The moderating role of NTSR1 polymorphisms on personality and coping styles among breast cancer patients

Background: Some personality traits could be a predictor for coping styles. The objective of this investigation was to examine the correlation between personality traits and coping mechanisms in the context of gene regulation among breast cancer patients. Material and methods: This cross-sectional study was performed in 2021. A total of 200 breast cancer patients were included in this study. Data were collected using the Trait Coping Style Questionnaire (TCSQ) and the Eysenck Personality Questionnaire-Revised Short Scale for Chinese (EPQ-RSC). And individuals’ blood was tested for genotype. Results: The findings indicated that there existed an inverse relationship between extraversion and negative coping style while also demonstrating a significant association between extraversion and positive coping style. Neuroticism was positively correlated with negative coping style but negatively with positive coping style. The SNP of NTSR1 only moderated the relationship between neuroticism and negative coping styles in breast cancer patients. Conclusion: The present investigation delves into the correlation between personality traits and coping mechanisms at the molecular level in breast cancer patients.


Introduction
Coping style refers to the individual's response to uncomfortable or challenging situations [1,2].According to the theory of trait coping, when facing stressful situations, the individual used two different coping strategies (positive or negative) [3].Positive coping style refers to the capability of handling problems, adapting quickly to stressors, and managing stress responses [4].Negative coping style could cause people to engage in some avoidant behavior (such as drinking) that prevents them from dealing directly with stressful events [5].About 2.3 million newly diagnosed cases of female breast cancer were diagnosed in 2020, representing almost one in four cancer cases among women [6].Those who adopt a negative coping style and feel hopeless suffered from severe psychological distress, adversely affecting their prognosis [7,8].Consequently, it is necessary to investigate the factors influencing breast cancer patients' coping styles.
It is possible that individuals with different personality traits may adopt different coping styles [9].For example, neuroticism was positively associated with a negative coping style but inversely correlated to a positive coping style [10]; conscientiousness was positively correlated to an active coping style [11].Extraversion was correlated with the problem and emotion-focused coping style, while neuroticism and psychoticism had a positive effect on the avoidance coping style [12].Therefore, we proposed Hypothesis 1: Personality can predict the coping styles in breast cancer patients.
In spite of this, not all findings about the relationship between personality and coping have been consistent.Some researchers found that adaptive personality traits were positively associated with active coping style [13], while some found that openness was not associated with any of the coping strategies [14].In addition, genetics and the environment influence the predisposition to exhibit certain coping styles [15,16].Single nucleotide polymorphism (SNP) is a genetic marker formed by a single nucleotide variation, which occurs when a single nucleotide in the DNA sequence is altered [17].Recent studies showed that SNP played an important role in the mechanism of mental health and coping style [18,19].For FKBP5 (rs9296158, rs4713016), A-allele carriers at both polymorphisms are the most susceptible to trauma [20,21].Prior studies found that the polymorphism of 5-hydroxytryptamine (5-HT) was associated with an individual's personality and contributed to the coping style [22,23].Nedic Erjavec et al. found HTR2A polymorphisms were related to irritability and impulsivity traits [23].G carriers of rs2070040 and rs4142900 in individuals with conduct disorder tended to be aggressive [23].Besides, the functional polymorphism in the promoter region of the 5-HTT gene was reported to moderate the influence of stress events on depression [24].
Neurotensin plays an important role in the central and peripheral systems [25], acting through the neurotensin receptor [26].Human neurotensin receptor 1 (NTSR1) is a G protein-coupled receptor with seven transmembrane spanning regions, a subtype of NTSR [27,28].Several studies demonstrated that NT and NTSR1 played a role in psychiatric disorders [29].Patients with schizophrenia present low levels of NTSR [30,31].There is evidence demonstrating that affective disorder patients showed significantly decreased NTSR density [32].An animal experiment also found that NTSR1-knockout mice presented more anxiety and less sleep than wild mice [33].However, the SNP phenotype of NTSR1 remains unclear in breast cancer patients.The association between the SNP of NTSR1 and patients' personality or coping styles needs further study.Based on the previous kinds of literature and the effect of genes, we proposed Hypothesis 2: The SNPs of NTSR1 moderated the association between personality and coping style.

Breast cancer patients and procedures
From November 2021 to December 2022, five researchers in our group recruited the participants in the wards of the Department of Breast Cancer Oncology in the Fourth Hospital of Hebei Medical University and distributed paper questionnaires face to face.The inclusion criteria were (1) diagnosed with breast cancer and (2) volunteered to join the study.Exclusion criteria were: (1) history of cognitive impairment or psychiatric disease, (2) history of other chronic diseases and (3) inability to communicate verbally.This study obtained approval from the Ethics Committee of the Fourth Hospital of Hebei Medical University (No.2021075).All breast cancer patients were informed and signed the informed consent.
Before the formal dissemination of the questionnaire, every researcher underwent training to ensure their ability to effectively address patient inquiries posed during the distribution of questionnaires.

