCTAGGGGACAATGGCCCTGGGCAAAAGCACTGCTTTCTGGGCCCC CTGGAGGGTAGGGCATGACACTGTGGTGCTAGAGGCAGGGGGGACATGAGGAGGTAGACGAATGTCCTGA GTCACCAGGTACAACCACTGCACCCCATTGTGACTGCCCCTGTGTCCGACACAGATTTGGGGGGTCTGGAA TGTCCCTTGAGCTCCTTAGGCACTCATGAATTTCTAGTTGAGCAAATAAAATGTCCAAGATGCCCAGTTTGAT CTGAATCTCAGATAAACAACAAATAGTCATTTTTACCAATGTTTGGGACATACTTATGCTAAAAATTGTTTATC TGAAATTCATATTTAATTGGGCATCTTACCCCATTGACCCTAATTAGCAAATCACATCCAGGAGTACAGGGCA TGGCTGGGTGGTGGCTGTCACACTGTGCAGTCAGAGGGACCAGGACGTCTTGGATGCAGGCGTCTGGCCA GCGTGGAAGGCCAGGACCTGGTCACGAGATGTGGCTCCCGGCATGGCAGGTCAAAGACTGCTGGTCGTCT GAGCCCAGCCCTGACTCCAGGCTGAGATGGCCATTCACCCAGACCTTGAGCCCTGCCCTGACTCCAGGCTG AGACGGCCGTCCACCCAGACCATTCTGAGCCCTGCCCTGACCCCAGGCTGAGATGGTCATCTATCGGGGGA AATTCACCCCCGATATTTCATGTAGGTTCTTTTCTATTTTCCCTAAGTGTCGGCCAGTCTGAGAAATAAAGGG ACAGAGTACAAAAGAGAGAAATTTCAAAGCTGGGCGTCCGGGGGAGACATCACATGTCGGCAGGTTCCGTG ATGCCCCCTGAGCCGTAAAACCAGCAAATTTTTATTAGTGATTTTCAGAAGGGGAGGGAGTGTACAAATAGG ATGTGGGTCACAGAGATCACATGCTTCACAAGGTAATAAGATATCACAAGGCAAACGGAGGCAGGGCGAGA TCACAGGACCACGGGACCGGGGCGAAATTAAAATTGCTAATGAAGTTTTGGGCACACATTGTCATTGATAAC ATCTTATCAGGAGACAGGGTTTGAGAGCAGACAACCAGTCTGACCAAAATTTATTAGGCGGGAATTTCCTCA TCCTAATAAGCCTGGGAGCACTGCGGGAGACTGGGGCTTATTTCATCCCTTATCTACAACTGTAAAAGGCAG CCGTCCCCAAAGCAGACATTTCAGAGGCCTCCCCTTAGGGATACATTCTCTTTCTCAGGGATGTTCCTTGCT GAGAAAAAGAATTCAGCGATATTTCTCCTATTTGCTTTTGAAAGAAGAGAAATATGGCTCTGTTTCACCTGGC TCACAGGCAACCAGAGTTTAAGGTGATCTCTCTTGTTCCCTGAACGTTGCTGTTACCCTGTTCTTTTTTCAAG GTACCCAGATTTCATATTGTTCAAACACACATGCTCTGCAAACAATTTGTGCAGTTAACACAATCATCACAGG GTCCTGAGGCAATATACATCCTCCTCAGCTTACGAAGATGATGGGATTAAAAGATTAAAGACAGGCATAGGA AATCACAAGGGTATTGATTGGGGAAGTGATAAGTGTCCATGAAATCTTCACAATTTATGTTCAGAGATTGCA GTAAAGACAGGCATAAGAAATTATAAAAGTATTAATTTGGGGAACTAATAAATGTCCATGAAATCTTCACAAT TTATGTTCTTCTTCCATGGCTTCAGCCGGTCCCTCTGTTTGGGGTCCCTGACAATAGCCGTCCACCCAGACT GTTCTGAGCCCAGCCCTGACTCCAGGCTGAGACGGCCGTCCACCCAGGGCGTTCTCAGCCCAGCCCTGACT CCAGGCTGAGACGGCCGTCCACCCAGGGCGTTCTCAGCCCAGCCCTGACTCCAGGCTGAGACGGCCGTCC ACCCAGGGCGTTCTCAGCCCAGCCCTGACTCCAGGCTGAGACGGCCGTCCACCCAGGGCGTTCTCAGCCCA GCCCTGACTCCAGGCTGAGACGGCCGTCCACCCAGGGCGTTCTCAGCCCAGCCCTGACTCCAGGCTGAGAC GACCGTCCACCCAGGGCGTTCTCAGCCCAGCCCTGACTCCAGGCTGAGATGACCGTCCACCCAGACCATTC TGAGCCCAGCCCTGACTTTCTGTCCAGAGTGCAGTCCTGAAGGAGCCCCTTTCTCCCACCACCAGGGGAGA CCAGTCTAACCCCTGACACGGGGCATCATGGGAGCCCTAGCCAGGGCACAGAAATGACAGGCCTGGCCCA CACCACAGGATGGCTGGTGGCCGCAGGAGCTCTGCCTCTTGGTGACCATCCAGAGGCTCTCTGACAACCCT ACTCAGATGTTCCCTGGAGCCACATGGCAAGAGTTCTGCCAGAAGAGCCATGAGCCTGGCACTCTGTGGGT TCTGGTCCCTGGGGGGGCACCCGTGCCCCAGGAGCACCTCCCCAAGGCCATGGTGCCAGCAGGATGTTGC AGAGGCCTGAGGTCAGAGTGGCAGGTGGGCGGCCTCAGGAATGGTGAGGAAGAAGCCGAGTGCTGACGC TGAGTGGGTCAGCAAAGGGCTGTTTGTTGACCTTGAGAGGGAGGCCCTCATCCTGTGTGGGAGGCCAGCA GGGCCCCCTGGGACCACGTTCCCTCCTTGCTTGGATTCAGGTAGCCCAGAGAGCTGAGGGGTTGGCCCTGG CTCCTCGGCAAGTTAGTGGCAAGCTGGATGGAGCTGGGGACTCCCGCCCTGGGCCACGGTTTCTCTATGAC CCTAGGTCCCAAGCAGCCAGTGGTGGGGCCTGGTCTCTGAGGCCCAGCTCTGAAAAAAGTGGTGTTGTTGG GGGGCATGGGCTGGAGGGCCTGGGCCCAGAGGACAAGGAGTTTAAGCAGGGATATGTTCCCTGACAGGGA AGCCCCGACCTCTGCCCTGGCCACATCCTTCCCCTGATCTCATGGCTGAAAAGCCACACCAGAATTCCTGCA ATTCAGTGAGGCAGCTCTTCAAAACCAAGCAGCTGGTGACGCACAGGGACAGAGGGTAGGCCTGCGGGGG AGAGGGCGGGAAAGGCCCTCACAGCAGGGCCCAGAGGAAGACTGACCCCAGGCTGAGCAGGAGCTGTAC GCACAGGGCCCAGGGGCTGCTGGCTGCCAGGACCACCGCATTGCAGAGGTCTCCCTTGCAGCAAGAGATC TGGGAGTCCACAACGGCACTTGTAAGTTTCAGCAGGGGGAATCCGACGTCCTTCTGGCAGGAGAGGAGGG AGAGCTTGTTCTCCCCTCTCACTTTACCTGCGAGAGAGCGGGGTGGGGGATGAGCAGGGCCCTGCCCCGG GGCCTCAGGGGCTTTCTGCAGGGAGGCTCCGCACCCCAACATCTGCTGGCTGGGAGCCTGAACATCAAAGC CATCAGGACCAAGGCCATCGGAGCCCTTTCACTTAGGGGACGCTGAGGCCACCCAGGGAACCAGGGTTGT GGCAGAAAGGGCAGCAGGGTTTGAGTTCTTTCCCCAGGCAGCCCTCACCCTGCCCCGGCCTTGGGACCCCC GAAGCGGGAGCTGGGTGGGCTGTGGAGGGGAGCTGGGTGCTGCTTTTGGGAAACAGCTGCGGGCTCCAG GCTGGC I I I I I I I I I I I I AAATATC I I I I I GAGATGGAGTTTTGCTCTTGTGGCCCAGGCTGGAGTGCAATGG GGCGATCTCTGCTCACCACAACCTCCACCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCCAGTAGC TGGGATTACAGGCATGCCCCACCAGGCCCGGCTAATTTTGTATTTTTAATAGATACAGGGTTTCTCCATGTTG GTCAGGCTGGTCTCCAACTCCCGACCTCAGGTGATCCCCCCGCCTTGGCCTCCCAAAGTGCTGGGACTACA GTCATGAGCCACCGTGCCCGGCCAGGGCAGGCCTTTTCCTGCCTCACCTCACTGAAGGGTGTGGCCTGGG GACAGATGCAGCTCCGAGAGCTGAGGGTGCTGAGGGTGCTGAGGGTGCTGAGGGCCTGGGCAGGCTGCA GCCCCTCCCCAGTCCTCCCATGGCCTGGCTGATATCCCACCATTCCAGAGTCATTCCTTTAGCTTGAAGCTAA CTGTCTCCAGGTTCTTCCAGGCAGGTAACTCACTGAATGCTCACAGCCTGCGCCTCTCCTGCATGGAGCTGGTGAGACCCGCGGCATGAAGTGGGGACAAGGGGCAGAGGTCACTGGGGTCCCCAGAGTAGCTACAGCAGCT GGCGTTGGGAGGCTACTGGGAAGTTGGTGTGGGCCCTGCTGCTGGGCACTGGGAGGGGCTGGGATGGTTT CCACAGTAATGAGGGCCCTGTGGGCGCCCCCTGCTCTAGAACCCGGAACATCCCCTGTCCCTGCAGACCCA CAACCCTTAGGCTCAGTTTGGTGGCCCAGGCTCCTCAGCCCTGCCGCTCTTCCACCCTTCCCTGCACAGGTC CTGCCCGCCATGCCCACCCCACCCCACCCCCCACCTCAGGAGCCACAGACCGGCCCCCAAAGTGCTGAGGG CTTGTAGGGGGACCTTGGTCCATCATGCAGCTATCTAAGAAGTGCCCCTCCCTCCCCAGAACCTGCCCTCTG CCCTGGCACCCCAGGGACTCACTGCCAAAGACGCTCACTTTCTGGGAGACACAGACCCCATCCAGGAAGGG GCACGAGACCACGCTGCAGGAGGCCCCTTCCAAGACCGCCAAGCATCTGTAGCAGCGCAGACCCTGAGCT GGGAGAGAGGGCAAGGTGGGTGACTGCAGCACATGCCAGGACATACACCAATGCCCTGGTGAGAGTCCCA TGCCCACAAGGGCACCCACTCTCACCACCACCCTTGTCCCAACCTGCCACCTGGACCTTTGCAATAGCCCCA CACACCTGCCTGCAACTGGCAGCCTGCAACTGGTGGCCTGCAACCCTCCAACACCACCTCTTTGTCCAGAG GCACCAGCAGCCACTGCCCAATGCCAAAGGCATACACGTGCCCAGGACTCAGTGAGTCCTCGGAAGTGAGC ACAGCCAGCACGCGCGGTAACAAGCACAGAGCAGGCACCAAACACACCAGTCACCTCTGCCACCGGAACAC CGAAGCCCGCCACCCCCAGCAGGCACGCCTTCCGAGCACACCCACCTGCAACTAGTTCCAGCACACACGGG GCAGCAGGAGTGCGCTGGAACAAAGTCCTCTAAAGGCCCCTCTGCTTCTCACCTCTCTCCATGCTCAGCAGG GCCACCAGCAGGACCAGGGACAAGGTCTTCATGGCCTGGAGACTGCTCATTCCAGGGTGCTAGTGCACAGA CAAACTTCAGCGAATCAGAGCAGCCCTCGGACTCCTCACCCTCCTGGGGATCGAATGAGTCCTGCACAGCA GCAAAGCCAGAACATCCGGCTCTCCATCTCCCACCCTGTCCTCAGCACAGTCACCCAAAGGAGAACTGTCTG CCTCACGCCCCCACCCCAGTCCCCATTCCTCCCTCCCATCCTTCCCTGCAATTCTCACCCAGAGCCCCATGG CTGTACTGCACCTCCTCTCCTTAGCGCCCCCACCCAGGACCCTCCCAGGCCTGGTCCACCCCTCCTCACTTT CCTCCCTCTGGCTGATTCTCAGCCACTTGCCCTGCAGGCTTGCCAGAGAGAGGAAGGTGGGGCAGGGAGA GGCCCTGCCCTCAGCGGCTCCTCCCCTCAGGCTCTTCCTGACTGGCCCAGAACAAGGGTGGGTTCCTGAAT TTCCATTTCACAGGAACTTGCTACTGGAGCAATAGGTGACTCCAGGCCAATTTACTCATTTTTTTTTTTCTTTT GAGCTGGAGTCTCACTGTTACCCAAGCTGGAGTACAGTGGCACAGTCTTGGCTCACTGCAACCTCTGCTTCC CAGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCGAGTAGCTGTGACTACAGGTGCCCACCACCACACCCAGT TAATTTTTGTATATTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGACCTCAAGT GATCCTCTCACCTTGGCCTCCCAAAGTGCTACGATTACAAGTGTCAGCTACCACGCCCAGCCCAATTTACTCT TTATACTAGAATTGGCAGCAGGTGGGCCTGGTGGCTCACATCTGTAATCTCATCACTTTGGGAGGCCAAGGC AGGAAGATAGCTTGAGGCCAGGAGTGAAACCACTTTTGCAAGATTATAACAATGAGAGGAGGCCAGGCACG GTGGCTCACGCCTGTGATCCCAGCACTTTGGGAGGCTGAAGTGGGCGGATCACAAGGTCAGGAGATCGAG ACCATCCTGCCTAACATAGTGAAACCCCATCTTTACTAAAACTACAAAAAAATTAGCAGGGCGTGGTGGCAG GTGCCTGTAGTCCCAGCTACTTGGGAGGCTAAGGCAGGAGAATGGCATGAACCCGGGAGGCAGAGCTTGC AGTGAGATGAGATTGCGCCACTACACTCCAGCTTGGGTGACAGAGTGAGACTCCAGCTCAAAAAAAAAAAA AAAAAATGAGAGGAATCTAACATAACTGACTCCATCTTGCTTTCTAATCTCACAAGCTAACTTGCCTTTGCTC AGGTGGCATAGGCCAAGCTAACTATGGGAGGAATTTAGTTCATAGTTTAGAGTAAGGATGGGCCAGGCATG GTGGCTTACACCTCTAATACCTTTGGGAGACCGAGGCAGGTGAATCACCTGAGGTCAGGAGTTCGAGATCG GCCTGGTCAACATGACGAAACCCTGTCTCTACTGAAAAAAAATAAAAATTAGCTGGCGTGGTGGCGGGCAC CTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCACTTTAACCCGAGATGTGGAGGTTGCAGTGA GCCGTTATTGTGCCATTGTACTCCAGCCTGGGTGACAAGAGCGAAACTCCATCTCAAAACAAAAATAAATGA ATACAAATAAAAAATAAAGTAAGGATGATAATAGTGTCTTCCCAAAACTACTCCACTCCTTGAGACCAAAGCC GCCTTTGTAAAACTAACGAAAGACCACAAGGTTAGCATTATGGTAGGGGCTTGATTTTTTTTTTTTAAGATGG AGTCTTGCTCTGTTACCCAGGCTGGAGTGCAATGGTGCCATCTCAGCTCACTGCAACCTCTGCCTCCCGGGT TCAAGTGATTCTCCTGCCTCAGCCTCCGGAGTAGCTGGGATTACAGGCGCCTGCCACCACGCCCGGCTTTTT TTTTTCTTTTGTATTTTTAGTAGAGACGGGGTTTTACCATGTTGGCCAGTCTCAAACTCCTGAGCTCAGGTGA TCTGCCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCATGAGAGGCACGGCGCCTGGCCCAGGGGCT TGACTTCTGCTCATGAGCCAGGATAGGTAGTCAAGGAAGTGACCATATCCTTGGGATGCAGCCACCGTGGC CACTGTACAGTCAACACAGTAAGCCTTTGCATTTGCGCTGTGGTCCAGCTCATCCAAGCAAAGCTAGCTCCA GGAGAGAAATCCCCCCTCCCAACTACAGAGCAGGCATATTTGATTTTCCCTGTCCTCAGACTGACCCTTTGC TCATTATAATAGAAAACACACCCCTGGTGGAGATTTAAGATGCTAATGACACATGCGACGTATGAGCAAGCA GGTGCAGCTACTGCACGTGTGCACCCAGAGGACCCCCCCAGAACATGCTTCCTAGCAACACCTCTGCCCAC CCGCTGTGAGTAATCATGGAAGACTCCCATGGAGGAGCCTCCCTGGTGCCAGTCTCTGCTGTCTCGCCCTTA CAAGCAGCCGGCCCTGAATCCTCTCTCTCAGGGCGTCCTATCTCTTCTGCATCAAACTTTCAAAATGTTCTTT CTCCTTTGCAATACATTGCTCTGTGCTGCATCTCCTTGGCTGTGTGTCTCTTGTTTAAATTCTTTTAAACTAAG AAGACAAGAATGGAGGCCTCACAGCAGCCGCCAACGTTTCTGGTGCTGTGACTCGGATGGAGGTTCGTCTG CGTCATTCACTCCAGTTTCCCTTCCCCGCAGCGGGTACCGTGGCAGTGCCAGGCTGCCTGGTTGGCTGCCG CTGGTTTGCCCAGGGCTGTTTCAGTCAAGCTCCGGGGAAGGTTTCTAAGTTGCCTGGACCGTTTCTGTGGAT ATATGTGCTGCTCTCCTCTGGCTGCTGCCTCTGCACCACTCATCATTATCCAACCACATCGGTTGCTCTCAAC ATTCAGTATTTGGGCTGTTTCGCTGCTTGGTTTCACGCCTTTCTGGCCTCGTTTCAGACACAGCCCGTTTGCT GTCCCTCTGGTAGCACTCGGCCGCCACCTCGTGGCCATTGTGATTTATTGTCTGATCGGGTTTTCTGTTTTAC AAAATTTTC I I I I I I I I I I I I I I I I I I I I GAGACAGAGTCTCTTTTGCCCAGGCTGGAGTGCCATGACACCACC TCAGCTCACTGCAACCTCCACCTCCTGGTTCAAGCGATTCTCCCGCCTCAGCCTCCTGGGTTGCTGGGACTA CAGGGGTCTGCCACCACGCCCGGCTAATTTTTTGTACTTTTAGTAGAGATGGGGGTTTCACCATGTTGGCCA GGCTGGTCTCGAACTGCTGACCTCAAGTGATCCATCCACCTTGGCCTCCCTAAGTGTTGGGATTACAGGTGTGAGCCACCGTGCCTGGCCGAAATCTGTTTTGAGGGACACATTAAGAGTGCGCTGTCCCTCCAGACCTTATGC ATTTTCCACCTCCTTCAGATGGTCCTTCAACTGGCGTTCTTTGCTGAACAAAAGATGCTCAGTCATGTATCTG CCAGGCTGTGGGGACTGCATCGGGTATTCCAGGCACTGTAATCGGGCATCACCAATGGCCAACCAGTGAGG CAGGGAAAGGCTCGCTGATGAGACGTTGGGCCCCCCAGCCAGCAGCGGGGGCCACCTCAGTCGGGCCTGG AGACTTCCAGCGCCCGTGAGACCCAGGACGGTGTATGCCAAATGCCCGTGACCTCCTAGCGCCCTGATTTC ATGGGGGTTCAAGGGGATGTCCCACACCCTGTCATGGTCCAGCTTGGCTCGGGGACACCTGTGACCTCCTG GACTTTGGTATCTGTTTTTGTCATTGCAGGATTCTCTCGGCACCACGGGAACCACCTTCTCTACTCTCACTGA ACACCCCTGGGATGTATCTGTAAAAATTGGAACACCTTTCGGCTAGGTGAGCTAAAACGGAAAAGACATCTT TTGTTACACTGTTTGTCCTGGGTACAAGTTAGCTGACAATGAAAAGTGGTCAGAGAGTGGAACTGTGAGCTT CATCGCCATCCCGCAGCTCCGTCTTTCCAGAGGAATCAGGGACAATGGTCAGAAGTACCCATGAGTAGGTA TTTATGGCCTTACAACGGAACCCGGCTTTATGCAGCACCTGCAGGCTAAAATCCAGTAAGCCAGAAAGCCCC CCAGACCCATGCGAAGACCCTCTTTTATTAAAGGGAAGGGACTCCAGGCCCCATGGCCCAACTCCAAGTCC AGATAGGGGTTCTCAGGGCTCCACACCTCCTTCCGACTCCCCAGCATCCCCCGCACTATCAGAGTCTCCTGT GGAATCTAATCCCATTTCACCCCCTCCTTATGCTCCTCCTCCTTTGCAGGTACAATAGGGACTTGCCCAGCTG GAGCTGCTCGCACTGGGACTTCACACCATCCAGGGCCAGAGAAACTGCTCCCCTTATGGGAGGTCCCAAAT GGAGAGGGGACCATTATGACACATGTCCCATAAATGATCTAATTTATAGACTCACATCTATAAATAATCTAAT CCAATGGAAGCAAAAAAAACAAAAACTCATCTTCTGTAGCAGAGAAATGGAGAGTAGCCCTAGGAGTCCTCC CGAAACTGGGAGAAACACCCTGTCTGGTTGTGGGAGAAACACCTCGTCCAGTTGTGGGAGAAACGCCTGGT