Genetics of Self-injurious Behaviour
Condition: Self Injurious Behavior Without Suicidal Intent; Self-Injurious Behavior
MeSH terms: Self-Injurious Behavior
Study type: Observational

Summary: 
      Nonsuicidal Self-Injury (NSSI) is being increasingly regarded as a separate psychiatric
      disorder. Since the latest Diagnostic and Statistical Manual of Mental Disorders - DSM-5 from
      2013 defined NSSI as a separate diagnosis under section III - Conditions for Further Study,
      the knowledge about this field has increased considerably; however, the aetiology of this
      behaviour has still not been explained. There are many psychological explanations for the
      development and the continuation of NSSI. Researchers have identified the most common
      comorbidities (depression, borderline personality disorder, anxiety). The causes of NSSI are
      not known, although studies that have been carried out so far indicate both genetic and
      environmental factors.

      The research included 95 adolescents with NSSI (participants were diagnosed based on the
      DSM-5 criteria), an original control group consisting of 21 people without NSSI, and 118
      individuals from the general population as an additional control group for genetic research.
      For all participants we carried out the genotyping of polymorphisms for the TPH1 (rs4537731,
      rs1799913, rs7933505), SLC6A4 (VNTR STin2), OPRM1 (rs1799971), GNβ3 (rs5443) and DRD2/ANKK1
      (rs1800497) genes. The participants with NSSI and the control group without NSSI completed
      translated questionnaires for the Barratt Impulsiveness Scale (BIS-11), State-Trait Anxiety
      Inventory for Adults (STAI), MacLean Screening Instrument for BPD (MSI-BPD) and the Early
      Trauma Inventory Self Report-Short Form (ETISR-SF). The participants with NSSI also completed
      the questionnaire for the Inventory of Statements about Self-Injury (ISAS), and the
      Self-Injury Craving Questionnaire (SICQ). The investigators carried out an association
      analysis and G x E analyses.

      The aim of the research was to carry out the first G x E study on the etiology of NSSI in
      Slovene adolescents. We have hypothesized that NSSI could be associated with one of the
      candidate polymorphisms or a combination of candidate polymorphisms. Further we have
      hypothesized that the genetic polymorphisms associated to NSSI are the most connected to NSSI
      in traumatised individuals and that NSSI is associated with higher impulsivity.
    

Detailed description: 
      Theoretical background The etiology of Nonsuicidal Self-Injury (NSSI) has still not been
      explained. Studies that have been carried out so far indicate both genetic and environmental
      factors. It has been suggested, that NSSI is a polygenic disorder. Two neurotransmitter
      systems, the serotonergic system and the endogen opioids system, are the main candidate
      systems in the etiology of NSSI.

      The serotonin system is important in comorbid disorders of NSSI (depression, anxiety…). There
      is also growing importance of its direct involvement in NSSI. Deficiency of tryptophan, a
      precursor of serotonin, has been connected with increasing frequency of NSSI. Variable-number
      tandem repeat polymorphism of the serotonin transporter gene - SLC6A4 (VNTR STin2) has been
      associated with borderline personality disorder, a disorder with frequent NSSI, in one study.
      The are some important polymorphisms of the gene for the tryptophan hydroxylase enzyme, the
      main enzyme in the synthesis of serotonin. The polymorphism rs1799913 has been associated
      with depression and with parasuicidal behavior. This polymorphism is, together with some
      other polymorphisms (e.g. rs4537731, rs7933505), a part of a haplotype. This haplotype has
      already shown some connections with borderline personality disorder.

      There is also growing evidence that the changes in concentration of endogenous opioids (EO)
      could be associated with NSSI. Significantly lower β-endorphin and met-enkephalin values in
      the liquor of individuals with NSSI had been demonstrated. A recent study has shown
      significantly lower basal β-endorphin levels in adolescents with NSSI, when compared to
      healthy controls. Naomi I. Eisenberger is a leader in studying polymorphisms of endogenous
      opioids in conjunction with "emotional" pain. People with a lower threshold for physical pain
      also described a greater sensitivity to emotional pain. There is numerous evidence of the
      significant effect of the polymorphism of the gene for the mu-opioid receptor OPRM1 (A118G;
      rs1799971) on altered sensitivity to physical pain, pain perception and response to opiate
      analgesics. A significantly increased sensitivity of G allele carriers to social rejection
      (i.e. on a model for "emotional" pain) has been demonstrated. A number of studies have been
      conducted on the impact of the rs1799971 polymorphism on craving and addiction. Given the
      theoretical addictive background of NSSI and the interconnectedness of physical and emotional
      pain and the role of the rs1799971 polymorphism, this polymorphism is an important candidate
      polymorphism in NSSI. For individual addictions, different, partly conflicting results
      appear. Various studies have confirmed the G allele as a risk factor for various addictions,
      while others have not demonstrated any impact or have found a reduced risk of addiction in G
      allele carriers. Thus, in 2016, the first collaborative analysis was carried out in which
      9064 subjects of the Caucasian population were included. In this large, European sample, the
      G allele proved to be a significant (OR = 0.90; confidence interval 95 % (0.83 - 0.97); p =
      0.0095) protective factor for addiction in general. Direct studies of craving and the impact
      of rs1799971 on craving are rare and contradictory. There has been little research on
      potential genetic influences on the development of NSSI and rs1799971 does not appear to have
      been studied in this field.

