Associations of the serotonin transporter gene polymorphism, 5-HTTLPR, and adverse life events with late life depression in the elderly Lithuanian population

Late-life depression (LLD) is a multifactorial disorder, with susceptibility and vulnerability potentially influenced by gene-environment interaction. The aim of this study was to investigate whether the 5-HTTLPR polymorphism is associated with LLD. The sample of 353 participants aged 65 years and over was randomly selected from the list of Kaunas city inhabitants by Residents’ Register Service of Lithuania. Depressive symptoms were ascertained using the EURO-D scale. The List of Threatening Events Questionnaire was used to identify stressful life events that happened over the last 6 months and during lifetime. A 5-HTTLPR and lifetime stressful events interaction was indicated by higher odds of depression in those with s/s genotype who experienced high stress compared to l/l carriers with low or medium stress, while 5-HTTLPR and current stressful events interaction analysis revealed that carriers of either one or two copies of the s allele had increased odds of depressive symptoms associated with stress compared to participants with the l/l genotype not exposed to stressful situations. Although no significant direct association was found between the 5-HTTLPR short allele and depression, our findings demonstrated that lifetime or current stressful life events and their modification by 5-HTTLPR genotype are risk factors for late-life depression.


Results
Descriptive data of Kaunas Healthy Ageing Study are presented in Table 1.There were twice as many females (67.1%) as males.Nearly half of the responders were married or had stable partners (43.4%) and the majority of the remainder were widowed (41.1%).Over half of the participants visited church sometimes (58.9%) and most of them reported having children or friends (73.1% and 86.4%, respectively).According to the EURO-D scale, 160 (45.8%) had case-level depressive symptoms (EURO-D score ≥ 4).Those with depression had significantly lower education levels and were less likely to attend church.They also presented a higher number of stressful life events than those without depressive symptoms.The genotype frequencies did not deviate from the HWE (P > 0.05) and reflected frequencies previously observed in white European populations 14,29,44 .The frequency of the 5-HTTLPR genotypes and alleles in elderly population did not differ significantly according to EURO-D scale score.
In univariate age-adjusted logistic regression analyses (Table 3), a 5-HTTLPR × LSE interaction effect on depression was observed in that highest odds of depression were found in participants with both high stress and s/s genotype for LSE; for CSE, this highest-risk group was observed both for the s/s genotype and when analysed for presence of the s allele (i.e., s/s and s/l combined) with a dose-response pattern from the low stress × l/l combination to the high stress × s/s combination.

Discussion
This study investigated the association between 5-HTTLPR polymorphism, its interaction with stressful life events, and risk of late-life depressive symptoms in a sample of older people from a Lithuanian catchment.Consistent with previous studies [33][34][35] , we found no significant direct association between the 5-HTTLPR s allele and late-life depressive symptoms.On the other hand, our data replicated the results of two meta-analyses 34,35 , in the strong associations found between stressful life events and depressive symptoms, strongest for more recent stressful events.One of the most recent meta-analyses of this polymorphism 45 concluded that 5-HTTLPR and stress interaction is a dynamic process, producing different effects at different time points.Data show that the effect of the 5-HTTLPR x stress interaction emerged only in the case of chronic stress, i.e. the risk of depression by 5-HTTLPR genotypes emerges with time and the difference is seen only following chronic stress.It is widely acknowledged that chronic stress, more than acute stress, is a key factor for the onset of depression 46 .However, our 5-HTTLPR and stressful events interaction analyses show that both lifetime or current stressful life events remain important factors for the onset of depression, and that 5-HTTLPR genotype potentially modulates the development of depression.Specifically, analyses show a dose-dependent effect of number of stressful events experienced by elders and the role of HTTLPR genotype.These results support previous conclusions 14,47 that 5-HTTLPR genotype modifies the strength of association between stressful events and depressive symptoms.In our sample, the odds of depression were increased in s homozygous participants who experienced high lifetime stress compared to the l homozygotes with low or medium stress.Our results also confirm prior findings 14 that increasing numbers of 5-HTTLPR s alleles confer higher vulnerability to depressive symptoms associated with recent life events.These findings support the 5-HTTLPR × stress interaction hypothesis and suggest that the effect of the interaction on depression risk is dose-dependent on the number of stressful events.
The prevalence of depressive symptoms varies according to geographical region, with rates being lower in northern Europe and higher in southern and eastern Europe countries 2 .The prevalence of late-life depression displays considerably higher rates in the oldest compared with the youngest age group.Studies that have used the EURO-D scale to assess depressive symptoms in European countries have reported prevalence rates, between 17.8 and 38.3% in older population aged 50 and over 2 and 15.8-41.4% in people aged 65 and over 48,49 .The prevalence of depressive symptoms (EURO-D ≥ 4) in our sample (45.8%) also support previous findings.
Considering other findings, depressive symptoms were more frequent in older people with lower educational attainment.This is consistent with conclusions from other studies that higher education is associated with decreased risk of late-life depression 2,50,51 .Practice of religion may also serve as a protective factor against depression in older people 2,52 and our study replicated prior findings showing a strong association between church attendance and reduced likelihood of depression 53,54 .Previous studies have found that intergenerational contact frequency is independently and inversely associated with depressive symptoms in the elderly 55,56 .Our findings show that the odds of depressive symptoms decreased with increasing frequency of meetings with children or friends; however, the associations failed to reach statistical significance.
This study has several limitations.First, depressive symptoms were ascertained as an outcome according to a cut-off score on a screening instrument, albeit one that has been widely used and validated in previous studies, and findings cannot necessarily be applied to diagnostic categories.Second, we did not seek to ascertain or analyse stress-related factors experienced in childhood and adulthood separately.Third, potentially important factor such as use of medication was not included in the analysis.Despite these limitations, the strengths of our study are that we used EURO-D scale which was originally developed to compare symptoms of LLD across European countries 41 and has been validated and used in many epidemiological studies.To our knowledge, this is the first study investigating the impact of current and lifetime stressful life events on the occurrence of depressive symptoms in an elderly Lithuanian population.This population-based study is homogeneous by genetic ancestry and adds further informative evidence not only about the impact of stress-related life events as risk factors for depression onset, but also presents effect of sociodemographic factors on the development of depression in the elderly.In addition, the evaluation of the 5-HTTLPR and traumatic stressful events interaction demonstrated the significance of genetic variation in phenotype as LLD.
In conclusion, although no significant association was found for elderly carrying the short allele of the 5-HTTLPR and being particularly susceptible to depression, our findings demonstrated that lifetime or current www.nature.com/scientificreports/stressful life events and their interaction with the 5-HTTLPR genotype significantly increased odds of late-life depression in elderly population.Because of inclusion difficulties (not possible to find at home after repeated visits, not having a telephone, refusing to participate), other participants were included using a snowball sampling method and recommendation from study subjects.In total, 353 persons participated in the study.Of these, 298 agreed to provide blood samples for genetic study.All the participants were Caucasian and Lithuanian.
The study was approved by Kaunas Regional Biomedical Research Ethics Committee (Lithuania) (permission number BE-2-49/2008).In accordance with the Declaration of Helsinki, written informed consent was obtained from each participant and aims of the study had been fully explained.All clinical and laboratory data was coded and entered into a secure computer database only accessible to the research study team.