CCGGTTGTGGGAGAAACGTCTCATCTGGTTGGCTCCTTTCCAAAGCAGCTGGACTCTGCGCCCTGCAGGTG GCCTGGCTGCCTTAGGGCAGTTGCAGCCATCGCCATTCTAGTAGAAGAAGCCCAAAACCTGACTTTTTTTTT I I I I I I I I I I I I I I GAGACGGAGTCTTGCTCTGTCACCAAGCTGGAGTGCAGTGGTGTGATCTCGACTCACTG CAACCTTTGCCTCCCGGGTTAAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGAATTACAGGCATGCA CCATCACGTCTGGCTAATTTTTGTATTTTTAGTAGACGCGGGGTTTCACCATGCTGGCCAAGGTGGTCACAA ACTCCTGACCTAAAGTGACCCACCCGTCTCAGCCTCCTAAAGTGCTGGGATTACAGGAGTGAGCCAGCTTGC CCGGCCCCAAAACCTGACTTTCAGACACACCTTCGAGGTCCTCACTCCCCACCAGGTGCAGGGAGTTTTAGA AATAAAGGGCCATGTTGGCTCTTGGGAGGAAGACTTGCCGTGATCAGGCGCCCTCCTGGACTCCCTCAAGG TTACTATAAAAACCTGTGGTACCCTTAATCCAGCTTCTCTCATGCCAGCCTGTTCCTGGGAGAACCTAACTCG TTCCAGGGTTGCAACATGGGCCAGGCCTTCCACAGTAGAATAGACCTCAGGGGTGCACCCTCTAGAATCCT GGTGCTGAATGGCTCACGGATGGAAGTAGTTGCATGGAAAATAAAAAAAGAGGGGCAGGACATGCTGTGGT TAGTCTACACCAAACAACAGATGCCCACACTGCCACCTAATACCTCGGCGCAAAAAGCAGAGTGAGTCCCCT GACCAGGGCCCGGCTCGGGGTCGGGGAAAGGCGCTTCACGGGTGTGCGGACTCCAAGGGCGCCTCCCTT GTCCCTCGTGCACACACAACATTTGGAAAGAACAGGTCTTCTAAATGCAAGGAAATCCCCCATTACATATGC AAAAGAAATACTGCGGCTTTTAGAGGCTGTGCAGGAGCCTGAGCGAGTGGCAGCCCTTCACTGCCTGGGAC ATCAAAGAGGAAACTCCTCAGTGACATTGGGAAATGCAAGGGCTGACAGGAAAGCTAAGAGGGAACTCCGG GAGCAGTGGCCCAGCTAGCCTTGTTCCCCTGTGCCCATTCTCTTGACGTAAATCCATCTTAACTACAAAAGAA CTAGCAGCTCAGCGTGGTGGCTGTAATCCCAGCACTTTAGGAGGCCAAGGCGAGCGGATCATCTGAGGTCA GGAGTTCGAGACCAGCCTAGCCAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCTGGCCGT GATGGCATGCACCCGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAGTCACTTGAACTGGGGAGGCA GAGGTTGCAGCGACCCAAGATCGCACCATTGCACTCCAGCCTGGGTGACAGAGCAAGACTCTGACTCAAAA CTAATAATAATTAAAACATCAAAACAAGCATAAGCTAAAGAAAGTCAGGGGACTCTAACACCTGAAGGTTAG TGGCTCATAGGTCAAAAGCTCTTGCTTCCTCAGCCAGACCAGTAGAAAATGGTTAAAAGTTTACATGATTCCC AGCTGGGCACAGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCGAAGCACGTGGATCACTTGAGGT CAGGAGTTCTAGACCACCCTGGCCTACATGGAGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGC ATGGTGGCACCCACCTGTAATTCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCACTTGAACCCGGGAGGC AGAGGTTGCAGTGAGCCGAGATCGTGCCACTGCACTCCAGCCTGGGTGACAGAGTGAGACTCTGTCTCAAA AAAAAAAAAGAAAAGAAAAGAAAAAAGCCGGGCGTGGTGGCCCAAGCCTGTAATCCCAGCACTTTGGGAAG CTGAGGCGGGCAGATCACGAGGTCAGGAGATGGAGACCATCCTGGCTAACACGGTGAAACCCCGTCTCTAC TAAAAATACAAAAAATTAGCCAGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCA GGAGAATGGCGTGAACCCGGGAGGCAGAGCTTGCAGTGAGCCGAGATCGCACCACTGCACTCCAGCCTGG GGGACACAGCGAGACTCCGTCTCAAAAAAAAAAAATTTACATAATTATCTCCATATGGGGCAGGACACCATG ACCACCTGGGTAGACCATCTGTTTATGGGCAAGGGCCTGGCAACCACAATAGAGTCACTCGGGCCTATGAA CTTTGCTCCCAAAATAACCGTGGGGGCAAACAAAGACAAAGGCCTCTCACTAACCCCAGTCCAGCAGCGAG GAACCCAGGAACCCCTCCTGCTGAGGACTGGCAGGTTGACCGCACCCACACGCCCTCCTTTCGAGGCTTTA AATACCTTTTGGTTTTGTAGACACCTTTACCGGTTGGGTGGAAGCTCTCCCCCAAGAACAGAGAAAGCTACA GAAGTGGTCAAGGCATGTTTTGGCCTCCCCCGCTCCTTGCAGCTAGAACCCAGCAGGGAGCTCGAGCTTTG GGAATTAAGTATCGTCTCCGCTCCTCGTGGAGGCCGCAATCCTCCGGCAAGATTGAAAGGACCAGTCACAC TTTAAAGCAAACCCTGGCAAAATTATGCCAGGAAACTTCTGGGTCTTGGTATACTCTGTCATCTATCGCGTTC ATGAGAATACAAACGGTCCCCAAAGCGAAAAGGAAACTTAGTCTTTTTGAAATGACTTATGGGAGACCCTCC CTCACCTTAGACCTGTTAATCAAACCTGAAACTCAGTGTATTGTAAAATATATTCAAAATCTAGGCCAGGTAC GGCTGGCTGTCCAGGAATATGGCAACACAGGGCTGCCCTCGCCTGGCAAGTGAAACACAACAGCAAAATGC TGCCTGGAGACTGGGTCCTATTAAACACCTAGAAGGAAGGTTCCCCAGCAGATCAACTTCTTCCTAAATGGA AGGGACCTTACCAAGTGCTACAAACCACCCCCGATGGCTGTAAAGCTACAGGGAGTCACCAGCTGGGCCCA CATGACCAGAATTAAACTTTGCAATTAACCTTTACAGAAACCAGAGGAAGCCGCTCATGACACCCGGGAGCTCACAGAGGCTCTGAAGTTTCTATTCAAGAAAACAACTCCAGATGGGGCTGATCTGGAAGACAAGTAACATCT TCCTTTTCTTGGTGCTCGCCTGCCGTGCTCTAGGTGCTTTTAGAGATGCTGGTCTGTGTGCTAGGGAAGCGA TGAACCATACTGTTAGTCTATTAAACATAACTCAACCTGGCTGGCTTTCCATGACTACCAAACGCTGGGAATA TGCCAGACCTGTGCCCGTGGAAGACTCTGGCCTCCCAGCACACCTCCAGGAACAAACTCCCTGGCCCGCAG GATAGGGATAGCAGCCCCAACCGTCAATACAAAGGGATTTTACTGAGTGGGCTCATGGGTTCTTTAAGTTCC CCCTCTTAACGATCAACGGTAATTCAAACGTCCCAAAAGGAGAAATGCTTTCTTAGCATATCTTTTACCTGGA GTATTTCCCCTTCTGCCTTTACAGCAATCATGCCAGTTCTACCACTTTTGCAAGAAAGCTCCAAGAGAGTCAG TGTAGCTGAAACTTTGTCACTGGGTCACCTATCTAGTCCATATGTCTGCAGGAATTATACTGATAAATGGACA ACTCACCATGCAATCAATCTCCCGCTCTATCCTGTTCCCTGACCGGCATGGTTTCTACTTCCTGTCAGCACAA CTAGGACGTCTGGTTTCTGCTGGGCTGCCCTCTCATGGGAAGCCTTTAATCACTCTAGCTCAATATATCCTCT GGGGACCATCAACATTTCCTATTAAAGATAACTACACCTGGCCGAGCGCGGTGGCTAACGCCTGTAATCCCA GAACTTTGGGAAGCCAAGGCGGGTGGATCACAAAGTCGGGAGATCAAGACCATCCTGGCTAACACAGTGAA ACCCCTCCTCTACTAAAAATACAAAAAATTAGCCAGGCGTGGTGGTGGGTGCCTGTAATCCCAGCTACTTGG GAGGCTGAGGCAAGAGAATGGCGAACCTAGGAGGCAGAGTTTGCAGTGAGCTGAGATGGTGCCACTGCAC TCCAGCCTGGGGGACAGAGCTACACTCCATCCCAAAAAAAAAAAAAAAAAAAAAAAGAGAGAGAACTACACT TGTGGTGGGACCCTAAGAGGGGAACCCTATTTAAAGAACTTGTTAACACTACCATCCAACTCTACCATCCCC TGATGGGCCCAGTGACCACTGCCAACATAGCTGCACAAAACCTTTCACTTACAGGCCAGAAAATAGAAAACC GCCTCTTATAGAACCTCCACTGCTTTTACCACTATAATGAAAAATACTTTTGTAGCACAAATTTCACCACCACA CGGAGTTGTGGGGGGTTGTGGATCTCCAGTGTATCTACGACTCCCTCCACTCTGGAAGGGACAGCGCTCCA TCGCTTACAGCTCCCTCCATCTAACTCCACGCTTACAGCTCCCCCCATCTAACTCCACACTGGCTACTACGTC TCCCCCTTTTCCCACGTACCAACATTGCGCGAGCCGCCTCTGAGCAGGACCCCTTCTTCCCTTGGGTTTAGT ATTATCCTCTCTATTGGGATTAGCAGGGACAGCTACGGGAGACAGAGCCTTGGGAATCCAGCATAAACTGTC TTGGGAGACTGAATGGCCCTCCAGCAAACGGCAGAGGGCCTCATGAGTCTTCAACAGCAGCTGGACTCCCT GGCTGCCGTAGTCCTGCAAAACCGAGGGGCCTTAAATCTTCTCATGCTGGACAAGGAGGAACGTATCTATAT CTAAAAGGGGAATGCCATGTCTACGTCAATCAGCCCAGTTTGGTCGTAGAACGAATTAAAAACATTGTTACC CAGGCAGACAAAATGGAATCTTTAGGAACTTCCATGGGAACTTGGAAGCAATGGCTGTTGTCTGCCCTACTC CCTTTAATAGTGCTGATTATTACCACACTTTTAGCCTTAACTTTT^ GTTGTTGCTTTTCTGTTTTAAGATCATTTATTAGAACACAGTCATTCAGAAGCCATTGAGACATCAGGCAGCA GGAAGGAAGCTGGGGCGGAGCAGGCCCTGGGAAGGACCAAGGACAAAGTAATAGCCACAGTAATGACATT TCATTTTATTCTGATAAAGACTAATGTATGCCTGATAACCTAGTGAGAATCCATAAGTTTGGCAGTTCACAAC ATTTTTAGAAAGCACATACGATTAACATTCAAATAAGGCATTATAGAAAGTTTTGTAAAGAATGAAGTGTTTA CTGTCATTCTTTTAAAAAACCTTGGCTCATCTTGAAAGATCAATGAATTTTTAAAATATCAGAAGAAAAGAAAA ATAAAAATTTCCCCCCAAAATACGTAAGAACCACTTACTGGCACTGGTATTTTAAGTACCTGGAAAAAAACGG ACCAGATTTTTAAAGGCAATTAATAACAGCTTGTACGAGCGCTTGTTTCATTTGACTTGGCATCAAGTAAAGG AAGAGTAAATACGCCGTGAAAGACGCCATCGGGCTTTTCCTCCACTTCTCCCAGACCACACAGCACATCAGC AGCCACCCTTGCTGCCCACTCAGGAATCACTGTAGATTCCAATTTTTAAATGGCTGCTTAGACGACGCCAATA GAGTTCTTTCTCCTTACAGTAAGGCGGCAGTGAATGCTAACAGGTATCGAGTTCTCTGATCAGGAACAAAGA ACTCCTTCAGGAAACTCACTCACTTTCCTGGTCCTTGTTAACCTGTCACATAAATTCTTTTTATTGGCACACCT GTTTACTAATTATGATTGATTGCTATTTATGCCAAGGGAGCATTTCCCAGGCGTGCCTCATCTGTTTACTCAT GACACAGTGAGCCTACTTTATTTACACAGTGCCGCGGGTTTCCTCTTTTTTCTTTTCTGTTTTT GGTGGCACTATCACTTGTTATTTATCAGGGTAGATCATACATTTGGATCGAAAAGAGAAACCGGCAACTAGA TCCTAAAACACATTTATCAACCTGAGTCACATCGGGAAACATATAGACTTTAATTCCATTTTGTTGAAAATTCA TTCAACTTTGGTGCTTGTCCAAGGACTTATGATGTCAATTTCTGACATAAATCATAACCCCGAATATATATGTA TTTTCAAAAGAAACAAGTCATCTTAAAGTAATATTTTTCTATATGCTAATTGATACATTTTTATAGCAAATTGAA AATTCTGAGCAAACTGAAAGTATGCTTAACAACAAAATAAATACAGCATATATGGTTAGCACATACATTTCTT ACTGTAAAGGCAGAAGTGAATTTGTGTCTTACAATAAATCTGTAAATCCAGTTGTTTTCTTTCTGGAATTTATA TAATGTCTCACCATGTTCCACAAAAAGGCTAGAAATGCCTTTTTTTTGGAATGAATCTATGCAAAAATTTCTGA TTACATATTTTCCCCAAATGACATGTAACTTTTTTTAACTTTTCCAGAAAAATATGGAAACTTTATCAACCACTT ATTAACTGAACAAAAAGTGAGATTACTACAAAATGCTCGTTTAATTTTGCTTTAACAGATGTTTTAAAAGTTCA GGCATCGCTGATATTTTTGAGGATAACTGCATAAAACACACTAGATGATTTCAAAGGATGAATCTTAGTATCT GACTCATCTGGCACATCCTTAGTATCCAGAATAAAATCAGTAGAAATAAAAGTAATATAGTTTTCAAAGAATT CATACATATTGGAAGTCTTAGGAAAAGTGCTTCTAAATGCAGGGACTAGGAGGTTTGCCCATCTTCCTGTTA ATAGTTACACACATTTCTCCTCATGGAGTAACTGAAGTTTTCTGGCTTGTTTGTGCAACTTTAGTTGGTAGGA AAGTGTATATATAGGGCCAAATCTTGTTGGTTTCTGTTCCGGAGAATGTTTCCAACACCCCCTTTTTTCTGGT AATATTCCGGGACTGGCTTTGTTTGGCTTTATAAGCCTTTAGTCTCTTTTTTACTATTATTATTATTTTTATTTT TTTTTTTGAGGTGGAGTCTTGCTCTGTCACGCAGGCTGGAGTGCAGTGGTGCCATCTCGGCTCACTGCAATC TCCACCTCCAGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCTGAGTAGCTGAGACTACAGGCGCCTGCCACC ATGCCCGGCTACATTTTGTATTTTTAGTAGAAACAGGGTTTCACCATATTGGCCAGGCTGGTTTCGAACTCCT GACCTTGTGATCCGCCCGCCCCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACTGCGCCTGGCTG CCTTTAGTCTCTTGATAACCGTCTCTGGTTTATCATCCTCATGCTGAATGAGAGGGTCCCCAGTCAGGTCATC AATGCCCACAGTTTTGGGAGGGTTGAATTCAATGTTGAAGACTCAGCCGCTGGCGGGATGAATCCAGCAAG CAGTAAGGCGTTGTTTAATGACCTCAAAGGGCACATTCAGCTTAATCACTGTGTCGATCTGATCAGCTCTATC TAGGGCTTCTGCCTGTGGATGTGTTCTTGAAAAACCATCAACAGCTAGACTGGGTGAGACTTTTCGGCTCCTGCAGCTGGCCTGACTGGGGGCCTGTGTCGGGTGCCATATGAGAGATTTCAACCAGCCCATGCGCAACCAGA GGGATGCGGCCCACGGTGCGGGTGGTCTCAGCGTCGTCTCTGTCTGACCCTCCCTCCCTCTGCATCTCTCT CAAATCTTGGCAGCCCACAGACTGAGTCAAATTGAGAAGAAAGAAAGTGGAGGCAAGTTCATTAACGTCAGT GCAATTAATTTAACAGTATTCAATTACAAACAAATAGAGCATTTCAGAGAGTGATGAGGGTTCTGAGGATGC ACAAACAAGGGGTGTAGGGTCCAGCCCCACAGGGTAGGTGGGTCTCTCCCCGCGTGCGGCGACAAGAGAT TGTAGAAATAAAGACACAAGACAAAGAGATAAAAGAAAAGACAGCTGGGCCCAGGGGACCACTACCACCAA GTTGCAGAGACTGGTAGAGGCCCCGAATGTCTGGCTGCACTGATATTTATTGGATACAAAGCAAAAGGGGC AGGATAAGAAGAGTGAGCCATCTCCAATGATAGGTAAGGTCACGTGGGTTGCGTGTCCACTGGACAGGGG GCCCTTCCCTGCCTGGCAGCTGAGGCAGAGAGAGAGAGGAGACAGAGAAAGATAGCTTATGCCATTATTTC TGCATATCAGAGACTTTTAGTACTTTCACTAATTGACTACTGCTATCTAGAAGGCAGAGCCAGGTGTACAGG ATGGAACATGAAAGCGGACTAGGAGCGTGACCACTGAAGCACAGCATCACAGGGAGACGGTTAGGCCTCC GGATAACTGCGGGCCGGTCTGACTAATGTCAGGCCCTCCACATGCAGTGGAGGAGTAGAGTCTTCTCTAAA CTCCCCTGGGGAAAGGGAGACTTCCTTTCCCGGTCTGCTAAGTAGCGGGTGTTTTTCCTTGACACTTATGCT ACCGCTAGACCTCGGTCCGCCTGGCAACCGGCGTCTTCCCAGACGCTGGCATTACTGCTAGACCAAGGAGC CCTCTGGTGGCCCTGTCCGGGCATAACAGAGGGCTCGCACTCTTGTCTTCCGGTCTCACTTCACTATGTCCC CTCAGCTCCTATCTCTGTATGGCCTGGTTTTTCCTAGGCTATGATTATAGAGTGAGGATTATTATAATATTGG AATAAAGAGTAATTGCTACAAACTAATGACTAATGATATTCATATATAATCATATCTAAGATCTATATCTGGTA TAACTATTCTTGTTTTATATTTTATTATACTGGAACGGCTCATGTCCTCTGTCTCTTGCCTCGGCGCCTGGGTG GCTTGCCGCCCACATGCCTCAGCCTCCCAGAGTGCAGGGATTACAGTCATGAGCCACCGTATGCGGCCACA TCTTGTTATGAAAAGAAATTCTCAATGTTAATGAGGTCAAATTCATGTATATTTACTCTGATAGTGCTTTTGTG GAGTAGACAAAGATTACTTAAAATATTCCCATGTGTTGACTTCTAAATGTTTTATGGTTTTAAGTCCTACATTC AGGGCTAGGATGGTTTTGGGTAGGCTGTGAGGTTTGGGGTCATGATTTACTGACGGTTTTTTTTTTAGACAA ATATTCCATTGATCCAGCACCATTCTTCAAGCTAACTTATAACGATCCCAAATGGGAGTATGGAACTCTAAGA AGGCAAGAGAAGCAGAATAATAACTATTTGGATAAATATAAAATATCAATGGTGTAAAATAAGAATAACGTCT TGTAGGTTTATAATATATATGCAAGTAAATATATATGGGACTATAGGCACGCACGACCACACTCAGCTAATTT TTAAAATTTTTTTGTAGAAATGGAATCATGATATGTTGCCTGGCTGGTCTTGAACTCCTGAGCTCAAGTGATC CTCCCACCTCAGCCTCTCAAAGTACTGGGATTACAGTCATGAGCCACTGTGTCTGGCACAAGCAAATATTAA AACAATGAAAGTACAATGGAATAGAGGTAGGTAAATGAAGTTATGCTGTGTAATGTTCTGAATTGTTCATGA GTGTGGGCGGCAAGCCACCCAGGTGCCGAGGCAAGAGACCAAGGGCACAAGCTGTTCCAGTATAATAAAG AAAATATATAGAATAAGAAT (SEQ ID NO: 2)

