TPH2 5'- and 3'-regulatory polymorphisms are differentially associated with HPA axis function and self-injurious behavior in rhesus monkeys

Abstract

Tryptophan hydroxylase-2 (TPH2) synthesizes neuronal serotonin and is linked to numerous behavioral traits. We have previously characterized the functionality of polymorphisms (especially 2051A>C) in 3'-untranslated region (3'-UTR) of rhesus monkey TPH2 (rhTPH2). This study further assessed the functionality of additional polymorphisms (-1605T>C, -1491Tn, -1485(AT)n, -1454A>G, -1325In>Del and -363T>G) in rhTPH2 5'-flanking region (5'-FR), and evaluated the effects of rhTPH2 5' and 3' genotypes on central serotonin turnover, hypothalamic-pituitary-adrenal (HPA) axis function and self-injurious behavior (SIB) in 32 unrelated adult male monkeys of Indian origin. Haplotypes of the rhTPH2 5'-FR polymorphisms exert a significant, cell-dependent effect on reporter gene expression, primarily conferred by -1485(AT)n. The -1485(AT)n and 2051A>C polymorphisms interact to influence cerebrospinal fluid (CSF) 5-HIAA and plasma adrenocorticotropic hormone (ACTH) in the afternoon. While -1485(AT)n exerts significant main effects on the afternoon cortisol level and nocturnal HPA negative feedback, 2051A>C has significant main effects on the morning cortisol level and cortisol response to ACTH challenge, as well as marginally significant main effects on the daytime HPA negative feedback and self-biting rate. In addition, the genotype/allele frequency of the 5'-FR -1325Ins>Del differed significantly between the self-wounders and non-wounders, whereas 3'-UTR 2128S>L polymorphism differed significantly in genotype/allele frequency between the high- and low-frequency biters. This study shows the functionality of rhTPH2 5'-FR polymorphisms, and provides evidence for the differential association of rhTPH2 5'-FR and 3'-UTR polymorphisms with HPA axis function and SIB. Our findings shed light on the role of TPH2 gene variance in physiology and behavioral traits, and also contribute to the understanding of the pathophysiology and genetics of SIB.

Figure 1

Haplotypes of the rhTPH2 5′-FR and their effects on reporter gene expression. (a) Trace chromatograms of −1454A>G and −1325In>Del(TA) polymorphisms; (b) Illustration of 5′-FR polymorphisms in rhTPH2 and hTPH2; (c) rhTPH2 5′-FR haplotypes and their frequencies in the cohort of 32 monkeys; (d) Luciferase activity driven by rhTPH2 5′-FR haplotypes in RN46A and HEK-293 cells. Except for the deletion construct (M0), all haplotypes occur naturally for the loci between −1605 and −1325. M3 has the dominant prevalence in the population and is thus regarded as the wild-type haplotype. M4′/M5′ differs from M4/M5 only at the −1454 locus, with the former followed by a prime (') indicating a “G” substitution at −1454. Data are shown as Mean ± SEM.

Figure 2

Comparisons of CSF level of 5-HIAA (1997 sample) between rhTPH2 genotypes. Subjects were grouped as −1485(AT)6 carriers or non-carriers, and stratified by 2051A>C genotype (CC or AA&AC). Data are shown as Mean±SEM and sample size (n) for each group is indicated. *p<0.05, **p<0.01, ***p<0.001 for comparisons between indicated groups.

Figure 3

Comparisons of the plasma levels of cortisol (a) and ACTH (b), plasma cortisol response to ACTH stimulation (c), and DEX suppression of urinary cortisol excretion (d) between rhTPH2 genotypes. Subjects are grouped as −1485(AT)6 carriers or non-carriers, and stratified by 2051A>C genotype (CC or AA&AC). *p<0.05, **p<0.01, ***p<0.001, ^#0.05≥p<0.10 for comparisons between indicated groups.

Figure 4

Comparisons of self-directed biting rate between rhTPH2 genotypes. Animals are grouped as −1485(AT)6 carriers or non-carriers, and stratified by 2051A>C genotype (CC or AA&AC). *p<0.05, ^#0.05≥p<0.10 for the comparisons between designated groups.

Figure 5

Comparison of plasma levels of cortisol (a), ACTH (b), cortisol response to ACTH challenge (c), and DEX suppression of urinary cortisol excretion (d) between SIB groups. *p<0.05, **p<0.01, ^#0.05≥p<0.10 for comparisons between designated groups.