In the proposed study, three objectives will be pursued:

1. To develop a method to identify more effectively the acute and long-term risk of adolescents with the most threatening self-harm behaviours.
2. To identify the factors that influence the risk of self-harm behaviours and the success of treatment/treatment of these behaviours in the most at-risk adolescents (changes in these factors).
3. Develop guidelines for more effective treatment of the most at-risk adolescents.

For this purpose, a sample of approximately 200 young people who will be hospitalised for suicide risk (the most at risk in Slovenia) and an approximately equal number of healthy adolescents will be included. At inclusion, the presence of several factors will be assessed by reviewing demographic data, clinical diagnosis, self-assessment questionnaires and clinical psychological tests (CSSRS, B-NSSI-AT, ISAS, LPFS-BF2.0, BPFSC-11, TSCC, PAI, ECR-RS, DASA-YV, ASHRS), social assessment, and blood sampling for genetic analyses (DNA isolation, sequencing, nucleotide sequence recognition, quantification and evaluation of short tandem repeats, identification of methylation sites). Longitudinal tracking of autoaggressive events and heteroaggressive events during hospitalisation will be performed and recorded on an ongoing basis. The risk and protective factors of the adolescents most at risk will be compared with a control group of adolescents. The same factors will be reassessed in the most at-risk adolescents after 6 and 18 months of treatment as usual. The data will be collected in a data entry and storage system that will ensure the privacy of the data entered in accordance with the GDPR.

This will allow the investigators to identify young people at particular risk of severe self-harm behaviour more reliably, to target them for more intensive and effective treatment, and thus to improve their safety, quality of life and prognosis in the short and long term.

DESCRIPTION OF THE RESEARCH PROJECT "Influences on and prevention of self-harm behaviour among the most at-risk adolescents".

1. Scientific background Detailed in the attached Study Protocol
2. Detailed description of the work programme 2.1. Content and work programme The study will include a sample of approximately 200 young people who will be hospitalised for suicide risk (the most at risk in Slovenia) and an approximately equal number of healthy adolescents. At enrolment, the investigators will assess the presence of several factors by reviewing demographic data, clinical diagnosis, self-assessment questionnaires, social assessment, clinical psychological tests, rating scales and blood sampling for genetic analyses. They will longitudinally track autoaggressive events and heteroaggressive events in the patients during hospitalisation and keep a running record of them. The risk and protective factors of the most at-risk adolescents will be compared to a control group of healthy adolescents. The same factors in the most at-risk adolescents will be reassessed after 6 and 18 months of treatment. A detailed description of the study design is given in Figure 2 and Table 2 of Study Protocol.

2.2. Subjects: Subjects with inclusion and exclusion criteria are described in Table 2 (Study Protocol, WP2).

2.3. Methods: 2.3.1. Questionnaires and psychodiagnostic tools:

1. Enrollment questionnaire, General questionnaire1, General questionnaire2, General questionnaire3: The questionnaires will cover general and demographic data, information on previous treatments, medication received, mental disorder at discharge (ICD), psychosocial situations (ICD), physical illnesses (ICD), COVID status (vaccinated, recovered, tested), use of psychoactive substances, history of self-harm, suicide attempts, school performance, experience with peers, sexual orientation and identity, residence, family composition, family history of mental disorders, self-harm and suicide attempts.
2. Columbia Suicide Severity Rating Scale (CSSRS).
3. The Brief Non-Suicidal Self-Injury Assessment Tool (B-NSSI-AT).
4. Personality Assessment Inventory-Adolescent (PAI-A).
5. Trauma Symptom Checklist for Children (TSCC).
6. Experiences in Close Relationships-Relationship Structures (ECR-RS).
7. Dynamic Appraisal of Situational Aggression-Youth Version (DASA-YV).
8. Adolescent Self-Harm Risk Scale (ASHRS): this is the scale we developed for the present study.
9. The Level of Personality Functioning Scale-Brief Form 2.0 (LPFSBF 2.0).
10. Lifetime Incidence of Traumatic Events questionnaire (LITE).
11. Borderline Personality Features Scale (BPFSC-11).
12. Inventory Of Statements About Self-Injury (ISAS).

2.3.2. Genetic methods: DNA isolation: The genomic DNA is going to be isolated according to the established laboratory protocol using FlexGene DNA kit (Qiagen Germany) and 5 mL EDTA-blood sample. Isolated DNA will be stored at 4°C.