[0077] In the case of the C8orf31 region, nine differentially methylated sites were identified. The skilled person will understand that not all of the differentially methylated sites need to be identified in order to determine or measure the resilience status of a subject. As shown in the Examples herein, it is not necessary to measure or identify methylation in all of the sites that were identified for an individual to be classified as highly resilient. For example, the inventors have shown that methylation at four sites gives a good classifier of high resilience.

[0078] In some embodiments, the genomic region is CDH9. As shown in Table 1, by CDH9, we mean the region of sequence in chromosome 5, positions 26,880,709 - 27,121,257 (as determined by the GRCh37.pl3 assembly). This region is over 20,000 nucleotides long, and all of the differentially methylated sites were determined to be within the promoter region ENSR00001321802. Therefore, in some embodiments, by CDH9 we alternatively mean that this is region in chromosome 5, positions 27,037,001-27,039,200. The forward sequence of the promoter region is shown in SEQ ID NO. 3 below. In some embodiments, the genomic region as defined herein is defined in SEQ ID NO. 3.

[0079] Legend:

[0080] CpG site in classifier (bold underline)

[0081]

[0082] >NC_000005.9:27037001-27039200 Homo sapiens chromosome 5, GRCh37.pl3 Primary Assembly, forward strand TGTTCAAGTTCTTTGGTGCTATGTGCTGTAATAGCATGTTATTTCATTTTCCTCATCTTTCAAAAGATCTATGT TTCAAACCTGGATTAGCCAGTTTTATAGTTGCAAAAATGTATTTCACAATGGACACACATTGCCTAGGCAATG ATGTAGTGTCTGAATGTGGAAAATAGGATTCTTCTTCTGCAGAATGTAGGAGGAGGTTTTTTACAAAATTGAG AATCTTGAATAATCAAATTCATTCTTAACGGCTGTGCCTGAAGATGGGTAACTCTTCCAAATTCATAGATAAA GCAAAGGGAGTCGAAGGAAGATTCCCTTCTGTCTTGCAAAAGTTACCACTTTTTTTGATGTCACAAAGTCATT CATTCTTCATGCTAAAAAGATTTAGAAACCTTGTGAAAAAATCCCTATTAGGAATCTCATCTACCTATAATACA AAATATAAAATGACAACACATCTCCAGAGAAGACCACCAATTTCTCCTCCAATCTGTAGTCCATTATATCTTCA TCAAAAGTTGATGATACAAATAAATGTAAACTTATCTACTACAGTTAATAACTTTCTTTTTATTACACCTTTGTT ATTTAAAACAGATTATGAGAATTTCACCCTGGTAACAGTCTCTAACTGTCAATACTAAAGAATTTTGATGACCT TATAGCTAAAATCCAAGCCTTTATTGAGACGAGCCTACCTGCAAAAATTTATTTGATGTGAATGGTGATCTTG AGCATGCGATGGAAAACACAATAAAACAATGCTCCACCAAATCAATGCATTTCCCACATCACTTCAAATTTTT TAAATGCCAATTTTGGCTTAGAAACACTCTCCCTTTCTTGTGGCTTTCATCTATCTAGTCATCATTACATTAAG ACATATTTGCCTACAATAATAGAAAGCAATTATAGCTCACAGATGTATACATTCAGAGTGTCGACCTGAGTGG GAGCCACAAATACGTGAAGAAACCTCTTTTACCAGGTAAAGTGATCACCTTAATCCTGACATCCAATATGCAT CTGTGGCATTTTTACTATTCATATATTGTTTCATTTATAATATTGAAAATAAAGAAAGCATTTCTTATTCAATTA TTAATAAAGATGCCCTTATTTTTGACAATTTTGACTCAAGTGATTTAAACAAAAGTTATGAGAAGACCAGGAG TCCATTTCAAATTAATATAATAATCACATACATGAACACATAAATATGTGAATAATTGAAAGCCACCACAATCA ACTGATACAATTTTAAGTTTAGTTTTATTTTTACGACAATATTCCATATGCAAATATGAGGACTATAAAAAGAG CTTGAGTAGGTATCAGCTTTGCAAAAGTAAAATAAGAAGCAAATGTTGCAAGTTTTGTCACTGAGCATGTAAA CATTTTACTCAAATACTACTAAAATGTATTCTTATGTGCTTCCAAGTGTTGAGAGAGAGAGAGAGAGAGCAAA ATATAGCAACTAGGAGAAAAGCAATATTTGTACTACTTCGTTATAAGAAAGCAAAAACTGAATAGCTTACATT GTCAAGCAAAGTAAATAACACTTACCTTGCTTAACAATGGAACTGAGTTTAGCCCTACTCCGCACTGACAGTT CCGTTGTTGCTTCTGTCTCGGTCCTTCTCTTCCC I I I I I ATAGTATCTTCACAGCTGAATCTTCATGCATCTCA GTGTGGCTTTCTCTTCGAGCATCACTTTTAACTCAGACAGGCAGGAAGCTGAAGGCAAAGGAACTCTCTATC TGATTGGTTTCCATTCAGCGTTTCTGATTAATAAGAGACGTCCCTCAAATAGGAAGATATTGCCGCTGATGG CGCTGCAGGGCGCCAGGTTTGGGCTTGGGGGAGTAAGGACTTCTTTGGGCACAATGATTTGCAAGCGAAT GAAGTGGGAGGACTCAGAAAGGAGGGCTTTGTCAAAGACCCCCATAAGAGAAAACGACCCAGTGTTTTTAG TCTCGGTTAATCAGCTCTCCTTAATCTGTTTGCAATTGGGAGAGAAAGGTCACAGCATGTCTGTAACCTGTAA ATCATCTGTAGAAAATATATATCACAGGGAGAGTGCTGTTGCTCAAAAGAAATTTTGTTTATGGTTCCTGCAG AATTTACTACTACCTATTTCAATTCAGTGTCAAAATGTCAACTTTAAGAAAAAGAGGAGAAAAAAATACAGAT AATAT (SEQ ID NO: 3)

[0083] In the case of the CDH9 region, seven differentially methylated sites were identified. The skilled person will understand that not all of the differentially methylated sites need to be identified in order to determine or measure the resilience status of a subject. As shown in the Examples herein, it is not necessary to measure or identify methylation in all of the sites that were identified for an individual to be classified as highly resilient. For example, the inventors have shown that methylation at four sites gives a good classifier of high resilience.

[0084] In some embodiments, the genomic region is PF4. As shown in Table 1, by PF4, we mean the region of sequence in chromosome 4, positions 74,846,528 - 74,847,841 (as determined by the GRCh37.pl3 assembly). The forward sequence of this region is shown in SEQ ID NO. 4 below, and this includes an additional region downstream that includes the differentially methylated region. In some embodiments, the genomic region as defined herein is defined in SEQ ID NO. 4.

[0085] Legend:

[0086] CpG site in classifier (bold underline)

[0087]

[0088] >NC_000004.11:74846528-74848041 Homo sapiens chromosome 4, GRCh37.pl3 Primary Assembly, forward strand TTTTTTAAAAGCCATCTGTGTTACTGATATTTATTGGCAAAGGTAATAATAATGGTCAAGGTAAATATGTAGCT ATAAAGTACTGCAATCATGTAAATGCATGAATATGTATCAGCCAACATGTAACACCAAGCATAACCAGTATTC ACACCTTCCTTCAAAATACTTTTTGCTTAAAATACCGGAATTTTTTTCTTCCATGAAATTGTTATGTGTCAACAC AATTTTTGCACTATTATTAAGAAGTATTTTGACTATACTACAACTTGATTTATTTTGTTTATTTAAAATCATAAG GATAACACAAATATCAGAAGTTCTTTCACAGTTAGATTGAAACTGGAAAAAAGAAGTATGCTATATAGCAAAT GCACACACGTAGGCAGCTAGTAGCTAACTCTCCAAAAGTTTCTTAATTATTTTCTTGTACAGCGGGGCTTGCA GGTCCAAGCAAATTTTCCTTCCATTCTTCAGCGTGGCTCTGGCAGGGAAAAGAGAAGAGATGTGACTTTCAG TCCTTGGGTTTGCAAATGGCATTAGAAGGGGGAGGGTTGGGCAGAGGAGGCACGGAGCGGGAGCACTGAC AGATGCAGTGCAAGGACTCACATCAGTTGGGCAGTGGGGCAGTGGGGTCCGGCCTTGATCACCTCCAGGC TGGTGATGTGCCTGGGACGGACCTGGGAGGTGGTCTTCACACACAGGCACTGCAGGTCCCCATCTTCTTCA GCTTCAGCTGAGGGGGAAATGGAGAGGGTAAGAGAGGAGGAGGGGAGGGAGTGGTGACTCCATGAGTAG CCAGAGGTACTTTAGGGACTTTTTCCACTACCTTCAGCCTTCTTTTCCTTACCATAAATCACACCTCCCCCAG ACAGAAGTTGTTCTAACCAGGTGGGAGGTGCAGGTGCAGCGCCTGGCACTGTGGCCAGACACTCAGCAGG CTCTCCGTTAAGTGTGGCTGTGATCATGATCCTAGAGGATTCCTCCCCAGCCCCAGGGCTTCCCGGACTCCC CCTCGCCGCTGCCAGCCCTCACAGCCTGGCTTCTGCTCTCACCGCTGGCGAAGGCGACCACAAGTGGCAGG AGCAGCAACCCCAGGAACAGCAGCCCGGGGCGTGAGGCGCAGAACCCGGCTGCGGAGCTCATGCTGCGG CAGAGCTTCCAGCAGGATCTCAGTGCTCAGTGCGATGGGAAACTCGGGCTGGGTCTCTGTGGCCAATGACT CCTGAGCCTCGCCGGCACGTTTTATTCCCGGCTGTCCTTCCAGTCCGGAAGCCTGAAGTGGACACCGAGGA ACTGCGGTGCGGAAACTAAGATAGTACTGGGAAGACTTGGGTTCTGGCCAGCCAAGATTACCAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAGAAAAGAAAGAAAGAAAGAAAGAAAGAAAAG GCTCTGGGCAGCTGCCTTGGCTGGGACCTCCAACCTCAACTTACATACAAGCACGTTTGTAGAGTTAGAAAC AGAACTT (SEQ ID NO: 4)

[0089] In the case of the PF4 region, seven differentially methylated sites were identified. The skilled person will understand that not all of the differentially methylated sites need to be identified in order to determine or measure the resilience status of a subject. As shown in the Examples herein, it is not necessary to measure or identify methylation in all of the sites that were identified for an individual to be classified as highly resilient. For example, the inventors have shown that methylation at four sites gives a good classifier of high resilience.

[0090] In some embodiments, the genomic region is RP11-16E12.1 / RP11-16E12.2. As shown in Table 1, by RP11-16E12.1 / RP11-16E12.2, we mean the region of sequence in chromosome 15, positions 31,508,224 - 31,517,210 / 31,514,202 - 31,523,041 (as determined by the GRCh37.pl3 assembly). The forward sequence of this region is shown in SEQ ID NO. 5 below, and this includes an additional region downstream that includes the differentially methylated region. In some embodiments, the genomic region as defined herein is defined in SEQ ID NO. 5.

[0091] Legend:

[0092] CpG site in classifier (bold underline)

[0093]