      The main target of antipsychotics, the dopamine receptor D2, is also important in the
      research of addiction. The most famous polymorphism of the dopamine receptor 2 gene is the
      Taq1A (TaqIA) (rs1800497) polymorphism. Two alleles of rs1800497 are known - A1 and A2.
      Allele A1 has been associated with pathological gambling, a typical behavioral addiction, in
      one study. It has also been associated with impulsivity and addictive behavior in some other
      studies. The A1 allele has also been associated with borderline personality traits. G -
      proteins have been connected with the proper activity of numerous receptors. The polymorphism
      C825T (rs5443) of the G protein beta 3 gene (GNbeta3) has in some research been connected
      with depression. In one study, the T allele carriers had a statistical higher risk for NSSI
      as other depressive patients.

      Diminished pain perception and a higher pain threshold in people with NSSI have been shown.

      Ethics committee approval and project registration The study has been approved by the Medical
      Ethics Committee of the Republic of Slovenia (number 69/06/13). It has been chosen and
      registered as an internal scientific research and developmental project of the University
      Medical Centre Maribor, Slovenia, on 1.8.2013 (project number IRP-2013/01-07).

      Questionnaires All adolescents from the clinical sample and the original control group filled
      out the Slovenian version of the self-assessment questionnaire of the State-Trait Anxiety
      Inventory for Adults (STAI) and the following questionnaires, translated into Slovene:
      Barratt Impulsiveness Scale (BIS-11), MacLean Screening Instrument for BPD (MSI-BPD), Early
      Trauma Inventory Self Report-Short Form (ETISR-SF). The STAI measures two forms of anxiety.
      "State anxiety" measures the current state, i.e. the level of anxiety that the individual
      feels at a given event. "Trait anxiety" refers to the long-term state of the individual.
      BIS-11 is probably the most frequently used instrument for the measurement of impulsivity.
      The MSI-BPD is a screening questionnaire used to search for borderline personality disorder
      in adolescence. ETISR-SF measures the childhood trauma, including physical, emotional and
      sexual abuse, as well as general traumatic events.

      In order to identify NSSI, the investigators used the Slovenian translation of the Inventory
      of Statements about Self-Injury (ISAS) and the Slovenian translation of the Self-Injury
      Craving Questionnaire (SICQ) for the clinical sample. The ISAS is a questionnaire consisting
      of two parts. The first part consists of 12 questions about self-injurious behavior and the
      second part of questions about 13 functions of NSSI. The SICQ contains 7 questions about the
      craving for NSSI.

      DNA extraction and genotyping There is little research about genetics of NSSI. Some candidate
      genetic polymorphisms had been chosen with the help of this information. Predominantly, the
      candidate genetic polymorphisms in the study have therefor been chosen with the help of
      information about genetics of addiction and comorbid disorders (depression, borderline
      personality disorder, …).

      DNA for genotyping was extracted from peripheral blood mononuclear cells using TRI-reagent
      (Sigma, Steinheim, Germany) according to manufacturer's instructions. Quality and
      concentration of extracted DNA was checked using agarose gel electrophoresis and SynergyTM 2
      (Biotek, Winooski, VT, USA) spectrophotometer.

      For the genotyping of the polymorphisms, the investigators first performed the real-time
      polymerase chain reaction (qPCR) and then the high resolution melting (HRM) or the
      restriction fragment length polymorphism (RFLP) method. The qPCR was performed as follows:
      denaturation at 95 °C for 10 min, multiplication, 45 cycles of 95°C 10 s, 60°C 15 s, 72°C 10
      s. The HRM has been made at the following protocol: 95°C 1 min, 40°C 1 min, 60 - 90°C at 0,02
      °C/s and cooling at 40°C for 10 s. The HRM reaction has been made with the LightCycler® 480
      (Roche) and the LC480 HRM Master Mix (Roche) has been used. The investigators have analyzed
      the genotypes from the melting curves (TABLE 1). In the RFLP protocol, the product of the
      qPCR has been incubated with the right restriction enzyme from New England Biolabs (TABLE 1).
      Analysis has been done with agarose gel electrophoresis (2 % agarose gel, tris borate EDTA
      buffer). The DNA samples were mixed with 0.25% xylene cyanol and 40% sucrose. Electrophoresis
      has been run at 150 V for 20 minutes. At the end, the agarose gel has been analyzed under the
      UV light.