Trait coping style questionnaire (TCSQ)
The TCSQ was used to measure the coping style of breast cancer patients, including two dimensions, positive coping style and negative coping style [34].Each dimension consists of 10 items on a Likert scale of five points, from 1 (absolutely not) to 5 (absolutely yes).A higher score in one dimension indicates that breast cancer patients are more likely to use this type of coping style.In China, the effectiveness and reliability of TCSQ have been validated [34].In this study, the internal consistency coefficient of positive coping style and negative coping style was 0.775 and 0.787, respectively.

Eysenck personality questionnaire-revised short scale for Chinese (EPQ-RSC)
The EPQ-RSC is a personality trait measurement method which has been used in early studies, and its reliability and validity have been demonstrated [35].It consists of four dimensions extraversion, neuroticism, psychoticism, and lie detector scale; each dimension has 12 items.The Cronbach of EPQ-RSC was 0.992 in this study.

SNP genotyping
In this study, we analyzed the SNP sites of NTSR1 (rs6090453, rs6011914, rs2427422) in breast cancer patients.Genomic DNA was extracted from the whole blood using the Genomic DNA Purification Kit (Tiangen, Beijing, China).The NTSR1 SNP polymorphisms were genotyped using Sanger sequencing (Sangon Bioengineering, Beijing, China).DNA fragments of NTSR1 were amplified by the polymerase chain reaction (PCR).The PCR products were subjected to sequencing commercially (Sangon Bioengineering, Beijing, China).All the sequences were determined in both forward and reverse directions.The primers were listed in Table S1.

Statistical analysis
For the SNP locus of the case group, the Hardy-Weinberg equilibrium (HWE) deviation was tested by chi-square (χ 2 ).Continuous variables were expressed as either mean (standard deviations [SD]) or median (interquartile range [IQR]).Categorical variables were presented by using frequencies and percentages.A univariate analysis was performed using the Chi-square test, Student's t-test, analysis of variance (ANOVA), or the Wilcoxon rank sum test where appropriate.Correlations between personality and coping style were examined using Pearson's correlation test.Finally, we used the SPSS macro, PROCESS (Model 1) developed by Hayes to test our hypotheses.All models were adjusted for age.All statistical analyses were performed using SPSS statistical software 26 (IBM SPSS Inc., Chicago, IL, USA), and the significance level was defined as P < 0.05.

The moderating role of NTSR1 polymorphism
To explore the association between personality and coping styles, we used hierarchical regression to analyze the moderating effects.Personality was the independent variable, coping styles were the dependent variables and NTSR1 SNP types were moderators.First, we entered age into Model 1.Second, we put the independent variable and moderating variables into Model 2. Finally, we put the interaction of an independent variable and moderating variables into Model 3. We found that NTSR1 SNP types (rs6090453, rs6011914, rs2427432) only moderated the relationship between neuroticism and negative coping style, and the regression results were presented in Tables 3, 4, and 5.We used PROCESS macro implemented in the SPSS software (IBM Corporation, Armonk, NY, USA) and 5000 bootstrapped resamples to examine the moderation effect and picture corresponding simple plots.Results showed that neuroticism could predict a negative coping style for G-allele carriers (CG simple slope = 0.394, t = 6.705,P < 0.001; GG simple slope = 0.239, t = 4.219, P < 0.001).However, neuroticism could not predict a negative coping style for rs6090453 CC homozygotes (simple slope = −0.061,t = −0.496,P = 0.621) (Figure 1).Neuroticism also can positively predict it for G-allele carriers in rs6011914 (CG simple slope= 0.425, t = 7.163, P < 0.001; GG simple slope = 0.236, t = 4.312, P < 0.001) but cannot predict negative coping in rs6011914 CC homozygotes (simple slope = −0.158,t = −1.277,P = 0.203) (Figure 2).Meanwhile, neuroticism was associated with negative coping in G carriers (AG simple slope = 0.425, t = 6.780,P < 0.001; GG simple slope = 0.261, t = 5.125, P < 0.001), but was not linked to negative coping in an AA carrier with rs2427411 (simple slope = −0.188,t = −1.496,P = 0.136) (Figure 3).