[0094] >NC_000015.9:31508223-31523065 Homo sapiens chromosome 15, GRCh37.pl3 Primary Assembly, forward strand GCGGCCGCCGCCAAGCAGGTGCGAGCCGTCGGGCGGGGCGCGCGGGAGGCGGAGAGGATGGGCCCCTG AACCACCCCGCGCGCCCCACCTCAGCCTCCCGGGGCGAGCCAGGTTTCCGGGGCGGGGGCCGCGAGGAG GAGGAGGAGGGCAAGGCGGGGGAACGGGAGGGAGAGGACCCGGGGAGGGGGAGGGGGAGGCGGAGGG GAAGGGGGAGGCGGAGGGGGAGGGGCAGAAGAAGGGGGCAAGGAGGGCAGAGAGATGAGCGAGAAAGA AAGGAGGGGTGCGGGAAAGGAGGAGAGGAAGGAGCGGTGGGAGGAGGAGGAGGGGTGCGGGAGAGAA GGGCTGAGGGAGAGGCGGAGGGAAACCGCACCCCTGCCTCTTTCCTGGGCCGAGGCCAGCCGGGGAGGA AATGTTGAAATGGGCGAAGTGCACATCCGCCTCCAGGCCTAAGGGGTGTAGGGGTCAGGCCGCACCGACCCCGCCCTTCCCAGCCCTTCCTGCCAGACCTCTGCCTCCGATGGGCGAGCCCGGTTGCTGGGAGGCGGTTCG CTGTGGAGCCTGGACCCTCTCCACAGTTCAGCCCGCCGGAGGCGTGTGCGCTTCCAGATGAGAGCACTTGT TCACTGAATGACCGTGCGTACTTGGGGGGACATCTGGCCCCAGAACCTCCAGGAGCAGGGTGGCTAATGCT GAGTGCCGTGGCCTGGCACTGGGAAGCGCCGGTCCTCAGTCCCTTTCCCTGCTCCTGGGCATCCCAGGCCC CTTGGGGCGTTCTTACACTGCCTGAGTTAGACCTGGAAGAGTCCCCTGTGCCAGGGCAGGGGCTTTGCCTG CTTGGTCCTTGTTCACCCCTGTGTATAGCTACTACCTATTACCTACCCACCTGTCCACAAGTGTCAGTGCCTG ATGTAGGAAGTTACAGTAATTCACAAATAAATCTATCACAAAGGGACATGGATACTCACACTTCCTTCTTACC CCTACTTCCAGGCAGCCATTGTTAATGGGTGCGTGTGTACAGTTCTGGGCACATACCAACATGCACGGATGC ATGTTAACCTGCTTTGTATGGAAGAAACGGGGTCAGGATATCATAAAAAGAATTAATGGGTAATCAGTATTC ATCAAATGCCAAGATCTAGTTCTGAGCACTTTGCAAATATTAATTTAATCACCACGAACCTCAGTAAAGTTTAT TACTATTACTCGCATTTTTCAGATGAGGCGCCTAAGGCACAGAGAGGTTAAGTAGCCTGCCCAGGATCACAC AGCTACTAAGTACAGAAGTCAGAATTTGAGCCTGACTCCAGAGCTCATAAGGTTAACAACGAGGCCTCCCAC TTTTATATGCGTATTATTCCATGAATTACTGTTTACACTAAGAATATGTTCTTTTCTGCAACCAATGGAAGATT CCTAGAAGAGGAACTACTGGCTATGTGGCATTGAGA I I I I I C I I I I I I C I I I I I I I I I CTGAGATGGAGTCTAG CTCTGTCGCCCAGGCTGGAGTGCAGTGGCACGATCTCAGCTCACTGCAACCTCCGCCTCCTGGGTTCAAGC GGTTCTCCTGCCTCAGCCTCCCAAATATCTGGGATTACAGGCCTGCCACTATGCCCAGCTGATTTTTGTATTT TAAGTAGAGATAGGGTTTCACTGTGTTGGCCAGGCTGGTCTTGAACTCCTCACATTGTGATCCACCCGCCTC GGCCTCCCAAAGTTCTGGGATTACAAGCGTGAGCCACCGCGCCTGGCCGTGTGTTTGAGATTCCTAGAGAC ATTAGTAAAACCCTCCAAAAGAGTGGGGACCTTACACCAGCCTGCCGGTTCAAGCTCCATACTGGACCTCCA CCTCCCCAACCCCCACCCCCACCCCACCAGAAAAGGACCCTCCATCCCCTTCAGGTCATAGAGTGAGGAAG AGGGAAGCTTCAACAGGCCCAGATGGCCCTGGTAATGGGCACGGTGACATTTGGGCAGAGAACCCCAGGG CCCTGCATCTGCAGACCTGGGACAGGCAGAGAATGTCCAATGGGGGTGCTAGTGGCATTGCCTGGCCATGA CTCTGTCCAGCTGTGGCATCATGTCCCATGGCCAAAGTGTCCAAGCAAGCCTGCTTCTGCCATTGACCTAGG GAGGCCTCTTGACACCTGCTCATTTTCCAAGCCTGTTCCTCCTGCATCCTCAAGATTCGGAGTGCCCTCCAAA ATAAGATCTTTTTCTGTAGTTGTTACTATGGGTTAAGAACCCTCCTGTCTGATACATACAACTGATTACAAACT TTTAATTTTTGCCAATCTGATAAGCAAAAAAAAAGAAAGTAGTTCATGGATTTAGTTTACATTTCCTTTTTAAC AGGGAGGTTGGGTAGAGACTTCTTGGTTTGCTCATGCCTGTAATCCTAGCACTTTGGGAGGCTAAGGTAGG AAGATCGCTTGAACTCAGGAATTCAAGCCAACCTGGGCAACATAGCAAGACCTCACCTCCACTAAAGGGAAA AAACAATCCATGTTCCTTTTCTTCCAACCCAACAGATCTGTGGCTGGGCAGCAGCTGCTGAGCTCAACTACA ATTCCAGCCTGCTTTACAGTTAGGCTTGTCCACAGGACCAAGTTCTCAGTGACAGAGTAAGAGCAGGCCTGT GATAGGAGCCTCTTCTGTACCTCGGCCTCCAAGCTGCCTAGAATCTGGCTGTGGCAGTGCCCAAGGGGAGG CGAGCAGCAGGAGAGAAGGGTCCTGGGGTTTGGGGGGACCAGGAGGAGCAGAGCTGCTGCTCAGCTCAG ATCCATAACCTGAGAAAGAAAGAGGGATGGCACATTTTATGTGTTAACTTAAATATCTGGCTACATCGCTCAG ATATTTGATTAGACACTATTCTAGATGTTTTTATGAAGGTATTGATTAATTGATTGATTGATTGAGACAGTCTC ACTCTGTTGCCCAGGCTGGAGTGCGGTGGCACAATCTTGGCTCACTGCAACCTCCACCTCCCAGGTTCAAGT GATTCTCGTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCATGTGCCACCATGCCTGGCTAATTTTTGTA TTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCCTGACCTGAAGTGATCTGCCCACCTTG GCTTCCCAAAGTGCTGGGATTACAGGCCTGAGCTACCACAACCGGTGAAAGTACTTTTTAAGATAAGATTAG CATTTAAATCAGAAGATTTTGAGTAAAGCAGATTATTCTCCACAATGTGGGCAGATCTCATCCCATCAGTTGA AGACCTTAAGAGAAAAGAGACTGACCTGCTCTGAGCAAGGAGGAATTCTGCTGGCCAACTGCTTGTGAACT CCAACTTGTAATATCGGTCTCCAGCCTACCGGCTTACTCTGCAGATTTTGGATTTGCCAAGCCTCCACAACCG CAGAAGCCAATTCCTTAAAATGAATCCCTTACAACAGTTCCCTAAAATAAATTCTTCAAAATTCCTTAAAATAA ATGTGTGTGCATGTGTGTCTGTGTATATATATAGCCTATGTAATGTCTAATATAGATCTATAATATATATTAGA TAAATATACACATACTTATCATATATATATGTGTGTGTGTGTGTGTGTTTCTCCTATTGGTTCTGTTCTCTGGA GAACCTAATACAGAAACAAATCTGTTTATTCTTCTAGCCTCTGTATCATCAGGCCTCTTTGTTAGAGCAGGTG AGCTTTACCCTAACTAATTCAGAGGTTGATTCGTTTTCATGCATGTAGATACTTGTCATTTAGCTTCTATTATC TGCCTGTATTTTAAGGTTCTTTGTCTTAAAATGTTATGACTCCAGGAATGTTATATATTAAGTATATTAACCTTT TGACTATTATATATCTTGCAAACATCTTGTAATCAGTCACTTGTCTCTTTATTTTATTTGGAGCGGCTTTCAAC CTAGAGCAGTGGTCCCCAAACCCGGGGCTGCAGACTATTCCCTGTCTGTGGTTTGTTAAGAACCAGGCTACA TAGCAGAAGATGAGTAGCAGGCGCGGGAGCATGACCACCTGAGCTCCGCCTCCTGTCAGACCAGCAGCGA CATTAGATTCTCATAGGAGCACAAACCCTACTGTGAACTGTGCGTATGAGGGATCTAGGTTGCACATTCCTT AAAAGACTCTTAATGCCTGATGATCTGAGGTGGAACAGGTTCACCCTGAAACCATCCCCCGCTCAGCCTGTG TCTGTGGAAAAATTGTCTTCCATGAAACTGGTCCCTGGTGCCAAAAAGGCTGGGGACTGCTGACCTACAAGA TATATTAGTTATGACTCTTGGTGCATACTCTTGACAGAAATCCCACTCAACCTAGTATAATGCAGAGGACAGT TCACGGAGCAACTCAAATAATTGCCACTTTTACAATATTGAAGCTTCCTACGGGAAACATAATACATCTCTCA ATTTATGTCCTTTAATACGATTAAACATTTTTCTTCATGTAGGTTCTCCAGAATTTTGGTTATGCCTATGCCTA GATTATTTATGATTGTTGTTGGTATTGCAAATGTCCTCTCCTATTGATTATTTAATGGTATCTATGGAAAGTAT TGATTTTTGTGTATTTGCTCTGTGCCCACTTGCCATCTAACTGGTTGTTATATTTTATTGAACCTTTCAAAGGA TATGAATTATGCTGATTTTAATGTCTTCTTCTGATTCCTGCATTATTATCCTGTTTTCTCCAGCGTTCTGTCTAC TTCTTCTGCAGGCTGTTGGTTTCCTTCAAATGTCTGGGGATTTTTGATTGTCTGATCCCATTTGTAAATGAAC GTCCAGAGAGTGGTTTTCAAAGTGAGATCCATGGACCCCTAAGGGTTCTTAAGACTCTTTCTGGGTGTCTGC AAAGCCAAAACTATTTAATAGGAATAAAAAAGACTTGTTTTTCCTAATGGGTGTTCAGGTGCTCACTTCAGCA GCACATATACAAATGGGTGTTCAGTGGAGTTTTCCAGAGTCTACATGATGTGTGTTATCACTATAGCTTGAAAGAAGGAGGTATGAAAATCCAGCTGTTGGCTGGGCATGGCGGCTCCTGCCTGTAATCTCAACACTTTGGGAG GCTGAGGCAGACAGACCACTTGAGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAACGCCATCTC TACTAAAAATACAAAAAAAAAAAAAAAAAAAGCTAGGCGTGGTGGTGCATTCCTGTAATCCCAGCTACTTGG GAGGCCGAGGCAGGAGGATTGCTTGAACTGGGAGGCAGAGGTTGCAGTGAGCCAAGATTGTGCCACTGCA TTGCAGCCTGGGTGTCAGAGTGAGACTCTGTCTCAATAAATAAATTTAAAAAATTAAATACATATGAATACAT AAAAATTAAATGCATAAATACATAAAAATAAAAAAATACAACAACAAAAAAGAAAATCCAGCTATTAAGCCAG ACATTGGGGAGTTTTTTTGTTTTGTTTGTTTTGTTTTGAGACAGAGTTTCACTCTTGTTGCCCGGGCTGCAAT GCAGTGGCACCATCTCAACTCACTGCAACCTCTGCCTCCCGGGTTCAAGCGGTTCTCCTGTCTCAGCCTCCT GAGTAGCTGGGATTACAGCCACGCACCACCACACCTGGCTAATTTTTTGTATTTTTGGTAAAGATGGGGTTT CACCATGTTGGTCAGGCTGGTCTCAAACTCCCGACCTCAGGTGACTCGCCCGCCTCGGCCTCCCAAAGTGC TGGGATTACAGGCGTGAGCCACCACACCCAGCATGTATAAGTGTTATTTATGTTAACATGCCATTATTATTTT AAGATAAATGAATATTTATTTATTTTTCACTTGAACTTCTACTACCGTAAATATTGATAGATAAACTCATATGAA TGAAAGCCCGCTGAGACCCTCAGCCATTTCTAAGAGAGGGTGAAGGGTTCCTGAATGCAGTGTGTTTGCGA TCTGTCAGTCTCAACCAATCATTCTTGCTGTGCAGAGTAGTTCTTGCACACAACTAAACCACCACTGTCTGGC AACCCTGGCACCACTGGGCACCTGTGGTCATGGAGACAGGCCAGGACGTTGCAAAGGCAGAGTGCCTTCCT GGGCCTCTCATCTGGGCCGCTCTCCCTGTCAGTGTGGCTGACAGTGCCTGGCAGTCACCTGGGCGGGCCCC TGCTTCTCTCTTTGGGCCAGTGTTGGCTCTGGAGGCTCCCGGTGCATCTTTGTCTTCAGTCTGAATGCTGCA CTCAGACTTTACCCTGGGGACAGGCAGGACACTGCCTCAGCCACCTGGGTAGCCGTTTGTGTGATTACCGT ATCCAAGCTCCATGACTCATTCCTTCTCCACTGCCCCCACCCTACCCCATTTCCAGACTTGAAATGGTCTCCC ACCTGCAGGCCATTGGCTCTATTGCGTTTCACCACGAATCTGATCTCCTCCGCTTTATATCTTCCAGAAATTC TCCAATGTTCTAGTCTGTTGGTGGCATTTTTCCTGGTTTTCAAGGATTTCTAAGTGTTTTTATATTTATCCTAC TACATCAGAGGGTGGGGGAGATAGGTAGGAGAGGTGCAGTTGAGCCTTGAACAATGTGAGGGTTGGAGAG CCAACCCCTCACGCAGTCAGAGCTTGTATAACTTCAGGCTCCCTCCAAACTTAACTAATAAATAGCCTACTGT TAACTGGAAGCCTTACTGGTAACATTAAAAAATCAGTTAACACGTACTTTGTATGTGTATTTATACTGTATTCT TACAATAAACTAAGCTTGGGAAAATAAAATGTCATTAAGAATATCATAAGAGAAAGCATTTACTAAGTGGAAG TGGATTATCATAGAAGTCTTCATCCTCGTTGTCTTCATGTTGAGTGGCTGAGGAGGAGGGGCTGGTCTTGCT GCCTCAGGGTGGCAGAGAGGAAGACATGGAGGAAGTGGGAAGGGAGGCAGGAGAGGCAGGGACACTTGG TATAACTCTGATTGAAAAACATCCCTGTGGGTGGACCTGCACAGTTTGAACCTGCGTTGTTCAAGGGTCCAC TGTGCTTGAAAGTGTTTAGTCAGCAGTCCTGAACGACATGTAAACAATGGTTGAATGAAGGAGCAAGGGAG CCTAAGGGTGGGGAGAGGCGGGTGTCGGCAAGGACCTTCCTCCAAGACAGGGTTGCTGCGAGGGGCAAAT GGTGACTGATGATTCACAAGAAGGCGTACCTGTGTGACAGCACCTGTAAGCCAGGTGGGATCAGTGCGGCC TGTCGTCTGCTGTTGTCATGTGGAGCTCAGCAAACGGTGGGAGTCCTAGGGGACAACATACACAGCTTAGC AGCAGGTGCCCCTCTCTTCGAGCCTAATTTAGCCAATGGGAGTTTAATTTAGCCACGGGGGCGTAATTGCTA GAAGAAAAGAGAAGAAAAAAAACCTGATGCCCTCACCGCCTTCCAATCCTGACTCTTTAGGAAAAGCCTGGA TCTCCGACATCTCCAGAGCGCCTGACCAAGCGTGAGAAGGCGGGATGTAGCCGGGGTGTGTGTGTCGGGG GGTGGCAGGATGCTCCCCAGAGGCGGAGAGCGGCGCGTGGGGGGTGCTGCAGGCGTCCTGGGAGTTTGG GGGCGCCGCCCTTCCTCCTGCCTTGGGCCCAACCGGTGGATCCTGTCGAGTCTGTTGTGGTCCCCTTCTTA GGAAAAGCACAGAGAGCCCTTTAAAGATGTTGACCTGACAGCAGCAGGGGGCCCAGGGTGACCACGGAGC AAATCAGGGAAAGGAAACTTAGGTCGCCACACATTTGATTTAAAAGAAAATAACTTCCCTCCTGAAGCAGGG GCTGGGGTAGGTGCCCTGGCACAGGCACAGAACCAGAGAGAACCCTTGCCGCTGCTCCGGAATGTCGGGC CACACCCTCCGGATACGGGAAAGGCTTTCTTCTCCATTTCAAATGACCCTGTTTCGCC I I l l i IAGATACAAA TGTTTATTTGGTCTGACTTTGAAATACCTCGGAGCAGCTGAAATAATTGAAGGCAGCCCTGTAAGAAGCAGC ACTTCCAGACCTTCTCACGGGCAAACCCTCTGCTGGGACCGCCAAGGGCCCCCGCGAGACACCTGAGGGTC AGCACAGCCCCTCCCTCCCTGTGCCGTGCTGGCCGTGCCGCAGCTGGTCACCCGCACGAGGACCTCGAGAT TGCCCTGCGGACTGGCACTTCTCGAGGGAAGTCATCGCAGCTGCCATGGAGCCAGCATTTGTCGGGGACCT GGCACGCAGGCCACCGGCTCTGGGAGAGGGCTGAGGAGCCGTGGGGCTCACGTTTCAGGGATTAGCTTGT GTAACTTGACTTGTTCCCCCAAATTCTACAAGTACAGAGAATATGCAGAAATCTGTGAGAGAAACAACCTTCT GCAGCTCGTTGTTGTGGGGATTATATTAGTAAGATGCCTAGAAAATACGATTCAGAAGGAATGTGGGTGCGT GTGACTTGGTGGTGGGTGGGCTCCACTCACCACCCTCCCTCCGCACCTGTCCGAGGCATTTGAACCAGAGC AACTCCATTTCGAATAGGGGCCGGTAAAATAAGGCTAAGACCTACTGGGCTGCATTCCCAGATGGTTAAGGC ATTCTAAGTCACAGGATGAGATACGAGGTCGGCACAAGATACAGGTCATAAAGACCTTGCTGATGAAACAG GCTGCAGTAAATAAGCTGGCTAAAACCCAAAATGGCAACAAGATGTGTCCTCTGGTGGTCCTCACTGCTACA CTCCCACCAGCACCATGACAGTTTACAAATGCCATGGCAACATCAGGAAGTTACCCTATATGGTCTGAAAAG AGGAAGCATGAATAATCCACCTCCTGTTTAGCATATCATCAAGAAATAACCATAAAAATGGGCAACCCACAG CCCTTGGGGCTGCTCTGTCTATGGAGTAGCCGTTCTTTTACCCCTTTACTTTCCTAATAAACTTACTTTCACTT TATTCTGTGGACTCGCCCTGAATTCTTTCTTGCCTAAAATCCAAGAACCATCTCTTGGGGTCTGGATCGGGAC CACTTTCCTCTAACACACCCACTGAGAAGTGCTTGCTGATGTTAGGGTTCACAGTGTCAGGGGCTGCCGTAG TGTCACCCCATTCTACAGATGAGAAAACAGCCTCAGAGTTTTTTGTTGTTGTTGTTTGAGATGGAGTCTCACT CTTTCCCCCAGGCTGGAGTGCAGTGGCGTGATCTTGGTTCACTGTAACCTCCGCTTTCTGGTTTCAAGCGAT TCTCCTGCCTCAGCCTCCCGGAGTAGCTGGGATTACAGGTGCCCGCCACGCCCGGCTAATTTTTGTATTTTT GGTAGAGACGGGGTTTTACCCTGTTGGCTAGGTGCCTCTCAAACTCCTGACCTCGTGATCCGTGGGCCTCT GCCTCCCAAACTGCTGGGATTACAGGCATGAGCATCAGAGTTTTTAAGTCAGAACTGGAGCCTGAGCCCGA CAGGTGACACAACCCGGCCTTCCCTGAGGGGCTGGCACGCCGCAAAAGGTCGCCAAGCTCTCCCTCTTCCGGTGTGAGGTGCAGCTCGCCGGTGGGTATGCACAGGGAGTCCTCTATTTTGCTCGGTTTTGCCTGGGACCAT GTGCTCTGTGTACTGCATCATGGTCTCCACAGTGGATCTCGGTAGGCACAGCTGGACTTGGTCCTCAGTTGC CTGCTGTGCCCTGCTGGGGATCTTTTTGCACGGGCCATTCCCCCTCGCACTTGCCTTCCCTCCCCCGCTCCT GGCTGGCTCCCACTCTTGGCCGGGGCTCAGGCAGGATTCTCTACTAGACTCAGCAAAGCACCTGCAGTGGG CCTGTAATAGGAGGGAGTGGTGGGTCCGGAGTTGGTTCCTTCCAGTGGGATTGTGGTCTTGCTGATTTTGA GAATGGAGCCCCAGACCTTCCTGGTGTTACAGCTCTCAAAGAAGACAAGGACCCAAAGGGTGAGCAGCAGT AAGAGCTATTGTGAAGAGCAAAAGAACAAAGCTTCCCCACCACGGAAGAGGACCCCAGCAGGTTGCCTCTG CTGGCTCCGTGTGGAGAAGGGTTGGGGGGGAGGCAGCTTTTATTCTCTTATTTGTCCCCGCCCATGTCCTGT TCCTGTCCTATCAGAATGCCCTTTTCTCAATCCTCCCTGCGATTGGTTACTTTTAGAATCCTGCTGATTGGTCC ATTTCACAGAGCGCTGATTGGTGTGTTTTACAAATCTCTTGCTAGCTACAGAGCACTGATCGGTGAGTTTTTA CACAGCACTGATTGGTGCATTTTACAATCCCTTGCTAGCTACAGAGCACTGATTGGTGCGTTTTACAATCCTA GCTACGGAGTGCTGATTGGTTCGTTTTACAATCCTCTTGTAAGACAGAAAAGTTCTCCAACTTCCCAGTGGAC CCAGGAAGTCCCGCTGGCTTCACCTTTCAGTGGGGCTGTGTTTAGGAGACAATTCCACTCAGGCATAGTGG CCTGATCATGGGGTGCAAAGCTGGCGGCCAGACAGCCTTCCAGGTGGTGACTTTTACTATACTGGGGATGT GGCTTTATGGAGCTATGCTGGCCCACTCCCTCCAGAAGGAGCCTGATGGATCAAAGATGGAAACCAAAAAA ATACCGGAAGAAAATGATTAAAAACAAAAAACGAACAAAACAAAACTGTTGGCCAGTTGCGGTGGCTTAAGC CTGTAATCCCAGCACTTTGGGAGGCCAAGGCAGTCAGATCATTTGAGGTCAGAAGTTTGAGATCACCCCAAC AAACATGATGAAATTCTGACTCTACTAAAAACACAAAAGTCAGCTGGGCATGGTGGCGTGCTTGTAGTCCCA GCTACTCAGGAGGCTGAGGCAGGAGGATCACTTGAACCCAGAAGGTGGAGGCTGCAGTGAGCCAAGATTG TGCCACTGCAGTCCAGCCTGGGTGACAGAACAAGACTCTGTCTCAAACACACACACACACACACACACACAC ACACACACACACACACACACCCCTGTAATCTTGCAAAGGGGTGAACTCTTTAATGATCTTGCAAAACCCTGT GGCCATAAACAACATAATTGATTTATTCTACCCGATAAGAAAAAAAATATATCTGCATGGCAGAAAACATCCT GCTTAAAGTTAAAAAAAAAAAAGATAAACAGAAAAAAATTATGGCACATTTGTAAGATGGAATTTACCCTGCA GTCATGAAAAAGAATAAGGTATACTTATGTCTATGTGAAAACAGAATCATATGCATAATAATATATGCAACTT ATGTCACAGGCAAAGGGCCACTAAAAATCAAAGAGAAAGAAATCCAACAACTGAATTGGAAAATGGGCAAT GGGAAGAAATCCTTCCCAGAAAAGAAAAATGCCTCTAAACATTCAAAAATATGCTGACTGTCATCACAATAAC AGAAACGCAAACTGAAATATCAATGAAATCCTACTTTCTGCTATCAGATCAGCAAGTTCCAAACTTTGACAAT CGCCTTTCAAGTGAGCTGAAGGAACATGCTCCATGGGGGAATGTAGTCACATTTACCAGAACTACACACATA TGTGCTCTGGCCCAGCAAGCCCACTTCAGAGTCTTGTCAAGACATGTAATACAGAGGCTCATCAAAGCATTG TAGTGATTGGAAAAGGATAGGAACAACTTAAGCACTATCAAGATTAATTACATCACATCCCTAGGTCTGCATT ACCATGCAGTCATTAAAAAAGAGGGAGGTGGCCATGTGTGGTGGCTCTCAAGGTGTAATCCCAGCACTTTG GGAGGCTGAGGCAGATGGATCACTTGAAGTCAGGAGTTCGAGACCAGCCTGACCAACATGGTGAAACCCCA TCTGTGCTAAAAATACAAAAACTAGCCAGGTGTGGTGGCGGGCACCTGTAATCCTGGCTACTTGGGAGGCT GAGGCAGGAGAATCACTTGAACCCGGAAGGCGGAGTTTATAGTGAGCCAACATCACGCCCCTGCACTCCAG CCTGGATAACTCCGAGTGAAACTTCAGAGTGAAACTCCGTCTAAAAAAAAAAAAAAAAAAAAAGAAGGAGGT ATATTTAGGTCTATGAAACACAGCAATCTTCAGAGTATATTGTTTCATGAAAAAGAGGAAGCTAGAGGGTAT GTGTATAGATTTATCTCATTTGTTCGAATGAGTGTATGTGTGTGTGCACACAGAAACTCCTGGTAGGATGCAT AAAATATGGACGTTAGTGTGTTTTCTCTGGTGCTATGAACTGAATGTTTGTGTACTCCTCCCCCCAAATTCAT ATGTAGAAATTTTCGGCCCCAGTGCGATGGCATTAGGAGGTGGAACCCTTGGGAGTTTAGGTCAAGAGGAT GGAGACCCCAAGCTCTCTTCCTCTTTCCCTGCCACGTGGCCATCTGCAACCTGAAGAGAGCCCTCCCCATGA CCTGACCATACTGGCACCTTGATATGGGACTTCCAGCCTCCTGAACTATGAGAAATAAATGCCTGCTATTTAT AAGCCACCCAGTCTATAGTCCTTTGTCATAGCAGCCTGAGCTGACTGAGACCGTCAGGTAAGTGGCCTGGG GTCTGGGGTGGTAGCATTCCTATTGTCTCCTTTATTCTGTTGGAATGTTTGACTCCAAGCATAAAGGACATTT TTAGTTCTTTTG I I I I I I I GGG I I I I I I I I I GAGAGGGAGTCTCACTCTGTCACCCACGCTAGGGTGCAATGG TACAATCTCCACTGCAACCTCTGCCTCCTGGGTTCAAGCAATTTTCCTGCCTCAGCCTCCCGAGTAGCCAGG ATTACAGGCGCCTGCCACCATGCTCAGCTAATTTTTGAATTTTTTGTAGAGATGGGGTTTCACCAAGTTGGCC AGGCTGGTTTTGAACTCCTGACCTCAGGTGATCCACCCACCTTGGCGTCACAAAGTGCTGGGATTACAGGC GTGAGCCACCATGCCCGGCCATTTTAAGTTTTTTAAAACACTCAAAGCCATGCCTTGGGGAGGCTGCAGTCT GGCTCTGTCCCACCCTTGCCTGCTGCTGGTTGGAAGTGGGACCAGCCCAGCTGCTCCTTGGGTCAGGGTCT ACAAGGCCAGCCCTGGAAAGGCAGATCCACAACACAGCCAGTGCAGCACCAGGAAGGGTGCCCTGGGCTG AAGTCTGCATGGGGCAGCAGAGGCAGAGCCTGCCAGACCTGCAGACATTTCTGCAGTTGATTATGTTCTTAC TTGTCCCTAAGATGAGCACTTACCTAGGCATAGCTGATGCCCTCTGGTGGGGCCGGGGGGGGGGTGGTGT CCCAAATCTGATGGTGGAGCAGCAGGGGATCCCCTGTGGGTAGAGACCAACAGGCAGGCTCTGTGCCGTG GGCTGGCTGGTGTACCCAGGAGACCTCAGCTCACACCCTCCTGGGAAGGACTGCCGGCCACTTCATAGGTG GAGCACAGAGGTGCTGGCTCCACCCCCTGTGTGTGCGTGGCACTCTGTAGTCACCAGCCTGGCTGTCTGCC TCCTTGTTCCAGGCATGGTCTCAATGGCCTCAGACCCTCCCTCCTGAGCCTGATTAAGAAGCAAAGCACCCT GCAGTAGGGGACACTCTTCTTAGGAGGAGGCGGCAATTCTCACTAATAAGTGCTTTTCTTCTACCACATCAT GTACATTAAGTGG I I I I I I I I I I I I GTTTTGC I I I I I I G I I I I I I I GTTTTG I I I I I G I I I I I G I I I I I I I GGATA CAGAGTCTTGCTCTGTTGCCCAGGCTGGAGTGCAGCGGTGCAATCTTTGCTCACTGGACCCTCCGTCTCCCG GGTTCAAGCGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGAACTACAGGTGTGCGCCACCACACCCAGCTA ATTTTTGTATATTTAGTAGAGACAGGCAGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGACCTC AAGTGATCTGCCTGCCTCGCCCTCCTAAGTGTATA I I I I I CTTTTC I I I I I I I I I I I I I I GAGACGGAGTCTCG CTCTGTCACTAGGCTGGAGTGCAGTGGTGCCATCTCAGCTCACTGCAACCTCTGCCTCCCGGGTTCAAGCGATTCTCCTTTCTCAGCCTCATGAGTAGCTGGGATTACACACGTGCGCCACCAAACCCAGCTAATTTTTGTATT TTTAGTAGAGACGGGGTTTCACCATGTTGCCCAGGATGGTCTAGATCTCTTGACCTTGTGATCCACCCACCT CGGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACCGCGCCTGGCCGTGTATATTTTTCTAAATAGACA CCTGACCATGCTTAGGTCATCCATCGAAATTCCCTAGCACTTGCTGGCTTCCCTGAATTCAACTGAATTGCGC GTGCCCACTGCTGCTGCATAACCTATGGCGGGCACAGCCATGGGATAAAGTGGGCAGTGTGCACACATTCC CCGCACAGTCTGTGGCCTGGCCAAGCCCCTGGGCATTCGAGGCTCCCGGCAAGGCTTCGTGTCCCGACAGT TTTGGGTAGGGGCACGAGGTGGGTGAGCCCGGATCCCAGCAGCAGGCATGGCTCCCTGGGTTTTCATGTC ATCTGTGCCCCCTTCTACCAGGATCCTGCAGCAGCTGAAGGGAGACCCCTCTCCTTTTAAGGGTTAAACCCC CAGTGGCACAGAGCAACTTACCTGCCCTTGCATGGTGAACACTCCCATCTTTATCACATCAGTAATTCTTGCT AAAAGCAGATGTAATTTTGAAGGTGGGTAAAAGTAACTTGAAAAGTTGGTGGGCAGATGGCAGAGAGCCTG CTCATCCTTCTTTCAAACTGTCATCAGCTGCAC (SEQ ID NO: 5)