Mental health and psychological status assessment.
A questionnaire, which covered health, psychological, social support, and economic variables, was administered by trained interviewers in the respondents' homes.Depressive symptoms were assessed using the EURO-D scale 41 , which consists of 12 items: depression affect, pessimism, wishing to die, guilt, sleep, interest, irritability, appetite, fatigue, concentration, enjoyment, and tearfulness during the last month.Each item receives a binary present/absent score, creating an ordinal scale with a maximum score of 12. Scores of 4 or more were classified as case-level depressive symptoms, as previously reported to indicate the presence of major depression 41 .The List of Threatening Events Questionnaire (LTE-Q) 42 composed of 12 items was used to identify traumatic stressful life events that happened in the last 6 months (current stressful events, CSE) and during lifetime (lifetime stressful events, LSE) related to relationship breakdown, financial difficulties, illnesses/injuries and death of family or friends.In addition, all respondents were asked questions related to physical and sexual abuse, violence at home and at work, expulsion from school and homelessness.For each event, binary present/absent codes were allocated and the sum of scores was calculated as the number of stressful events and used for statistical analysis.For the 5-HTTLPR × traumatic stressful events (LSE or CSE) interaction analysis to predict depression, the LSE scores were grouped into two categories (low/ Table 3. Univariate logistic regression analysis of the 5-HTTLPR and traumatic stressful events interaction to predict depression*.Significant values are in bold.* Having high levels of depression symptomatology according to EURO-D (score ≥ 4); 5-HTTLPR polymorphism in the promoter region of the serotonin transporter (5-HTT) encoding gene (SLC6A4), aOR odds ratio adjusted for age, CI confidence interval.a The two categories of lifetime stressful events (LSE) were included in analysis: low/medium stress-0-2, high stress-≥ 3 number of negative life events.b The two categories of current stressful events (CSE) were included in analysis: not exposed to stress-0, exposed to stress-≥ 1 number of negative life events.Statistical analysis.Quantitative variables were described as median (minimum, maximum), because variable distributions did not satisfy the normality assumption (Kolmogorov-Smirnov or Shapiro-Wilk tests).Nonparametric Mann-Whitney U tests were used to determine differences in the distributions of continuous variables between two independent samples, and Kruskal-Wallis for three independent samples.Categorical variables were described using frequencies and percentages.The χ 2 test was used to determine differences between the categorical variables and for the assessment of the Hardy-Weinberg equilibrium (HWE) for the distribution of genotypes.Univariate and multivariate binary logistic regression analyses were applied to evaluate the associations between adverse life events, 5-HTTLPR genotype and the odds ratio of depression according to EURO-D scale (score).Univariate binary logistic regression analysis was applied to evaluate the 5-HTTLPR × Traumatic stressful events (LSF or CSF) interaction to predict depression.Adjusted odds ratio (aOR) with 95% confidence interval was calculated.Statistical analysis was performed using the statistical software package IBM SPSS Statistics version 27 for Windows.P value < 0.05 was considered statistically significant.
Ethics statement.The study was approved by Kaunas Regional Biomedical Research Ethics Committee (Lithuania) (permission number BE-2-49/2008).In accordance with the Declaration of Helsinki, written informed consent was obtained from each participant and aims of the study had been fully explained.All clinical and laboratory data was coded and entered into a secure computer database only accessible to the research study team.

Table 1 .
The basic characteristics of study population.Significant values are in bold.5-HTTLPR polymorphism in the promoter region of the serotonin transporter (5-HTT) encoding gene (SLC6A4), EURO-D depression symptom scale.

Table 2 .
Univariate and multivariate logistic regression analysis to predict depression*.For the Kaunas Healthy Ageing Study, a sample of 400 participants of both sexes over 65 years old was randomly selected from the list of Kaunas city inhabitants between 2006 and 2009 by the Residents' Register Service of Lithuania.From this list, 197 persons agreed to participate in the Study.