Sequencing: Sequencing will be performed on available in-house long-read sequencing technology nanopore PromethION (Oxford Nanopore Technologies). The ability to detect DNA modifications via differences in the electric current intensity produced from a nanopore read of an unmodified base and that of a modified base, enables the detection of DNA methylation in different CpGs from human natural DNA at epigenome-wide level.

Libraries will be prepared using ligation sequencing kit (SQK-LSK 109, Oxford Nanopore Technologies) in following stages: 1. NEBNext Ultra II End repair / dA-tailing, 2. ligation of the native barcodes, 3. ligation of sequencing adapters, 4. flow cell priming and DNA library loading (Flow Cell Priming Kit, Oxford Nanopore Technologies).

The analysis will be conducted using barcoded participants' DNA, enabling the simultaneous analysis of multiple participant's, by pooling the equimolar amount of DNA into a single pool, based on the observation groups. The sample pooling will reduce the effect of individual epigenome variability and enrich the cohort-specific epigenetic signals, potentially associated with the clinical phenotype.

Bioinformatics Base Calling: From the raw sequencing signal, the nucleotide sequences will be determined using the software tool Guppy, which is based on a predictive neural network model. The identified reads and their reliability will be stored in FASTQ files. For subsequent alignments of long reads, the human genome (GRCh38) will be used as a reference.

Variant Calling: Megalodon software (Oxford Nanopore Technologies) will be utilized for determining nucleotide sequences from the raw signal, alignment of readings on human reference genome and variant calling. The reliability of the findings will be additionally confirmed using the software tool Medaka (Oxford Nanopore Technologies). The tool uses a neural network model to identify genetic variants, based on constructed consensus sequences. Variant data will be stored in VCF files.

Quantification and evaluation of short tandem repetitions: Tandem-genotypes and STRique will be used to detect changes in length of tandem repeats, from aligned long DNA reads. In addition to the aligned reads input, the software tool STRique accepts the raw sequencing signal, based on which a hidden Markov model quantifies and evaluates the likelihood of a change. The validated changes in length of tandem repeats will be stored in TSV files.

Methylation sites detection: Different methylation-calling tools will be utilized using the METEORE software tool. The reliability of the methylated CpG islands found in the results will be demonstrated by the prediction models (random forest and multiple linear regression). Additionally, the detected methylated sites will be compared with the results of the Remora software (Oxford Nanopore Technologies). The locations of reliable methylation sites will be stored in BED and TSV files.

2.4. Conduct of the research "Influences on and prevention of self-harm behaviour among the most at-risk adolescents" The detailed survey process is described in Figure 2 and Table 2 of Study Protocol.

Detailed definition of the work packages of the research "Influences on and prevention of self-harm behaviour among the most at-risk adolescents".

2.4.1. WP 1: Establishment of a working research module on "Influences on and prevention of self-harm behaviour among the most at-risk adolescents".

Lead organisation: University of Ljubljana, Faculty of Medicine and Centre for Mental Health, University Psychiatric Clinic Ljubljana (UPKL) Participating organisations: the Clinical Institute of Special Laboratory Diagnostics of the Paediatric Clinic of the UKC Ljubljana (KISLD) and the Faculty of Electrical Engineering, Computer Science and Informatics of the University of Maribor (FERI) T1: At this stage, all questionnaires were prepared in sets with the corresponding survey numbers.

1. Enrollment kit (Screening questionnaire, CSSRS, B-NSSI-AT)
2. Time1 kit (General Questionnaire1, PAI-A, TSCC, ECR-RS, DASA-YV, ASHRS, LPFSBF 2.0, LITE, BPFSC-11, ISAS), venous blood sample collection for genetic analyses
3. Time2 kit (DASA-YV tracing, Incident Report (commited and prevented), General Questionnaire2)
4. Time3 kit (General Questionnaire3, CSSRS, B-NSSI-AT, PAI-A subscales, TSCC subscales, ECR-RS, DASA-YV, ASHRS, LPFSBF 2.0, LITE, BPFSC-11), venous blood sample collection for genetic analyses.

T2: Development of a protocol for subject enrolment and data acquisition, storage and analysis. This work included the preparation of information materials for subjects and parents/guardians, informed consents/assents and a detailed protocol that will be accessible to the investigators throughout the study.

T3: Preparation of the data entry and storage system: The data entry and storage information system will be prepared so that it will enable data entry via pre-prepared templates for initial and intermediate reporting of measurements. The system will ensure data privacy, as access will be restricted to anonymized data and allowed only for the purposes of analysis (in line with the General Data Protection Regulation (EU) 2016/679 (GDPR)). The system will ensure that data is safely deleted after the project is completed (according to the GDPR). The information system will be physically located in the Republic of Slovenia, and physical access to the hardware will be limited only to administrative activities by qualified personnel.