[0095] In the case of the RP11-16E12.1 / RP11-16E12.2 region, nine differentially methylated sites were identified. The skilled person will understand that not all of the differentially methylated sites need to be identified in order to determine or measure the resilience status of a subject. As shown in the Examples herein, it is not necessary to measure or identify methylation in all of the sites that were identified for an individual to be classified as highly resilient. For example, the inventors have shown that methylation at four sites gives a good classifier of high resilience.

[0096] In some embodiments, the genomic region is ZFP57. As shown in Table 1, by ZFP57, we mean the region of sequence in chromosome 6, positions 29,640,169 - 29,648,929 (as determined by the GRCh37.pl3 assembly). The forward sequence of this region is shown in SEQ ID NO. 6 below and this includes an additional region downstream that includes the differentially methylated region. In some embodiments, the genomic region as defined herein is defined in SEQ ID NO. 6.

[0097] Legend:

[0098] CpG site in classifier (bold underline)

[0099]

[0100] >NC_000006.11:29640169-29649191 Homo sapiens chromosome 6, GRCh37.pl3 Primary Assembly, forward strand TTCCTTCCTTATCCACGCACCTGTCTCCCTCTACTCCAGCCTCATTACCCCAGAGGTCAGTCCTCAGGAAAACT AAACACAAAGAAAGAGCTCAGTCAGAAAGGCCATTTATTTATGTTTCAAGATGCTCACTGCCTCCTTTGTTTTGT CTCCTTTGCAGGCCTTCTCTCTTAGGCCTCTTCTCCTGGGGGTATGGATCCTGGGGGGAGATTGATCACCTCC ATGCTTCCATTCCTCCCCAGCCATAGTGGGGACATCATGAGAGAAGCCAAGCCACTGGCCCAGGATCACCCG GCATTTATGGTGGCTGCTCTGGCACAGGTCCTTGCCTTTATAGCCCCTCCAGTGATCCATAAGGCCCTCTTTCT CCCCAAAGGAGAGGTCACAGATAGGGCAAAGGTAGCTCTTCTGCTTCCAGTGGGTCTGCTGGTGTCTGACCA GCCTGGAAAATGAGCTGAAAGACTTGCTGCAATGGAAGCAGTAGTTGGGCGGCTCTGTGAGGTGGGCCTTCT GGTGTCTGGAGAGATAGGATTTCTTGCTAAAAGTCAAAGAACAATGGGGGCAACAGAAGACATTGAGTCTTG AGGGCTTCACTGGATGAGAGTTGGATCTGGCATCCTGACAGAGGGTTCCAGTGATGGGTGCCTGGGTCCTG GTCACAGGTGCTTGGTTCTTAAGTACAGATGCCTGGTTCTGGGCCATAGGACCCTCAGTTCTAAATATGGGTTC CTGGGACCTGGCCACTGGTGCATGGTTCACATCCAAAAGCCCCTGGATGGACCTCTGGCTTCTGGCGATGGG TGTCTGGAATTCAGCCTGGGTGCCTGGAATCCTCAAAGTACACTCCTGGTTTCCATCCACTGGCTCCTGGTTTT GGTGTATCTTCTGGTGGCGTTTGAGCTCAGACTGGTCCCGGAAGCTCTTCCCACACACAGAGCATGAATGGG GCCGGTAACCCAGATGGACGCGGCGGTGACGACTTAGTCCAGAAGCATCACAGTAGGTCTTGTCACAGAGC GTGCAACAGAAGGGCCTCTCCCCAAGATGCATGCGTCTGTGATAGCTGAGGGACTTGGGGCTCCGAAACAA CTTCCCACACTGACTGCAGCTGTTAGTCAGCTTGGGATTGTGAACAAACTGGTGGCTATAGAGGTAGGAGCG CCTGCTGAAACATTTGCCACAGGTGTAGCAAAAAAAGGGTGGCCCAGCCTGGGATGCTTGAAGCACCCGGG TCCTGTCCATAGTCCCAGCTGGGGCAGATAGGGGGCACTGGCCGGCCCCTCTGCATGCAAGGAAGACCTTG TCATCACTAGTCCCCTCATCTCTCAGACTGGGATGTTGTTCTCGAAGCTCTTTCTTCTTGCCTTCTACAGTGAAT GAGGAAGAATAACACAAAATTCACTGTAAGAACTCCAACAGAGGCTTGGCATGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCGAGGCCAGCGGATCACCTGAGGTTAGGAGTTCGAAACCAGCCTGACCAACATGG TGAAACCCTGTCTCTACTACAAATACAAAAATTAGCTGGGCGTCATGGCATCTGCCTGTAATCTCAGCTACTAG GGAGACTGAGGCAGGACAATCACTCGAACCCGGGAGGCGGAGGTTGCAGTGAGCCAAGATGGTGCCACTG CACTCCTGCCTGGGCAACTAGAGTGAAACTCTGTCTCAAAAAAAAAAAAAGAAAGAAAGAAAAAGAAGAAGA AGAAGGAGAAGGAGAAGAAGGAGAAGGAGAAGAGAAGGAGAAGAAGAAGAAGAAGGAAGAAGAAGAAGA AGAAAAGAAAAGAAGAAGAAGAAGAAGACGAAGACGAAGAAGAAGAAGAAGAGGAAGAAGAAGAACTCCA ACACAGCACTCCATTCAGCCTAACACACTTCTTGTCTCTGCCCTTGCTCTCCCACCCAACACATTCATCCTTACC CTTGGGCCTCATAGGCTAGAAATAAGAAGAAAAAAAGAAAAAATTGGCTTTTCAAATTAGAAGCAAATAAAAAG TTAACTGGAATCTTTCAACACTGTCAGAAATGTAAATTTTAACTTACAACAACACTTCTTGAAATCTATCTTATCT CATTCTCAATATTGCTCAAACTCCCATAGACAATCCACAGACACCCACATAATAATGCATCATGAACACTGGGC CACTTGAGGGTGAAAAGAGGTGTTATTAATAATCAAGCTGGGATGAGAAGTATAAACCAGGACTGTCCTGGAA AACCAAAAAGTGTATCAGCCTGGCTTGATATCTCTCTCAACTATTTACTACCAGGGACAAGCCTCCCTTACTCC AACCCAGCATGAAACCTATCTCCTTTGCTTCTCTTTTCTCTTGGAAAGAACATTTTAATCAGAGCACTATCATGG ACATAAGCAACTTTCATGTCATCTCTCAATCTCTAGAAACTGAAGACATCTACTTCTCCTGAAAGACTTAGATCT TCAGCCAGCCAGGCACGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGATGGATAACCT GAGGTCAAGACATCAAGACCATCCTGGCCAACATGGTGAAACCCTGTCTCTACTAAAAATACAAAAATTATCTG GACACGGTGGCACATGCCTGTAGTCCCAGCTACTCGAGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGA AGTGGAGGTTGCAGTAAGCCAAGATTGTGCCACTGCACTCCAGCCTGGCAACAGAGCGAGACTGTGTCTCAA AAAAAAAAAAAAAAAAAAAAAGAGAGAGAGAGAGAGACTTGGATCTTCAACTTGAAGTCAAGGGACTTGAGC CTATGATATTAAGCTCTCTTTCAACTCCAAGTCTGACCAGGCTGGACAGAGGTACACTAGGAGAGCATCTATAG AGCATTCATCCTCTTCATCAGCTCTCCATCCTTTCAGGGGTTATCCTGGGCCCTTTTCCCCTTCCTCCCTGCTTG GCAATTCTTACCTGAAAGGCCTTCTGTGTTTGGGAGATGGACAAACTCTCTCCACTGTTCCTCTTCTTGCTCAA GCTTGGTGATTAGCTCTGGCTTATGCAGAAAGATTCTGGCTGATGTGTGGGAATGAGAAAGAGTTGAGTTGGT CCCAGGTATGGCCCCTTCACATCTGATGGGGACAACAGGCTACCTCCTGTAGCCTTTGTTTAAGAACCATAAC CTGGGACATGTAGATGCGGAAAGGAGACATTAAAAGGCCAGCTGCTAGCAAAGTACCTGGTTCTCAGGAGTG ACTTAGTAAATATTTGTTTGATGAATGGAAAAATTTGCATATTTTGAGAACACTGTCATCATGTTACAAGTGTTAT CTTTGCCTTCATGCAGGCTATCATTTCTTCTCTTTACCACTGAGCTTAGTGACTCAGATCTTTCACACCTGGAAA GCATAGAACCAGGGGTCAGTGAAACTAATTGTAAGCTGATCTACCTGTCCAGGGAAACCAGATGTTCCAGGG CCCTTAGGACAGGGGGCTTGCTGAGGGAAGCCCAGCCTCTTACCCACAGATGTTAGATTCTTAAAGGTTTCCG ACATAACATCCTGGTAAAGGACCCTCTGGCTGGCATCTAGACAGTCCCACTCTTCCTGGGTGAAATTCACTGC CACATCCTCAAAGGTGACTGGCTTCTGGAAGAACAGGAGAGACTCAAGAAGTTTATATAAATATATATGTGTGT GTGTGTGTGTGTGTGTACAAGATTAACATCCAGTCTCAAGATTCAGAGAATTAAAACCTAAGAGAAAGATAAAA CCATGGAAGGAAGAGAGAAATATTAAAAGACAGACACAAGGCCAGCAACTGTGAAGTATAGAAAGGAAAGGA GGCCGGACGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCAGGCAGATCACGAGGTC GGGAGTTCGAGACCAGCCTGACCAATATGGTGAAACCTGGTCTCTGCTAAAAACACAAAAATTAGCTGGGCAT GGTGGCGCATGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATTGCTTGAGCCCGGGAGGCAG AGGTAGCAGTGAGCCAAGATCGCGCCACCGCACTCCAGCCTGGGTGACAGAGCGAGACTCCGTCTCAAAAA AAAAAAAAAGAAAAAAAAAAAAAGGAAAGGAAAGATGAAGAGAAAGGGAGAAAGATAAGATGTGGGGGAGA GGAAAGAGGATATGCAGATATGCAGAATATAAACAGGAAAGCAAAGCGAAGGAAAAAATGCTGCCACTCTAA CAAATTTCAGGAAGTACTCCATGAAGGATGCCAGGATGGTGCGGGAGATGGAGAAAGGTCTTGCAGCTCCTT TTTCTGGATGTCGTTCAGTCTGGAACAATCTGAGATTTCATTTGACCTGCAGGCAGGAGTATGTATGAAAGAGC TCCTGGAGTCCAGGACCTGGACCCCACCTCTCTCTAGCTTAGTCTCCTCACCTTCTTCACCCGTGCCTCCCTC CAGCAATCTCTCTTCATGGCTTCCTGCAGGGTGGCAGCTACCTCGCCCACCCATGGGAGCGTCTTCTGTACAG GTTCGATTGGCTTCAGCTGTTCAAACATCTTCTCTTCTGTGGTGTCTCTTTCTAGCTTTATCCACTCCTGGCCTG GTGCCCAGGCCTGACTGGATTCCTTCCTGGGGCTATCTACCTCCCAGTAACTGGGCAGATGGAGAGGCCCAG CAAAGGCCCCAGGGTTTGATGTGGCTTCCTGTGACAAATGTATCTGCTCCAAGAGGCTGTCTTCCTTTTTTGTT CTGCTGTCCAAATTCTCCTCTTCCACAATTGAGAACAATTTTGCTTCCCTCAAAGCTGGGCCACCGAGTTCAGG GCCCTGGTCACCCTTGGCTCACCAGCTGCCATTGTTTAGTAACAACACCAGCCTGGGCTAGGTGTCTGCCGTC TGTTCTACCCTGCTTCTAGAAACCTGAGGTCAGAGAAAAACAAAACATATCAGCAAGAGGGAGGGTAAGAAA CAGCTTCCTTATTTGGTCAGGGAATGCCAGCAGTTACTAAACCCCTACAGTGTGCCACTGGATGCTCTCAGCA ATGAGGTAACAATTACTGGCCCTGTCTTAAGGACCTAATGCAGAGATGCTAAATAATTTTCCAAGGACAAGTGG ACATTCTTGATCTACAAAAGTTAATGTTTAAACCTAATGTTAATGTTAGACTCAGTACCATTGGAAATCATGTAGC TGGGGTAACCAGGCTAGGATCTGTCACAGATCACCTCGAGTGAGTCTCTTTATTCTTTCTGACTTGGTTTCATC AGAAATGTGAGAATAAAGGAGACACTCTCTAAGATCTCTTCCATGACCAAAATTATACACACACACACACACAC ACACACACACAATTCTGTGATCTGGATTTTCAATACATGTAGTAGTTCCCCTTTATCATGGTTTTGCTTTCCAATG CTTCAGTTACCCATGGTCAACCATGGTTCAAAAATATTAAATGAAAAATTCCGGAGGACAGGCACAGTGGCTC ACACCTGTAATCCCAGCATTTTGGGAGGCTGAGGTAGGCAGATCATCTGAGGTCAGGAGTTCGAGATCAGCC TGGTCAACATGGTGAAACCCTGTCTCTACTAAAAATACAAAAAGAAAATAGCTGGGCATAGTGGCACACATCT GTAATCCCAGCAACTCAGGAGGCTGAGGCAGGAGAATCACTTGAACCCTGGAGGTGGACGTTGCCATGAGC CAAGACTGCGCCACTGCACTCCAGCCTGGGACATAGAGCGAGACTCCGTCTCAAAAAAAAATCCAGAGATAA ACAATTCCTAAGTTTTAAATTGCTTGACATTCTGAGTAGTGTGATGAAATCTTGTACCTTTTCTCTCTGGCCTGC CCAGGATGTGAATCATCCCTTTGACTAGCATATCCACACTGCAGACAATACCTGCCCATTAGTTCCTTAGTAGC TAGCCATCTCAGTTACCAGGTTGACTACTGTAGTATAGCAGTTGCCTGTGCTCAAGAATGCCTTATTTTACTTAATAATGACCCAAAAGCACAAGAGTAGAGACGCTGGAAATTCAGATATGCAAAGAGAAGCCATAAAATAAAAAGG TAAAAATTCTTGTCTTAAGGAAAGAAAAAATAATCATATGCTGAGGTTGCTAAGATTTACAATATAAATTATTTTG AGAGAGATACCACATTCATACAACTTTTATTACAATATATTGCTGTAATTGTTCTATCTTATTACTAGTTATTGTTGT CAATCTCTTACCATGCCTAATTTGTAAATTAAACTTTATCATTATTATGTATGTATAGAAAAAGAAAACCATAGTGT ATACAGGGTTTGGTACTATTCATGGTTTCAAAGTATCCACTGGGGTGGGGCGCGGTGGATCACTTCAGGGCAG GAATTTGAGACCAGCCTGGCCAACATGGTGAAACCCCGTCTCTACTGAAAATACAAAAATTAGCTGGGCGTGG TGGCACGCTGTAGTCCCAGCTGCTCAGGATGCTGAGGCAGAATTACTTGAACCCGTGAGGTGAAGGTTGCAG TGAGCCAAGACTGTGCCACTGTACTCCAGCCTGGGTGACAGAGCGAGATTCTGCCTCAAACAACAACAAAAA CAAAGTATCCACTAGAGCTCTTGGAACATATCACCTGTGGATAAGGGGAACCACTGTATATACAGATCTTTGTG AAGAATACTGCTAACAACCCAAGAGCAATCACTTATTCAGGGCTCACAATGAGCCCAGCACTGGAGTTCCCTG CTCATCCTTGGAAATTTCCTGCTCAGATGCAAACATAGCTGAACTCTCACCTTTTCCTGCTGACAGCCACTCAC CCACATCTCCCTTACTAGAGATAGAAAGAAAAGAATAAAGACCAAAAAACCCTGTTGACTATTTTTTCCTTTCAC TTTTTGAGAAGTGTTAATAGAACTGAAAATACCAGCAAGGAAAAACGCCCTCGAGGAATAGAGTTAATTGGATC TCCAAAATGTTGTCATGAAAGGTGCATTCCTGGGATATGAATTTGATTTCCTTCCTTTCTTCCTCTCTCTTTCTTT CCTCTCTCTCCCTTTCCTTTCCTGTCTTTCAAAACCATTCGCACTCCTTTTATGAGGCATGCAGATCTTGGATTAT TCTTCCACTTTCCAGCCAACTGCACTTCAAAACAGCCTTAATAAGGCTGGGCACGGTGGCTCAGCCTGTAATC CCAACACTTGGGGAGGCCGAGGCGGGCGGATCACCTGAGGTCAGGAGTTTGAGACCAGCCTGACCAACATG GACCTCGTCTCTACTAAAAATACAAAATTATCCCGGCGTGGTGGCGCATGCCTGTAATCGTAGCTACTAGGGA GGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCAGAGGTTGCGGTGAGCGGAGATCGCGCCATTGCAC TCCAGCCAGGGAAATGAGAGTGAAACTCCGTCTCAAAAACAAACAAACAAACAAACAAACAAAAAAAAACGC CTTAGTAACAGTGCCCTCAAGAACCTGGCCTTCCAGTTCTCTGGCAGAGAAGACCTACTGCTGCCGCTAGTCC TCAAGATGGCATTTGCTGGAGGCGGTAGGCAGAGGCCCTAAGTGTGGATTCTAACCCCCGTGGGGACTGAAT CTCTGCGGCTGTTGCTTGCCCAGGCACGTTTGCCTCCCATGAACTTCCTTCATCCACAGGGCCCCAAACCTCA TGCCGGCGGGAGGAGGAAGGAGACTGGGCATAACTCATCAGACTTTCGACTGTAAGAGCTGGAGGCCGCCT GCGGGCTTATCTGTACCCGGGCCTGTCCCCACCCTTCCAGAATGTAAATCCTCTGAGGGAATGTGTCGTCGCC ATCTTTCAGTCCTTTGAGTGCACCCAGTCTCTCTCCAACCCAAAACCCTTTATCCACAGCAATTCTGAGAATGAT GAGAATCCCCCTCACCCCTCACACCGCAAACAGTTGCAATGCTTAGTGGGATTCACCCTTGTCGTCACCAACC CTGCTACTCCAGCCACGTGAGTTTTCCGCCTGTCAGCCAAGCAAAATGGCCTTCCTGCAGTCGCACGGCCCT TTGGTCTCTGCTCAGGGCTTCGGGGACCCTTTCCAGCCATTGCCCTGCACCTACCCACCAGATCGCCGCCCT GGTGGGCGCTCCTGGCCCTGTCCTCCGCGCTTAGTTTGTCATTGGGCGCCCAGATCCGGAACCCCAGCCTC GAAGCTTCCGGTGGCCGGGAACAAAGCCGGTTTTGCTCACTGTCGCCTGGCAAAGCAGGCGCTTGTTAGCA CCCACTGAATGCGCTTATGTGCTCAGAAACGGTCCCATTGGTTGGGACTACCTTCCCCGATGCCCATCCGCCC AGAATCTTCCTTCTGGGATGCCGACTTTTTCAACACGTGCCAGGAGCCCTTCCTCGGCCCGGAATCCCCAGA GTGCCCACAGTGGACAGGGCACCTGGATACACCCCAGACTAACCCACGTTTCCCCGGAGGACCCCAGAGGT TGGAAGCCCCTCCAAGATTAGGGGCGCAGTGCTCCCCTGGCCTGCGGAAGAGTCAGAGGAGTGGGGACAA CATCCAACATCAGCCTCTACTACCGCTAGCGCGACTCCCCGCCGCCGCTCTACTCACCTGACGCGCGCAGTG GACCGCGATTTAGGGGCACAGGGTCTCCCGGGGACCAGCGGCTGGAGCGCTCCGGCCGAGCACCCGCAGT CCCGGCGCCGCGGCCCCACCCCGGCCCCGCCCTCTTCCGCTCCCTCCCAGTCATCAGGCCACCGAGAATGT GCCCCTTGACCCAGATGAGAGGGTGAGCCCGCCAAGGTCAAGCTTCCCATCCTAAGAATCACAGACAGCCC GGCCATGCACCACCACTTCGAGCCTCCGACCAACTGATAGCTGCTGGTCCCAAGTAGCGCTAGGATTTTCGC TTTCCCAGTCTTAATTGACTCTAAAAGAAGAAGAAAAAAAAGCCTGGGCGCGAT (SEQ ID NO. 6)

