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Digital archive of theses discussed at the University of Pisa


Thesis etd-04122011-180341

Thesis type
Tesi di dottorato di ricerca
Thesis title
Separation Anxiety Disorder: neurobiological related and polymorphism studies
Academic discipline
Course of study
tutor Prof. Pini, Stefano
tutor Prof.ssa Martini, Claudia
commissario Prof. Fagiolini, Andrea
commissario Prof. Faravelli, Carlo
commissario Prof. Giannaccini, Gino
  • oxytocin
  • polymorphism
  • separation anxiety
  • TSPO
Graduation session start date
Individuals with a diagnosis of adult separation anxiety (ASAD) have extreme anxiety about separations, actual or imagined, from major attachment figures. ASAD might represent a psychological/behavioral model for research probably involving a dysregulation of those neurobiological mechanisms of attachment
Genes involved in biosynthesis of neurosteroids may be crucial candidates for this anxiety intermediate phenotype. Translocator Protein (TSPO) is the first-limiting step protein of neurosteroid biosynthesis. I tested whether the two allelic variants of TSPO gene, 439G>A and 485G>A single nucleotide polymorphisms (SNPs), were associated with adult separation anxiety. A case-control study was performed in 182 patients with mood disorders and 190 controls. Eighty patients fulfilled DSM-IV criteria for adult separation anxiety disorder and 102 did not. For 439G>A, a risk to develop adult separation anxiety was evidenced for A allele carriers. A carriers showed significantly higher total scores on the scale for adult separation anxiety than patients with GG genotype. Some degree of Linkage Disequilibrium was found between the study SNPs. Our findings suggest that A-allele of 439G>A SNP might be implicated in pathophysiology of adult separation anxiety.
The de novo production of steroids and neurosteroids begins in mitochondria by the conversion of cholesterol to pregnenolone through cytochrome P450 side-chain cleavage (CYP11A1) enzymatic activity. The C-terminal amino acid domain of the TSPO has been demonstrated to bind cholesterol, thereby determining its mitochondrial translocation. The goal of the present study was to investigate the effect of the Ala147Thr single-nucleotide polymorphism localized in this TSPO region on pregnenolone production in healthy volunteers. Pregnenolone production was evaluated in a peripheral cell model, represented by circulating lymphomonocytes. First, CYP11A1 expression, both at mRNA and protein level, was demonstrated. Pregnenolone production varied among genotype groups. Comparison of pregnenolone mean values revealed that Thr147 homozygous or heterozygous individuals had significantly lower pregnenolone levels compared with Ala147 homozygous individuals. These findings suggested a dominant effect of the minor allelic variant Thr147 to produce this first metabolite of the steroidogenesis pathway.
However, attachment theory suggests that anxious attachment styles are associated with risk to psychiatric disorder, especially anxiety disorders. Separation anxiety would appear to be a core form of anxiety that is associated with anxious attachment. To make a contribution to unravel the interlinked relationships between the two phenomena (separation anxiety and anxious attachment), I investigated whether the G439A SNP within the TSPO gene, previously associated with ASAD, could be implied in anxious attachment too. To this aim, Stepwise regression analyses were performed using the G439A SNP as dependent variable and ASAD diagnosis and the five Attachment Style Questionnaire factors as independent variables, controlling for possible confounding factors such as patient principal diagnosis (bipolar depression or unipolar depression). The results showed that the A allelic variant of G439A SNP, which has resulted recently to reduce the TSPO function, was significantly associated with ASAD diagnosis, but not with the other independent variables included in the regression model. The positive association of G439A SNP with ASAD was further confirmed by the case-control study including an extended sample described earlier.
A further objective was to explore how the reduced TSPO function could be related to the other TSPO parameter “expression levels”, as reduced densities of this protein have been found in patients with ASAD. The TSPO density values were stratified in two genotype groups (GG genotype= normal TSPO function and A-carriers= reduced TSPO function) in patients with ASAD and in those without ASAD. The ANOVA with Bonferroni’s corrected t-test demonstrated that the GG genotype of patients with ASAD exhibited statistically significant lower TSPO densities than each genotype group of patients without ASAD.
In conclusion, the reduced TSPO function doesn’t represent an overlapping mechanism underlying ASAD and anxious attachment, suggesting that these two phenomena could not reflect simple different aspect of the same construct. Moreover, these findings contribute to consolidate the important role of the TSPO in ASAD. Indeed, the reduction of TSPO function or expression levels appear to be a hallmark of ASAD. Such evidences could have therapeutical implications, as it has been recently documented that ligands targeting TSPO are able to stimulate TSPO function and determine anxiolytic properties in rodents.
Finally, seen much evidence of an association between specific attachment styles and depression I investigated, in depressive disorders, the potential role of polymorphisms within the gene encoding the receptor of the main neurohormone involved in attachment processes, oxytocin. For this purpose, two SNPs, 6930G>A (rs53576) and 9073G>A (rs2254298), within the oxytocin receptor gene (OXTR), were studied in a cohort of 185 patients with major depression (50.3%) or bipolar I or II disorders (49.7%) and 192 matched healthy controls. A positive association between the GG genotype of OXTR SNPs (6930G>A or 9073G>A) and unipolar depression was demonstrated. In this group, GG individuals showed high scores on Attachment Style Questionnaire factors that have been previously associated with depression. Moreover, the GG genotype was also associated with high levels of adult separation anxiety. These findings support the involvement of the oxytocinergic system in the mechanisms that underlie depression and specific adult attachment styles.