      Gene; genetic polymorphism; sequence of the primer; temperature; genotyping method;
      restriction enzyme:

        1. OPRM1; rs1799971; CGGTTCCTGGGTCAACTTGT, GATCGTGATGGCCGTGAT; 60 °C; HRM; /.

        2. TPH1; rs4537731; GTTTCATGCAGGTATTAGTG, TGGCATTGAAGTAAGAGCAC; 60 °C; RFLP; Sau3AI.

        3. TPH1; rs1799913; GTTAAGCACTGCAGCGTGAC, AAGCGGGACATGACCTAAGA; 60 °C; HRM; /.

        4. TPH1; rs7933505; AAACTGAGAGGAAAATGCTTGC, CATTGCCGTTGAACTTTTGA; 65 °C; RFLP; NlaIII.

        5. DRD2; rs1800497; CTTGCCCTCTAGGAAGGACAT, ACCTTCCTGAGTGTCATCAACC; 65 °C; RFLP; TaqI.

        6. GNB3; rs5443; CATCATCTGCGGCATCACG, ACGCTCAGACTTCATGGAGT; 60 °C; HRM; /.

        7. SLC6A4; VNTR; GGGCAATGTCTGGCGCTTCCCCTACATA, TTCTGGCCTCTCAAGAGGACCTACAGC; 65,5 °C; PCR;
           /.

      Statistical analysis The data was analyzed using IBM SPSS Statistics program package (IMB
      Inc., Armonk, New York). P ≤ 0.05 was considered as statistically significant.

      The comparison of the share of traumatized and abused adolescents in the clinical sample and
      in the original control group has been done with the Fisher's exact test. All continuous
      variables were first assessed for the normality using Kolmogorov-Smirnov test of normality.
      Correlation between the age at the onset of NSSI and the total number of NSSI in life was
      determined using Spearman's correlation. The relationship between the total number of
      self-injuries (NSSI) in life and the value on the SICQ questionnaire has also been assessed
      using Spearman's correlation analysis. Relationship of the value of the SICQ questionnaire
      and the time that passes from the thought of NSSI to NSSI and also genotype and the total
      number of NSSI in life both have been assessed using Kruskal-Wallis H-test. The Mann-Whitney
      U-test had been used to determine the relationship between genotype and the age at the onset
      of NSSI. Different logistic regression models with parameters of the ETISR-SF questionnaire /
      BIS-11 / MSI - BPD / STAI STETE / STAI TRAIT and the candidate genotypes as independent
      variables and NSSI as dependent variable have been made. Unadjusted analyses of the rs1799971
      / rs1800497 genotype itself and craving variable were carried out using Mann-Whitney U-test.
      The contribution of the rs1799971 / rs1800497 genotype to the level of craving for NSSI
      (SICQ) was studied using generalized linear models and was adjusted to STAI STATE and STAI
      TRAIT / ETISR - SF. Logistic regression models with candidate genotypes as independent
      variables and pain perception during NSSI as dependent variable have been made. The
      contribution of the rs1799971 / rs1800497 genotype to pain perception has been studied using
      logistic regression models that were adjusted to STAI STATE / STAI TRAIT / MSI - BPD/ ETISR -
      SF. The relationship between pain perception during NSSI and craving (SICQ) was assessed
      using Mann-Whitney U-test.

      Aim of the study and the hypotheses The aim of the research was to carry out the first G x E
      study on the etiology of NSSI, diagnosed with the proposed research criteria from DSM-5, in
      Slovene adolescents. The investigators wanted to make the first association study of the
      rs1799971 in adolescents with NSSI and study the connection of the OPRM1 gene with addictive
      properties of NSSI and craving for NSSI. The goal was also to replicate the results of Joyce
      and his colleges (association of rs5443 and NSSI in depressive patients) in a sample of
      adolescents with NSSI.

      The investigators have hypothesized that NSSI could be associated with one of the candidate
      polymorphisms or a combination of candidate polymorphisms. Further the investigators have
      hypothesized that the genetic polymorphisms associated to NSSI are the most connected to NSSI
      in traumatised individuals and that NSSI is associated with higher impulsivity.
    

Intervention name(s): DNA extraction and genotyping of candidate genetic polymorphisms; self-assessment questionnaires
Intervention type(s): Genetic; Other
Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        Clinical sample We have invited 96 adolescents (12 - 21 years) with NSSI to participate in
        our study. The adolescents have been patients of the Paediatrics Clinic - University
        Clinical Centre Maribor, or the Unit for Adolescent Psychiatry - University Psychiatric
        Clinic Ljubljana, or the Child and adolescent Psychiatry - Health Care Centre Dr Adolf
        Drolc Maribor or have been treated by Natasa Potocnik Dajcman, M.D.. DNA of 95 adolescents
        could be isolated properly.

        Control group We have invited 96 adolescents (12 - 21 years) with no history of NSSI. From
        those adolescents, we managed to create an original control group with 21 participants.

        General control group Because of the demands of the genetics analysis, we have formed one
        more control group - the general control group. This control group was formed from 118
        adolescents (18- 24 years) of the general population, that donated their DNA samples for
        research purposes.
      
Biospecimen description: 
      DNA isolated from whole blood
    
Keywords: Self-Injurious Behavior; genetic polymorphisms; TPH1; SLC6A4; OPRM1; GNβ3; DRD2/ANKK1; anxiety; borderline personality disorder; trauma; impulsivity