Discussion
Coping style refers to the various strategies people choose to use when facing with adversity [36].Coping style could affect the psychological distress and prognosis of breast cancer patients [8].In this study, we found that the neuroticism and extraversion of breast cancer patients were associated with the coping style, while psychoticism was not.The SNP types of NTSR1 (rs6011914, rs6090453, rs2427422) moderated the relationship between neuroticism and negative coping style in breast cancer patients.To the best of our knowledge, this was the first study about the moderated effect of gene polymorphisms on personality and coping styles in breast cancer patients.
As expected, the positive coping style was negatively related to neuroticism and positively related to extraversion.However, the negative coping style had a positive relationship with neuroticism and a negative relationship with extraversion, which was consistent with previous studies [10,37].The characteristics of extroverts are energy, cheer, and gregariousness [38].Hence, individuals with a high level of extraversion tended to see stress as a challenge and adopt a positive coping style.Individuals with a high level of neuroticism would interpret stressful events as a threat [39], avoid solving these stressful events, and be more inclined to adopt a negative coping style.
NTSR1 is located both presynaptically and postsynaptically at D2-expressing synapses in the striatum, NAc, and ventral midbrain [40].NT impacted the DA system through NTSR1 [26], playing an important role in some mental and psychiatric disorders [29].Previous studies indicated that SNP exerted functions in defense mechanisms and mental health.Jung et al. showed that T-allele carriers in rs2572431 had an association with high levels of neuroticism [19].Compared with the C-allele (rs1130864), the T-allele is more expressed in people who have higher suicidal ideation [41].For OXTR (rs6770632), AA carriers were more sensitive to depression [42].Ma et al. identified significant differences in the intermediate defense and projective identification in genotypes rs2427422 and rs6011914.In this study, we found that G-allele carriers of NTSR1 (rs609045, rs6011914, rs2427422) were more susceptible to neuroticism and further negative coping style (Figure 1, 2, 3).Instead, for rs6090453 and rs6011914 CC homozygotes as well as rs2427422 AA homozygotes, the neuroticism could not predict the level of negative coping style (Figure 1, 2, 3).Nevertheless, the role of NTSR1 in defense mechanisms and mental health was rarely studied.In this study, we proved that NTSR1 polymorphism could moderate Submit a manuscript: https://www.tmrjournals.com/psmrthe relationship between neuroticism and negative coping style, which may be ascribed to the following reasons.
First, a series of studies demonstrated that DA neurotransmission was linked to cognitive function and impulsive behavior [43][44][45].Meanwhile, NT and NTSR1 regulated the function of the DA system [26].As a result of increased Ca 2+ influx via NTSR1, enhanced dopamine release in NAc led to impulsive behavior [46,47].Some studies also identified that stress coping styles and behavioral flexibility were linked across vertebrate phylogeny [48].The above studies directly supported our findings.Second, the previous studies about NTSR1 polymorphism function in the brain also indirectly demonstrated our results.Li et al. showed that for rs6090453, G-allele carriers had a better performance at working memory than CC homozygote [49].Ma et al. found that for rs6011914 and rs2427422, G-allele carriers were more likely to develop alcohol dependence [50].That is to say, the G-allele in NTSR1 had a stronger impact on cognition function.Those with G-alleles were more susceptible to stress events and more likely to adopt a negative coping style (i.e., alcohol addiction), which was consistent with our results.However, a study concerning schizophrenia showed that the frequency of C-allele in the rs6090453 was higher in schizophrenic patients [51], which was inconsistent with our findings.It may have been caused by SNPs altering the original function of gene expression.For example, some studies show that heterozygotes of rs375804228 may trigger the effect of NF-kB, inducing depression.Thus, further mechanistic studies of NTSR1 SNP types are required in the future.

Limitation
Our study also had some limitations.First, this is a cross-sectional study, which does not allow for causal inference.Thus, in the future, prospective cohort studies are needed.Second, this is a single-center study and needs further validation in a multi-center, larger sample among breast cancer patients.

Conclusion
To the best of our knowledge, this is the first study to test the moderated effect of NTSR1 SNP types on personality and coping styles in breast cancer patients.We identified that neuroticism and extraversion were both associated with coping styles.Importantly, we also found that this association did not exist in any case.For G allele carriers of rs6090435, rs601191, and rs2427411 at NTSR1, breast cancer patients with a high level of neuroticism may be at risk for a negative coping style.This new finding provides a basis for future studies to further investigate the function of NTSR1 SNP sites and from a new perspective, explain the phenomenon that previous studies had some inconsistent conclusions about the relationship between personality and coping styles.

FFigure 1 Figure 2 Figure 3
Figure 1 The moderating effect of rs6090453 SNP genotype in breast cancer patients (n=200)

Table 3 Multiple linear regression analysis of neuroticism and interacting effect of NTSR1 genotypes of rs6090453 and neuroticism with negative coping style in breast cancer patients (n = 200)
*All categorical variables were set as dummy variables.N, neuroticism; *P < 0.05, **P < 0.01, ***P < 0.001