[0101] In the case of the ZFP57 region, 23 differentially methylated sites were identified. The skilled person will understand that not all of the differentially methylated sites need to be identified in order to determine or measure the resilience status of a subject. As shown in the Examples herein, it is not necessary to measure or identify methylation in all of the sites that were identified for an individual to be classified as highly resilient. For example, the inventors have shown that methylation at four sites gives a good classifier of high resilience.

[0102] In some embodiments, the methods described herein do not involve the measurement of ZFP57.In some embodiments, the genomic region is ZNF727. As shown in Table 1, by ZNF727, we mean the region of sequence in chromosome 7, positions 63,505,812 - 63,545,717 (as determined by the GRCh37.pl3 assembly). An upstream sequence and the first 2000 nucleotides are shown in SEQ ID NO. 7. In some embodiments, the genomic region as defined herein is defined in SEQ ID NO. 7.

[0103] Legend:

[0104] CpG site in classifier (bold underline)

[0105]

[0106] >NC_000007.13:63505612-63507812 Homo sapiens chromosome 7, GRCh37.pl3 Primary Assembly, forward strand GCCAGGCCCCCAACACAAGGAGAAATCGCTGCCTGAACATGCTGCAGGCAAAAACCTGTCACTCTTTCCTCA TTCAGCCCAGTGTCTCATTACATCTCCTGTCGATCAGGGTCTAAATGTGTGGGGCGGAGAACCCCAGCCAAT CAGTGGTGCTAGCGTGAAAACTGCCCAATCAGGTGCGCAGCTAGAGAGGAAGAGGCGGGCTCTTCAATAT GGCAAGGCCTTCGTCTCCTAGCTTCTAGGCTCTGAGTCCAGTACCCGTCTGTACTATTCCATCTCTTCCGCTC CATTAGCTCCTCGGTGACTCCACCATAGCCCCTGTTATCCTGTGACCTGCAGGTACTGGGAGATCCATAGGG AAGAAGGCGGAACATCCGGAGGCTGGGAAATGGTGAGTGCGCGGAGTGGGTGTCCCGAGAAGGGGGAAG AGGCTGTTTGAATCCGGTCGGAACTGGCTGCGGTGGGATCTTGGCCTCGCGGTCAGCTCTGCAGCAGCTCC GAGTCCCCGTGGGCACAGTTCAGTCCTCACTTCCCTCCGTCGCAGATTAGGAGCTGAGCCTGCAGCCAAAA CCCGAGCGTCTTGTTTTGTCCATAAACGCGAATTTCTTTCCAGCCCAGAGAC I I I I I GGGCAGCTCTGTGTCG CAATCCCGAGTCTCCTCCAGATTGTGCGGGGACGTCATAAGACGAGAATCCTCATTCAGGGTCTGGAGTTC CTCCGTGGAAGAAGCAGTGGGCCTTGGGGTCCCCAGTCCCTCCTTTCTCCTTTTAAAAATTGTGGCTTATTTT ATTTATTTATTTTGGAGACAGTGTCTCACTCTGTGCCCAGGCTAAAGTGCAGTGGCAAGATTTCGGCTCACT GCAGCCTCCACCTCCTGGGCTCAGATGATCCTCCCACCCCAGCTCCCCAAGTAGCTAGGACTAGGGAGACA TGCGCCGCCACCTCGCCTGGTTTTGTGGTTTTGTTTTTTGTTTGTTTGGTAGAGACGGGTTTTTGCAATGTTG CTCAGCCTGGTCTCGAACTCCGGAGCTCAGGTGATCCGCCCCCCTCGGGCCTCCTAAAGTGCCGGGATTAC ATGCATGAGCCACTGTCGTAGCCTAAATTGAGGCATCTTTAGGATATAGTTTCGAAGTGTTTTCCAGCCCAAC TCTCCTATTTAAATGTAATACCCCATGTTGGAGGTGGGGCCTGGTGGAAAGTGTTTGGATTATGGGCGTGAA TTTCTCATGAATAGTTTAGCACCATTCCTCTTGGTATTGACCTCTCGATAGTGACTGAGTTCCCTGAAGTTCTT ATTTAAAAGTGTATAGCAACCCCCTTGCCTTCTTTTCAAGCTCCTGCTTCACCTTGCACCAGGATTTTAAGCTT CCTGGGTGGGGCCCCCCCAGAAGCAGATGCTGGTGTTATGCTTTCTGTACAGCCTGTGGAACCATGAGCCA GTTAATCCTCTTTCTTATAAATTACCCAGTCTGAGGCATTTATAGTAATGCCAGAACCGATTAATACAAACAGT TTATTTGCACAAACAGCAATTTATGAATCAGAGAATATCCAACTATGGTTTGGGGGCTCAAAGGAGAGACTT GGAACAAAAGGCTTTTGTAAGAGGTATGAGGAAGCAAACCAGATTCAGTATTTGATTGGTTACAGTTATGTA TTGCATTTGCACCCATCCGGTGGAAATGTCCTGGTTATGTACTTAGAGCTTTATTGGCAGCTTGTGGTTGGTT AAGCCTAAGTTTTGTG I I I I I I I I I I I I I I CTCAAAGTTAGTAATTTGTAAGAAATTCTTTCGACGTAGTTAGGT TTCCTTAGGCAGAATCCCAGAGCATCATGGCCATTTCAGCCTAATTGCCAGCTATTTAGTTATTTTAATGCTC TACAGGGTCCTTGGTTTTGTCTGCAATTTTCAAATGTTTGGCAAGCAGGGTCTCAAATCCAAAACTTCTCCCA GCCTAACTGTTATAGGGACTGTAGAAAATTCTACATTTCCAATTTCTTTCCCACATTCCCCAATGCCGACTATC CCTGTCCAGATCACATTATCAACTATTAGTCCTTTATTTAATTTCAAAACGGACGTGGCATTTTAATTGTTTAT TTTTGTTTAAGAGAGCAGTAGGTGGCTCTTTTTAATTTCGTCTGTTCGTGAACATTTCACATAACAGGAAAGC AGAGAGTAATCACCTGACGCTCTGCGCATC (SEQ ID NO. 7)

[0107] In the case of the ZNF727 region, seven differentially methylated sites were identified. The skilled person will understand that not all of the differentially methylated sites need to be identified in order to determine or measure the resilience status of a subject. As shown in the Examples herein, it is not necessary to measure or identify methylation in all of the sites that were identified for an individual to be classified as highly resilient. For example, the inventors have shown that methylation at four sites gives a good classifier of high resilience.In some embodiments, decreased methylation of CpG sites is associated with greater psychological resilience.

[0108] In some embodiments, the methods of the invention involve a comparison of the methylation levels in the genomic regions between individuals that have been classified as high or low resilience.

[0109] By increased and decreased methylation of CpG sites we mean that these sites are methylated more or less than in subjects with a different level of psychological resilience. For example, "increased methylation of CpG sites" means that subjects have an increased level of methylation at CpG sites in this region compared to individuals with a lower psychological resilience. In some embodiments, psychological resilience in this context can be expressed as a CD-RISC score.

[0110] By appropriate selection of some or all of the genomic regions in Table 1, and some or all of the CpG methylation sites within those regions, the methods of the invention exhibit high predictive accuracy for predicting or determining the psychological resilience of a subject.

[0111] The predictive accuracy of the method, as determined by an ROC AUC value, may be at least 0.50, for example at least 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98 or at least 0.99.

[0112] Thus, in one embodiment, the predictive accuracy of the method, as determined by a ROC AUC value, is at least 0.70, or preferably at least 0.74.

[0113] Thus, in another embodiment, the predictive accuracy of the method, as determined by an ROC AUC value, is at least 0.90, or preferably at least 0.92.

[0114] In the methods of the invention, the 'raw' data obtained in step (ii) undergoes one or more analysis steps before a prediction / determination of psychological resilience is reached. For example, the raw data may need to be standardised against one or more control values (i.e., normalised).

[0115] The methods described herein may be performed using a classification algorithm, for instance. By "classification algorithm" we include any algorithm that is capable of taking the data from step (ii) of the methods of the invention and using it to determine or predictthe psychological resilience of the subject. Therefore, a skilled person does not necessarily need to ascertain whether or how many CpG sites are methylated / unmethylated in a particular population.

[0116] The skilled person will be aware of common classification algorithms used in the art. Common examples are, but are not limited to, the following:

[0117] • Linear Models (for example Ordinary Least Squares, Ridge Classification, Lasso, Elastic-Net, Logistic Regression, Generalized Linear Classification, Stochastic Gradient Descent, Perceptron)

[0118] • Linear and Quadratic Discriminant Analysis

[0119] • Support Vector Machines (SVM) (for example SVM with linear kernel, SVM with polynomial (degree) kernel, SVM with Radial Basis Function Kernel)

[0120] • Nearest Neighbours

[0121] • Gaussian Process

[0122] • Naive Bayes

[0123] • Decision Trees (for example Random Forest)

[0124] • Ensemble Methods (for example Bagging, Boosting, Random Forests, Extremely Randomized Trees, AdaBoost, Gradient Tree Boosting, XGBoost, LightGBM)

[0125] • Neural-Networks Classifiers (for example Multi-layer Perceptron, Artificial Neural- Networks, Deep-Learning)

[0126] • Top Scoring Pairs (TSP) (for example TSP, k-TSP).

[0127] In some preferred embodiments, the methods of the invention are performed using a Random Forest classifier. Random Forest is an ensemble learning method for classification, regression and other tasks that operates by constructing a multitude of decision trees at training time. Given a set of training examples, each marked as belonging to one of two categories, a Random Forest training algorithm builds a model that predicts whether a new example falls into one category or the other.

[0128] In some other embodiments, a support vector machine (SVM) may be used to perform the methods of the invention. The methods described herein may be performed using a support vector machine (SVM), such as those available from http: / / cran.r- project.org / web / packages / el071 / index.html (e.g. el071 1.5-24). However, any other suitable means may also be used.

[0129] Support vector machines (SVMs) are a set of related supervised learning methods used for classification and regression. Given a set of training examples, each marked as belonging to one of two categories, an SVM training algorithm builds a model that predictswhether a new example falls into one category or the other. Intuitively, an SVM model is a representation of the examples as points in space, mapped so that the examples of the separate categories are divided by a clear gap that is as wide as possible. New examples are then mapped into that same space and predicted to belong to a category based on which side of the gap they fall on.