T4: Education and training of the working group: This part will consist of several educational sessions for the different members of the working group: a. Patient involvement, involvement of pupils and students of the control group (assessment of the fulfilment of inclusion/exclusion criteria, information about the study, obtaining consent from adolescents and relatives), b. Collection of questionnaire responses, c. Collection of biological samples, d. Data collection during hospitalisation, e. Follow-up. Initial training sessions will be followed by more frequent follow-up of the study processess, possible complications, necessary adjustments of the protocol according to the initial experience (project manager Maja Drobnič Radobuljac) and re-training if necessary.

2.4.2. WP 2: Study and control group recruitment, data collection, patient follow-up.

Lead organisation: UPKL Participating organisations: KISLD and FERI

Objectives:

In this work package the investigators will focus on the involvement of patients and healthy adolescents. The work package includes capturing all patient data at Time 1 and tracking patients during hospitalization until discharge (Time 2) and recapturing data at Time 3 (Figure 2 of Study Protocol). On the control group side, only data at Time 1 will be captured (Figure 2 of Study Protocol). This work package has three main objectives: enrollment of hospitalized and healthy adolescents, data collection and follow-up.

Tasks:

T1: Enrollment of hospitalised adolescents. The Enrollment Kit and the Time1 Kit will be administered (see Figure 2 of Study Protocol), and the Time2 Kit will be administered during hospitalisation and at discharge.

T2: Enrollment of healthy adolescents (control group). The Enrollment Kit and the Time Kit1 will be administered (see Figure 2 of Study Protocol).

T3: Data collection. Data from hospitalised patients will be collected by administering the Enrollment, Time1, Time2 kits (see Figure 2 of Study Protocol). Adolescents in the control group will have the Enrollment and Time1 kits administered immediately on enrollment, no other data will be collected from these adolescents (see Figure 2 of Study Protocol). In the event that any of the adolescents approached appeared to be in need of help for self-injurious behaviour or other overt mental disorders, they will be referred for appropriate treatment.

T4: Tracking. Six and 18 months after the end of the hospitalization, the investigators will contact the patients again and administer the Time3 kit (see Figure 2 of Study Protocol). If any of the adolescents approached appear to need help for self-injurious behaviour or other overt mental disorders, they will be referred for appropriate treatment.

2.4.3. WP 3: Analysis of extracted materials, data entry and statistical analysis Lead organisation: UPKL, KISLD and FERI Participating organisations: UPKL, KISLD and FERI

Objectives:

The first objective is to identify the factors influencing the occurrence of the most threatening forms of self-injurious behaviour in adolescents.

The second aim is to assess the factors that influence change in risk of self-harm behaviour in the most at-risk adolescents.

The third objective is to develop an instrument (scale) for rapid assessment of short- and medium-term risk of self-harm in adolescents (ASHRS).

The details are in the Study Protocol.

CLINICAL GROUP:

Inclusion Criteria:

• Suicidality
• Self-harming with no intention to die

Exclusion Criteria:

• Confirmed acute psychotic disorder
• Intellectual disability
• Severe physical illness (e.g. cardiovascular or renal disease)
• Disease of the central nervous system (e.g. encephalitis, brain injury or haemorrhage, epilepsy)
• Acute poisoning (including with psychoactive substances)

CONTROL GROUP:

Inclusion Criteria:

- Age 13-19

Exclusion criteria:

• Suicidality
• Self-harming with no intention to die
• Known mental disorder (e.g. depression, bipolar disorder, schizophrenia, intellectual disability)
• Severe physical illness (e.g. cardiovascular or renal disease)
• Disease of the central nervous system (e.g. encephalitis, brain injury or haemorrhage, epilepsy)
• Acute poisoning (including with psychoactive substances)
• Mental disorder, history of suicidality or self-injurious behaviour in a first-degree relative (a parent or a sibling).

CLINICAL GROUP:

• patients hospitalized in the Intensive Child and Adolescent Psychiatry Unit or the Unit for Adolescent Psychiatry of the Centre for Mental Health University Psychiatric Clinic Ljubljana
• aged between 13 and 19 years.
• In the case of consent, parents will also be asked for consent for those under 15 years of age.

CONTROL GROUP:

• 8th grade students in primary school,
• 1st year students in secondary school
• 1st year students in college
• aged 13-19 years
• Attending a regular systematic review. These scheduled regular systematic examinations are accompanied by venous blood sampling.