[0130] More formally, a support vector machine constructs a hyperplane or set of hyperplanes in a high or infinite dimensional space, which can be used for classification, regression or other tasks. Intuitively, a good separation is achieved by the hyperplane that has the largest distance to the nearest training data points of any class (so-called functional margin), since in general the larger the margin the lower the generalization error of the classifier. For more information on SVMs, see for example, Burges, 1998, Data Mining and Knowledge Discovery, 2:121-167.

[0131] In one embodiment of the invention, the classification algorithm is 'trained' prior to performing the methods of the invention using DNA methylation profiles from individuals with known psychological resilience status (for example, individuals known to have high or low levels of psychological resilience). By running such training samples, the classification algorithm is able to learn what methylation profiles are associated with high or low resilience. Once the training process is complete, the classification algorithm is then able to determine the resilience status of a sample from a subject.

[0132] It will be appreciated by skilled persons that suitable parameters can be determined for any combination of the genomic regions listed in Table 1 by training a classification algorithm with the appropriate selection of data (i.e. DNA methylation measurements from individuals with known resilience status).

[0133] Preferably, the method of the invention has a sensitivity of at least 60%, for example 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sensitivity. The method of the invention may have a sensitivity of at least 67%.

[0134] Preferably, the method of the invention has a specificity of at least 60%, for example 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% specificity. The method of the invention may have a specificity of at least 72%.By "accuracy" we mean the proportion of correct outcomes of a method, by "sensitivity" we mean the proportion of samples from subjects with high psychological resilience that are correctly classified as high resilience, and by "specificity" we mean the proportion of samples from subjects with low psychological resilience that are correctly classified as low resilience. In some other embodiments, by "sensitivity" we mean the proportion of samples from subjects with low psychological resilience that are correctly classified as low resilience, and by "specificity" we mean the proportion of samples from subjects with high psychological resilience that are correctly classified as high resilience.

[0135] Further statistical analysis of the refined data may be performed using methods well- known in the art, such as PCA, q-value calculation by ANOVA, and / or fold change calculation.

[0136] In some embodiments, the methods defined herein also account for the age of the subject as an additional variable, as age is known to be associated with changes in DNA methylation.

[0137] In some embodiments, the methods and uses defined herein involve measuring the DNA methylation status of the one or more genomic regions defined in Table 1, and then administering one or more therapeutic agents to alter the methylation status of the one or more genomic regions.

[0138] In this embodiment, the subject is identified as having a DNA methylation status that is associated with low psychological resilience, that can be improved by altering the methylation status of one or more CpG sites within the genomic region.

[0139] By "alter the methylation status" we include both increasing and decreasing methylation levels of the genomic region of interest, depending on whether high or low methylation is associated with improved psychological resilience.

[0140] For example, highly resilient individuals have been shown herein to be associated with higher levels of methylation of the LY6G5C region. Therefore, in some embodiments, the one or more therapeutic agents act to increase methylation of DNA, in particular in the LY6G5C region.

[0141] In some other embodiments, highly resilient individuals have been shown herein to be associated with lower levels of methylation of the genomic regions disclosed herein.Therefore, in some embodiments, the one or more therapeutic agents act to decrease methylation of DNA. For example, these therapeutic agents may include one or more therapeutic agents inhibit the action of DNA methyltransferase to inhibit DNA methylation.

[0142] In some embodiments, the one of more therapeutic agents are nucleoside analogs or non¬ nucleoside inhibitors. In some embodiments, the one or more therapeutic agents are selected from: flavonoids; hydrazine; S-andenosyl methionine; cytidine.

[0143] A further aspect of the present invention provides a method of identifying a candidate agent for use in improving psychological resilience of a subject, the method comprising determining if a test agent alters the DNA methylation status in one or more of the genomic regions in Table 1, wherein the test agent is identified as a candidate agent for use in improving psychological resilience of a subject if it alters the DNA methylation status of the one or more genomic regions in Table 1.

[0144] In some embodiments, the test agent is identified as a candidate agent for use in improving psychological resilience of a subject if it increases the DNA methylation status of the one or more genomic regions in Table 1.

[0145] In some embodiments, the test agent is identified as a candidate agent for use in improving psychological resilience of a subject if it decreases the DNA methylation status of the one or more genomic regions in Table 1.

[0146] Of course, whether a test agent is a candidate agent for use in improving psychological resilience of a subject increases or decreases DNA methylation will depend on whether the genomic region to be targeted is associated with greater psychological resilience in a hyper or hypomethylated form.

[0147] The skilled person will be aware of how to determine whether a test agent alters DNA methylation status in the one or more genomic regions in Table 1. For example, this may include treating a population of cells in vitro with the test agent and then measuring the methylation levels of the relevant genomic regions before and after treatment. An increase or decrease in methylation levels (depending on which is associated with improved resilience) is an indicator that the test agent is a candidate for use in improving psychological resilience of a subject.A further aspect of the invention also provides a method of identifying one or more genomic regions that are associated with psychological resilience in a subject, the method comprising:

[0148] (i) providing two or more samples from subjects with known psychological resilience; and

[0149] (ii) identifying genomic regions with a differential DNA methylation status in subjects with differential psychological resilience,

[0150] wherein the genomic regions identified in step (ii) are associated with psychological resilience in a subject.

[0151] This aspect of the invention provides scope to identify further regions of the genome that are associated with psychological resilience, as has been shown is possible for the first time in the data presented herein.

[0152] By "differential DNA methylation status" we include that the DNA methylation status is significantly altered when comparing individuals with different levels of psychological resilience. For example, individuals with low resilience may be associated with low DNA methylation in a particular genomic region and individuals with high psychological resilience may be associated with high DNA methylation in a particular genomic region. In some other embodiments, the level of DNA methylation when comparing two individuals with known psychological resilience is relative. For example, the individual with higher resilience may have higher DNA methylation in the genomic region identified than an individual with lower psychological resilience.

[0153] As described herein, the individuals with known psychological resilience may be measured and classified according to one or more of the known psychological resilience scales. For example, in some preferred embodiments, the individuals may have low psychological resilience (i.e. a CD-RISC 25 score of about 30 or less) according to the CD-RISC 25 scale, or they may have high psychological resilience (i.e. a CD-RISC 25 score of about 70 or more) according to the CD-RISC scale.

[0154] A further aspect of the invention also provides an array for predicting or measuring psychological resilience in a subject comprising an agent or agents for detecting the methylation status in one or more of the genomic regions in Table 1.The agent or agents may be one or more nucleic acid probes that are capable of hybridising specifically to the genomic regions identified in Table 1, or fragments or reverse complements thereof. In some embodiments, the one or more nucleic acid probes are provided as pairs of probes, wherein each pair of probes binds to the same region of genomic DNA, but one of the probes binds to the methylated form and one of the probes binds to the demethylated form (or a derivative thereof, for example DNA treated with sodium bisulfite).

[0155] Therefore, in some embodiments, the array comprises one or more pairs of probes capable of discriminating between methylated and unmethylated CpG sites in the genomic regions described in Table 1. The array may also comprise one or more pairs of probes capable of discriminating between methylated and unmethylated CpG sites in the genomic regions identified according to the aspects of the invention described herein.

[0156] Brief description of the Figures

[0157] Figure 1. Study outline. Flow of patients and data sets generated for the analysis of the association between methylation patterns and resilience status.15More than six of 25 items missing;2)Two samples failed QC;3)One sample failed QC.

[0158] Figure 2. Principal Component Analysis overview of methylation profiles in the Discovery set. A) Percentage of variance explained by the first 15 components, B - D) PCA scores plots of PCI vs. PC2, PC3 and PC4, respectively, with samples color-coded by low (red) and high (blue) resilience status.

[0159] Figure 3. Association of methylation levels at individual CpG sites and the resilience status. A) number of CpG sites identified as differentially methylated probes (DMPs) between high- and low-resilient samples when comparing all samples in the Discovery set and across its ten subsets; B) Volcano plots showing the statistical significance of DMPs relative to the magnitude of difference for every CpG site in comparison for three selected subsets (60%, 30%, 10%); C) Corresponding PCA score plots based on the CpG sites identified as DMPs for the aforementioned subsets, with samples color-coded by the resilience status; D) Proportions of DMPs overlapping with promoter and 5'UTR genomic regions, with horizontal reference line corresponding to proportions of all CpG sites probes in the methylation array, passing QC. *Proportions significantly increased / decreased (p < 0.05); E) Visualization of the strength of association (Iog2 fold-change) between resilience and methylation for the top 25 differentially methylated CpG sites, in the Discovery setand across its ten subsets; F) Bar plot showing the number of subsets in which individual CpG sites were identified as DMPs.

[0160] Figure 4. Association between methylation levels at genomic regions and the resilience status. A) Visualization of the strength of association (DMR area size) between resilience and methylation for the top differentially methylated regions, in the Discovery set and across its ten subsets; B - D) Examples of differentially methylated regions identified when comparing high- and low-resilient samples in the Discovery set and some of its subsets. The points show methylation measurements in samples obtained from subjects with low (red) and high (blue) resilience status. The curves represent the smooth estimate of the methylation profiles in the respective resilience groups.

[0161] Figure 5. Random Forest classifier for distinguishing samples from high- and low-resilient patients based on methylation measurements. A) distribution of the specificity (SP) and sensitivity (SN) obtained when testing the Random Forest classifier across the 500 runs of the splitting the Classifier set into train, internal validation, and test set. B) Area under the curve (AUC) as a function of number of features (CpG sites) used to construct the Random Forest model (top) and a visualization of the genomic annotations of the features used (bottom) showing the proportions of different genomic regions contributing to the classifier. C) A receiver operating characteristic curve, ROC, illustrating the performance of the final Random Forest classifier assessed on the Validation set.

[0162] EXAMPLES

[0163] The inventors sought to identify epigenetic markers of psychological resilience by correlating CpG methylation at various loci with known psychological resilience in patients with breast cancer.

[0164] MATERIALS AND METHODS

[0165] Study Design

[0166] SCAN-B Resilience, an amendment to the Swedish Cancerome Analysis Network - Breast (SCAN-B) initiative (40), is a multi-centre study conducted at four hospitals in southern Sweden (NCT03430492, clinicaltrials.gov), during 2016-2019. The protocol has been published (12) and the study was approved by the Regional Ethical Review Board, Medical Faculty Lund University, and the Swedish Ethical Review Authority. At the time they were informed about the breast cancer diagnosis, participants were also asked to provide a blood sample and their psychological resilience was measured using the Swedish version of the 25-item resilience scale produced by Connor and Davidson (CD-RISC) (41). Permission to use the CD-RISC scale was obtained from J.R.T. Davidson. The scale is aself-reported measure, consisting of 25 questions rated on a 5-point Likert scale. Scores range from 0-100, with higher scores representing higher levels of resilience. Mean imputation was performed for up to six missing items. The validity of the scale in the Swedish context has been confirmed prior to this study (42). Clinical variables were obtained from the Swedish National Breast Cancer Register (NKBC). Information on Smoking habits were collected during a 1-year follow-up visit.

[0167] Study subjects

[0168] Consenting subjects (n=1040) were enrolled into SCAN-B Resilience between 2016 and 2019 (Fig 1). Inclusion criteria for this study specified female patients with newly diagnosed primary breast cancer, aged 18 or older, and able to understand and speak the Swedish language. Further, subjects had to provide a 15 ml blood sample and to answer a minimum of 19 items of the CD-RISC questionnaire (n=934). For the Discovery set, 425 patients, enrolled between 2016 and 2018, were selected from the patients displaying the highest and the lowest CD-RISC scores. The Classifier set (n=123) was sourced from the Discovery set to train a classifier to distinguish between low and high-resilient patients. One sample of CD-RISC with a score under 50 was matched with two samples with CD- RISC score over 80. To eliminate potential covariate effects, samples were matched based on age, smoking, and tumor characteristics (PR, ER, and HER2 status). For smoking, daily, occasional, and previous smokers were combined into one group of 'ever-smokers', while never smokers where matched only to never-smokers. The Validation set (n=80) consisted of independent samples selected from patients enrolled during 2019 (Fig 1).

[0169] DNA methylation analysis

[0170] DNA was purified from whole blood. DNA-methylation status was assessed, using the Infinium MethylationEPIC vl.O BeadChip (Illumina Inc., San Diego, CA 92122). DNA was purified from whole blood. DNA methylation status was assessed, using the Infinium MethylationEPIC vl.O BeadChip (Illumina Inc.). Analyses were performed at the Center for Translational Genomics and Clinical Genomics, Lund University, Lund, Sweden (Discovery set) and at Eurofins Genomics A / S, Galten, Denmark (Validation set).

[0171] Data processing, annotation, and quality control

[0172] raw EPIC array methylation files (IDAT) were processed in R (version 4.2.1), using the ChAMP pipeline (version 2.21.1) for analysis of Illumina BeadChips (43). Briefly, probes were filtered based on (i) p-values >0.01, (ii) presence of at least 3 beads in at least 5% of samples per probe, and exclusion of (iii) non-CpG probes, (iv) SNP-related probes, (v) multi-hit probes, and (vi) probes located on the X and Y chromosomes. After quality control based on density and multidimensional scaling (MDS) plots, beta values were normalized(44) and batch effects associated with methylation date were corrected, using ComBat (45) . Cell type proportions were estimated, using the epiDISH package (version 2.18.0) (46). Probe genomic coordinates were obtained from the EPIC hgl9 manifest. The annotator package (version 1.26.0) was used to annotate probes in relation to genomic features, such as CpG annotations (CpG islands, shores, shelves, and open sea), genetic annotations (e.g., promoters, introns, exons) and FANTOM5 permissive enhancers. Cell type composition was assessed using three reference-based algorithms: robust partial correlations (RPC), robust penalized multivariate regression implementation (CBS) and Houseman's linear constrained projection (CP) as recommended by (13).

[0173] Differential methylation analysis

[0174] To identify differentially methylated probes (DMPs), i.e. probes with methylation signal statistically different between the two groups, ten sample subsets were created based on CD-RISC score percentiles starting with the full sample set of 423 samples (Discovery set), followed by stepwise removal of samples from the middle ranges in steps of 10%, i.e. subsetlO% would contain the 10thpercentile highest samples of the high group and 10thpercentile lowest samples of the low group, respectively (Table 3). Limma package (version 3.58.1) was used to fit a linear model for every CpG site accounting for age, menstrual status, detection mode and ER status. CpG sites were considered as DMPs when the methylation signal differed between the low- and high-resilient groups (p<0.05, logz fold change cut-off < -0.5 or > 0.5). Differential methylation regions (DMRs) were called using bumphunter algorithm (version 1.44.0) (22) with default setting, accounting forage, menstrual status, detection mode and ER status. DMR area was used to estimate the degree of differential methylation. DMR area corresponds by definition (22) to the absolute value of the sum of the estimated model coefficients in the region and, thus, it does not provide an intuitive representation of the underlying methylation signal in the region. However, given a model to compare high- and low-resilient samples, the coefficients represent the average difference between the two groups, and the reported area can be used as surrogate to indicate the strength of the difference in the methylation profiles.

[0175] Random Forest

[0176] The Classification set (n=123) was subjected to 500 iterations of stratified random partitioning into training (50%), internal validation (20%) and test (30%) sets, where each set preserved the inherent triplets of one low-resilient sample matched with two high- resilient samples. In each iteration the relevant features (CpG sites) were identified as those located in DMRs spanning over at least five CpG probes and distinguishing between low- and high-resilient samples. The internal validation set was used to further filter forrelevant CpG sites found in the DMRs regions by retaining only probes, which were consistently hyper or hypomethylated in both the training and internal validation sets (based on fold changes comparison). Ranger implementation (version 0.16.0) (47) of the Random Forest algorithm was then used to train a classifier distinguishing low- and high- resilient samples, using the relevant CpG sites as features and 70% of the samples (including both training and internal validation sets). Model parameters (mtry, number of trees, minimum node size, maximum depth, splitrule) as well as classification threshold were tuned, using nested k-fold cross validation with five folds (k=5) and three repeats (m=3), maximizing Youden's J statistics (J = Sensitivity + Specificity - 1) to account for imbalanced data. The tuned model was finally assessed on the test set. Across the 500 iterations, CpG sites were ranked based on the frequency of appearance. The final Random Forest classifier was tuned, using entire Classifier set (n = 123), again tuning model parameters and classification threshold using nested k-cross validation (k=5, m=3). The model's performance was evaluated on the Validation set (n=79), composed of completely independent samples.

[0177] Statistical Analyses

[0178] Assessment of differences in group demographics and clinical variables were carried out in R (cran.r-project.org), using the gtsummary package (version 1.7.0). P-values were obtained using Pearson's Chi square test for categorical variables with all expected cell counts >5, and Fisher's exact test for categorical variables with any expected cell count <5. For continuous variables Wilcoxon rank sum test was used. Unless otherwise stated p<0.05 was considered statistically significant.

[0179] RESULTS

[0180] Study description and cohort demographics

[0181] The outline and flow of patients for this study, part of the SCAN-B Resilience framework (12), is illustrated in Fig. 1. Briefly, samples for DNA-methylation (DNAm) analysis were first obtained from patients enrolled between 2016 and 2018, fulfilling the inclusion criteria (n=850). From those samples, a Discovery set (n=425) was selected from subjects characterized by the highest and lowest resilience levels, and, after DNAm analysis, used for portraying methylation profiles in respect to resilience status. Samples for a Classifier set (n = 123), used to train a predictive model capable of distinguishing resilience groups in breast cancer patients, were selected from the Discovery set. An independent, prospective Validation set (n=80) was also collected, using samples from patients enrolled during 2019, fulfilling the same inclusion criteria. Demographics and clinical variables of the study cohort and the sample-sets are summarized in Table 2:Table 2. Demographic and clinical variables

[0182] Discovery Set

[0183] Study Cohort High (n = Low (n = p-value3n = 93412122) 2132)

[0184] CD-RISC 71(13) 84(5) 56(8) <0.001 score

[0185] Age 62(11) 61(12) 64(11) 0.028 Menstrual 0.004 status

[0186] post 660 (80%) 147 (75%) 167 (87%)

[0187] pre 161 (20%) 49 (25%) 26 (13%)

[0188] unknown 113 16 20

[0189] Detection 0.28 mode

[0190] screening 547 (61%) 117 (57%) 129 (63%)

[0191] 354 (39%) 87 (43%) 77 (37%) symptomatic

[0192] unknown 33 8 7

[0193] Stage 0.41 46 (5.1%) 9 (4.4%) 12 (5.9%)

[0194] 564 (63%) 121 (60%) 131 (65%)

[0195] 266 (30%) 68 (33%) 58 (29%)

[0196] 15 (1.7%) 5 (2.5%) 2 (1.0%)

[0197]

[0198] 6 (0.7%)

[0199] Unknown 37 9 10

[0200] Histology 0.16 ductal 620 (79%) 136 (75%) 146 (80%)

[0201] lobular 93 (12%) 31 (17%) 19 (10%)

[0202] mixed 76 (9.6%) 14 (7.7%) 18 (9.8%)

[0203] unknown 145 31 30

[0204] ER status 0.038 negative 106 (14%) 33 (18%) 19 (11%)

[0205] positive 677 (86%) 146 (82%) 159 (89%)

[0206] unknown 151 33 35

[0207] PR status 0.18 negative 251 (32%) 63 (35%) 51 (29%)

[0208] positive 531 (68%) 116 (65%) 127 (71%)

[0209] unknown 152 33 35

[0210] HER2 status 0.61 negative 689 (89%) 155 (87%) 159 (89%)

[0211] positive 88 (11%) 23 (13%) 20 (11%)

[0212] unknown 157 34 34

[0213] Smoking 0.23 Ever 93 (13%) 26 (15%) 18 (10%)

[0214] Never 640 (87%) 152 (85%) 155 (90%)

[0215] unknown 201 34 40

[0216] 1Mean (SD); n (%);2Mean (SD) or Frequency (%);3Wilcoxon rank sum test; Pearson's Chi-squared test; Fisher's exact test; CD-RISC: Connor Davidson Resilience Scale 25-item; ER: estrogen receptor; PR: progesterone receptor; HER2: Herceptin receptor 2; Smoking: Ever includes daily, occasional, and previous smokers.

[0217] Data processing and quality control

[0218] During quality control (QC) assessment of the Discovery set, two samples were removed resulting in 423 samples available for the final DNA methylation analyses. A total of 714,959 CpG probes (approx. 84%) passed the QC assessment. In peripheral blood, variation in cell composition across different samples could potentially confound associations of DNAm with modelled outcomes (13). The inventors estimated cell type compositions using three different algorithms, i.e. Robust Partial Correlations, Cibersort, and Constrained Projection, but no differences in cell type composition between the high-and low-resilient samples were detected irrespective of the estimation methods used.

[0219] Psychological resilience associated differential methylation

[0220] Prior to investigating resilience-associated differential methylation, the inventors assessed the overall data structure by Principal Component Analysis (PCA) of the Discovery set (n=423), using the 714,959 CpG probes that had passed quality control (Fig. 2). The first two components, PCI and PC2, captured 13.51% and 10.57% of total variance, respectively, and the PCA plot showed a homogenous data set, with the majority of samples forming one large cluster (Fig. 2A and B). A second smaller cluster of 33 samples (7.8%) was formed along PCI but could neither be attributed to resilience nor any other available clinical variable (Fig. 2B). PC3 explained 6.48% of the overall variance and was found to be significantly associated with age, menstrual status, and smoking (p<0.05), as was PC4 (Fig. 2C). PC4, capturing 2.02% of variance was the first component to be significantly associated with resilience group. Overall, while resilience explained some of the variance in the data, the largest source of variation could not be explained by known factors.

[0221] Another challenge when investigating the association of DNA methylation and psychological resilience is the absence of a precisely defined threshold for categorizing high or low resilience in relation to the CD-RISC scores. Furthermore, nothing is known about the specific scores at which any differences in the underlying biological mechanisms would become detectable, i.e., the point at which low (or high) scores would be reflected by an altered molecular function. To gain some insight into this, the data set was dissected into subsets and performed differential methylation analysis. Starting with the Discovery set (100%), the inventors created ten consecutive subsets based on the CD-RISC score percentiles, i.e., 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% 10% and 5% of samples characterized by the highest and lowest CD-RISC scores (Table 3).Table 3: Subsets of Discovery Set

[0222] Group

[0223] ouoset

[0224] Low High p value All N 162 167

[0225] CD_RISC Mean (SD) 55.9 (7.5) 83.8 (5.5) < 0.001

[0226] Range 32 - 65 77 - 100

[0227] Age Mean (SD) 64.9 (10.7) 61.4 (11.8) 0.006 Range 34 - 88 35 - 89

[0228] s90% N 156 143

[0229] CD_RISC Mean (SD) 55.6 (7.4) 84.8 (5.2) < 0.001

[0230] Range 32 - 64 79 - 100

[0231] Age Mean (SD) 65.0 (10.7) 61.7 (11.8) 0.013 Range 34 - 88 35 - 89

[0232] s80% N 143 120

[0233] CD_RISC Mean (SD) 54.8 (7.2) 85.9 (5.0) < 0.001

[0234] Range 32 - 63 80 - 100

[0235] Age Mean (SD) 65.2 (9.9) 61.3 (11.8) 0.004 Range 38 - 88 35 - 89

[0236] s70% N 119 109

[0237] CD_RISC Mean (SD) 53.2 (6.9) 86.5 (4.8) < 0.001

[0238] Range 32 - 61 81 - 100

[0239] Age Mean (SD) 65.2 (10.0) 60.6 (11.9) 0.002 Range 38 - 88 35 - 89

[0240] s60% N 99 98

[0241] CD_RISC Mean (SD) 51.8 (6.7) 87.1 (4.7) < 0.001

[0242] Range 32 - 59 82 - 100

[0243] Age Mean (SD) 64.4 (9.7) 60.7 (11.2) 0.014 Range 40 - 84 35 - 89

[0244] s50% N 86 71

[0245] CD_RISC Mean (SD) 50.7 (6.5) 88.9 (4.3) < 0.001

[0246] Range 32 - 58 84 - 100

[0247] Age Mean (SD) 64.7 (9.9) 61.6 (11.4) 0.076 Range 40 - 84 39 - 89

[0248] s40% N 68 62

[0249] CD_RISC Mean (SD) 48.9 (6.1) 89.6 (4.2) < 0.001

[0250] Range 32 - 56 85 - 100

[0251] Age Mean (SD) 64.8 (9.7) 61.7 (11.1) 0.097 Range 40.0 - 84.0 39.0 - 83.0

[0252] s30% N 52 48

[0253] CD_RISC Mean (SD) 46.9 (5.7) 90.8 (4.1) < 0.001

[0254] Range 32.0 - 53.0 87.0 - 100.0

[0255] Age Mean (SD) 65.2 (10.0) 61.1 (11.1) 0.055 Range 40.0 - 84.0 43.0 - 80.0Group

[0256] oUDSet

[0257] Low High p value s20% N 33 29

[0258] CD_RISC Mean (SD) 43.9 (5.2) 93.0 (3.9) < 0.001 Range 32.0 - 50.0 89.0 - 100.0

[0259] Age Mean (SD) 66.1 (9.7) 60.0 (10.9) 0.023 Range 51.0 - 84.0 43.0 - 79.0

[0260] S1O% N 18 17

[0261] CD_RISC Mean (SD) 40.2 (4.1) 95.3 (3.5) < 0.001 Range 32.0 - 45.0 91.0 - 100.0

[0262] Age Mean (SD) 68.2 (11.1) 59.4 (12.1) 0.033 Range 51 - 84 43 - 79

[0263] s5% N 10 9

[0264] CD_RISC Mean (SD) 37.4 (3.3) 98.3 (1.6) < 0.001 Range 32 - 41 96 - 100

[0265] Age Mean (SD) 72.3 (11.1) 59.6 (14.2) 0.042 Range 52 - 84 43 - 79

[0266] In total, 27,881 differentially methylated probes (DMPs) (p<0.05, logz fold-change cut-off < -0.5 or > 0.05) were identified when comparing the high- and the low-resilient samples. Looking into the number of DMPs in the Discovery set and across the ten subsets, a sharp increase of DMPs when analysing the distal tails of the resilience scores was observed (Fig.

[0267] 3A). The first distinct increase in DMPs hypermethylated in the low-resilient group occurred in subset 60%, and for hypomethylated DMPs in subset 30%. The difference in methylation signal for many CpG sites became distinguishable first when average CD-RISC scores between samples derived from the high- and the low-resilient subjects differed by more than 20 (subset 60%, Table 3). In addition, PCA plots based on the different subsets also showed an increasing separation of high- and low-resilient samples (Fig. 3C), confirming that the differential methylation was attributed to resilience rather than other unknown factors. To further investigate the functional relevance of the identified DMPs, the inventors assessed their annotation in the context of functional genomic regions. Overall, DMPs were over- re presented in promoters and 5'UTRs across most of the subsets (Fig. 3D), and consequently, proportionally under-represented in the remaining regions (exons, introns or intergenic).

[0268] Next, the inventors analysed how often an individual CpG site was detected as DMP across the discovery set and its ten subsets. Of note, the majority of CpG sites were identified as DMPs in only one subset (n=20,846, 87.5%) or two subsets (n=2,251, 9.5%) (Fig. 3F), highlighting the challenge in performing many tests across numerous CpG sites, which increases the risk of identifying false positives due to multiple comparisons. On the otherhand, several CpG sites identified as DMPs in up to nine different subsets, of which up to eight were consecutive (Fig. 3E) was observed. Importantly, multiple DMPs exhibited a dose-response like pattern with absolute logz fold changes increasing corresponding to increasing differences in mean CD-RISC scores between the high- and low-resilient groups. Functions of the genes annotated to those CpG sites have only partially been elucidated but have been associated with basic cell functions like degradation of misfolded proteins in the ER (DERL2), promotion of cell differentiation and autophagy (CREG1), involvement in neural signalling by stabilizing the receptor of neuropeptide CGRP (RAMP1), as well as facilitating induction of hippocampal long term potentiation (CALB2) (14-17). Particularly interesting in the resilience context were E2F5, MAPT, and PCDH9, where E2F5 mRNA recently was proposed as a target for three anxiolytic miRNAs (IS), while plasma tau (MAPT) has shown association with measures of depression in older cognitively intact adults (19). Lastly, a large study discovered major depressive disorder to be associated with lower levels of PCDH9 in brain and peripheral blood (20). Overall, by dividing the Discovery set into ten subsets and performing differential methylation analysis, it was possible to narrow down the identified DMPs to the ones most likely representing CpG sites associated with the psychological resilience.

[0269] Differentially methylated regions (DMRs) are genomic areas that comprise several DMPs and are thus more likely to be biologically relevant than individual DMPs, since DNA methylation patterns may extend across multiple neighbouring CpG sites (21). Analogously to the DMP analysis, the inventors then used the Discovery set and its ten subsets to identify DMRs and regarded them as associated to psychological resilience when DMRs were detected across multiple subsets. A similar trend when estimating the numbers of DMRs per subset was seen, where a sharp increase at subset 20% was evident for the numbers of DMRs found per subset. To estimate the degree of differential methylation, the inventors used the DMR area (22) as surrogate to indicate the strength of the difference in the methylation profiles. Of note, a similar trend as with the DMPs, with larger area sizes of > 0.5 occurring in the smaller, more distal subsets (Fig. 4A) was seen. Also, similar to the DMPs, not all regions identified as DMRs could be associated with resilience. Some identified regions were detected across non-consecutive subsets (KCNAB2, MYCBPAP), or exhibited no increase (PER3) or even decrease (VTRNA2-1, AC023824.1) in area size. The PM20D1 region was particularly irregular. Methylation levels of this region have previously been shown to be associated with post-traumatic stress disorder (PTSD), although the result could not be replicated (23). When examining the results (Fig. 4), the inventors observed that PM20D1 was not only detected in a non-consecutive manner, but the direction of the methylation profiles was also reversed, i.e. hypermethylation in the low resilient group in subsets 100% to 60% changed to hypomethylation in the subsets10% to 5% (Fig, 4). This suggested that PM20D1 methylation status may not, after all, be linked to psychological resilience and illustrated once more the challenges involved with associating differential methylation data to psychological resilience. On the other hand, the same PTSD study (23) also reported hypomethylation of the ZFP57 region to be reproducibly associated with occurrence of symptoms, and a follow-up study showed that this hypomethylation was reversed in individuals responding to PTSD therapy (23, 24). In the data sets, ZFP57 exhibited the strongest increase in region area, spanning subsets 40% to 5%, and was also hypomethylated in low resilient subjects in concordance with the PTSD study (Fig. 4). Other identified regions included C8orf31, CDH9, and ZNF727, where the two latter have been linked to cognition (25, 26), while C8orf has an exon overlap with LY6S (27). However, the most consistent region by far, being detected as DMR in all but one subset, was a region annotated to LY6G5C, and the methylation profiles in the LY6G5C region showed a familiar dose-response-like trend (Fig. 4), making LY6G5C a strong candidate as marker for psychological resilience.

[0270] A LY6G5C-based classifier for high and low psychological resilience

[0271] Based on the promising findings of several regions likely to be associated with a psychological resilience profile (LY6G5C, ZFP57), the inventors built a classification model, using methylation at CpG sites to predict psychological resilience status. The Classifier set (n = 123) was used, in which each sample was matched with a CD-RISC score of <50 with two samples with a CD-RISC score >80. After initial interrogation, the inventors focused on building a classifier with a Random Forest, as this ensemble method tends to offer high accuracy, variable importance assessment and versatility in handling different data types. The training was focused on the CpG sites found in the regions identified as DMRs. To construct the final model, results were aggregated across 500 models trained by randomly repeating the data splitting. A total of 2,142 CpG sites contributed to the models across the 500 runs. Importantly, CpG sites located in the LY6G5C region were the most consistently identified features, occurring in 402 to 474 of 500 runs. Among the 50 most common CpG sites the inventors detected those in the ZFP57 region, which was consistent with previous findings based on the Discovery set. Across the 500 runs, low-resilient samples were correctly classified with a mean AUC of 0.7 (±0.19 SD), sensitivity (SN) of 0.67 (±0.11 SD), and a specificity (SP) of 0.71 (±0.10 SD) (Fig 5).

[0272] To build the final model, the inventors used CpG sites ranked by their contributions to the 500 models. It was noted that the best performing models in terms of high AUC were obtained when using between 6 and 9 of the top CpG sites. Strikingly, even here the top nine probes were exclusively annotated to LY6G5C (Fig. 5C). The final model included 9 features, 8 CpG sites and age, the only clinical variable that was accounted for due to thewell-established link between age and methylation status. The final model was tested using an independent validation sample set, containing samples collected during 2019 (n=79 passed quality control) and displaying CD-RISC scores < 50 (n = 12) or > 80 (n = 67). Classifying these independent validation samples yielded predictions of the resilience status, with an AUC of 0.74, a sensitivity of 0.67 and a specificity of 0.72, confirming the potential of the classifier for psychological resilience (Fig. 5C).

[0273] CONCLUSIONS

[0274] The data presented herein represents a multi-layered, genome-wide analysis of the epigenome on a large cohort of newly diagnosed breast cancer patients with known resilience status.

[0275] The inventors have identified several loci that display strong discriminating ability related to psychological resilience, which is a novel finding. In particular, identification of the LY6G5C region, which displayed the strongest discriminating ability related to psychological resilience is a very interesting finding. In this context it is important to recognize that the CpGs forming the LY6G5C DMR are not located in the promoter but rather in exons and introns, where methylation has been associated with increased translation rates (35) congruent with the finding that high-resilient individuals display higher methylation of the LY6G5C region. In concordance, another study showed that DNAm of LY6G5C in peripheral blood mononuclear cells is inversely correlated to cognitive decline (36). These findings also confirmed the relevance of ZFP57, the methylation pattern of which has previously been shown to be associated with both the development and successful treatment of PTSD {23,24), but herein the inventors have shown its importance in pshycological resilience. The phenomenon that increased methylation of LY6G5C seemed to be a characteristic of highly resilient subjects was also comparable to the finding that successful treatment and reduction of PTSD symptoms was related to increased methylation of ZFP57 (23). Taken together, these results point towards a role of LY6G5C in maintaining healthy brain function, as well as an improved physiological stress response, the latter being a signum for high resilient subjects.

[0276] In conclusion, the data herein clearly demonstrates that the CpG methylation status of several loci are associated with the development of psychological resilience. This provides support for the utility of such epigenomic patterns for predictive, diagnostic and therapeutic purposes to foster resilience in vulnerable individuals. In particular, the use of therapeutic agents to manipulate CpG methylation status is envisaged by the inventors.References

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Claims

CLAIMS1. A method of predicting or measuring psychological resilience of a subject, the method comprising:(i) providing a sample from the subject to be tested;(ii) measuring the DNA methylation status in one or more of the genomic regions in Table 1,wherein the DNA methylation status of the one or more genomic regions in Table 1 is indicative of the psychological resilience state in the subject.

2. A method of improving psychological resilience of a subject, the method comprising:(i) providing a sample from the subject to be tested;(ii) measuring the DNA methylation status in one or more of the genomic regions in Table 1,wherein the DNA methylation status of the one or more genomic regions in Table 1 is indicative of the psychological resilience state in the subject, and(iii) administering one or more therapeutic agents to alter the methylation status of one or more genomic regions in Table 1, thereby improving the psychological resilience of the subject.

3. A therapeutic agent for use in improving psychological resilience of a subject, wherein the use comprises administering a therapeutically effective amount of the therapeutic agent to the subject to alter the methylation status of one or more genomic regions in Table 1 which are associated with psychological resilience, wherein the therapeutic agent alters the methylation status to a status associated with improved psychological resilience.

4. The method or therapeutic agent for use of any one of the preceding claims, wherein the subject has cancer.

5. The method or therapeutic agent for use of any one of the preceding claims, wherein the subject has breast cancer.

6. The method or therapeutic agent for use of any one of the preceding claims, wherein the genomic regions are one or more of the following: LY6G5C, ZFP57, PF4, RP11- 16E12.1 / RP11-16E12.2, CDH9, ZNF727, and / or C8orf31.

7. The method or therapeutic agent for use of claim 6, wherein the genomic region is LY6G5C.

8. The method or therapeutic agent for use of any one of the preceding claims, wherein the psychological resilience of the subject is expressed according to a CD-RISC 25 score.

9. The method or therapeutic agent for use of claim 8, wherein the method is validated by comparing the CD-RISC 25 score determined by the method of any of the preceding claims with a CD-RISC 25 score calculated according to psychometric scoring of the subject.

10. The method of claim 2, wherein the psychological resilience of the subject is measured according to a CD-RISC 25 score, and wherein step (iii) involves administering one or more therapeutic agents to alter the methylation status of the one or more genomic regions, thereby increasing the CD-RISC 25 score of the subject.

11. The method or therapeutic agent for use of any one of the preceding claims, wherein the subject is identified as having a low psychological resilience if there is hypomethylation at the LY6G5C locus.

12. The method or therapeutic agent for use of any one of claims 8-11, wherein the subject is defined as having low psychological resilience if they have a CD-RISC 25 score of about 30 or lower.

13. The method or therapeutic agent for use of any one of claims 8-11, wherein the subject is defined as having high psychological resilience if they have a CD-RISC 25 score of about 70 or higher.

14. The method or therapeutic agent for use of any one of the preceding claims, wherein the sample from the subject is any biological sample comprising DNA, preferably a whole blood sample, optionally wherein the blood sample is a serum or plasma sample.

15. The method or therapeutic agent for use of any one of claims 2 or 3, wherein the one or more therapeutic agents inhibit the action of DNA methyltransferase to inhibit DNA methylation.

16. The method or therapeutic agent for use of claim 15, wherein the one of more therapeutic agents are nucleoside analogs or non-nucleoside inhibitors.

17. The method or therapeutic agent for use of claims 15 or 16, wherein the one or more therapeutic agents are selected from: flavonoids; hydrazine; S-andenosyl methionine; cytidine.

18. The method or therapeutic agent for use of any one of claims 2-16, wherein the therapeutic agent increases CpG methylation at the one or more genomic regions, optionally wherein CpG methylation is increased at the LY6G5C region.

19. A method of identifying a candidate agent for use in improving psychological resilience of a subject, the method comprising determining if a test agent alters the DNA methylation status in one or more of the genomic regions in Table 1, wherein the test agent is identified as a candidate agent for use in improving psychological resilience of a subject if it alters the DNA methylation status of the one or more genomic regions in Table 1.

20. A method of identifying one or more genomic regions that are associated with psychological resilience in a subject, the method comprising:(i) providing two or more samples from subjects with known psychological resilience; and(ii) identifying genomic regions with a differential DNA methylation status in subjects with differential psychological resilience,wherein the genomic regions identified in step (ii) are associated with psychological resilience in a subject.

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