Cortisol and behavior in fragile X syndrome
Introduction
Fragile X syndrome, caused by mutations in a single gene on the long arm of the X chromosome, occurs in 1 of every 2000 to 5000 live births and is the most common known inherited cause of developmental disability. The cytogenetic fragile site on the X chromosome from which the syndrome derives its name is typically caused by the presence of more than 200 cytosine-guanine-guanine (CGG) triplet repeats within the promoter region of the fragile X mental retardation (FMR1) gene, which prevents normal transcription. This “transcriptional silencing” of the gene and the subsequent diminished or absent production of the FMR1 protein (FMRP) results in aberrant brain development and function (Devys, Lutz, Rouyer, Bellocq and Mandel, 1993, Tamanini, Willemsen, vanUnen, Bontekoe, Galjaard, Oostra and Hoogeveen, 1997). Because females have two X chromosomes, production of FMRP is maintained to varying degrees by the presence of the unaffected X chromosome. Consequently, females tend to be less severely affected by fragile X than males.
In addition to cognitive impairment, individuals with fragile X typically demonstrate a neurobehavioral phenotype that includes stress-related symptoms such as hyper-arousal, hyper-responsivity to sensory stimuli, hyperactivity, impulsivity, gaze aversion, and social anxiety and withdrawal (Cohen, Fisch, Sudhalter, Wolf-Schein, Hanson, Hagerman, Jenkins and Brown, 1988, Lachiewicz, 1992, Freund, Reiss and Abrams, 1993, Lachiewicz and Dawson, 1994, Cohen, 1995, Mazzocco, Baumgardner, Freund and Reiss, 1998). Recently, FMRP expression has been linked to some of these phenotypic characteristics of fragile X, including social withdrawal, anxiety and depression (Hessl et al., 2001). Despite the relatively consistent links between FMR1 gene function and outcomes in fragile X, considerable variability in stress-related behavior problems exists, ranging from high levels of distress, often in novel social situations, to normal functioning. This variability can in part be explained by non-genetic factors, such as characteristics of the home environment and the effectiveness of educational and therapeutic services (Hessl et al., 2001). However, other individual characteristics of children or the environments in which they live may help to better account for these individual differences, leading to more effective methods of assessment and treatment of stress-related symptoms.
One such individual characteristic, the function of the hypothalamic-pituitary-adrenal (HPA) axis, may help to explain some of the variability in stress-related symptoms among children with fragile X. Regulation of the HPA axis is complex and involves feedback mechanisms occurring at the level of the hypothalamus, pituitary, hippocampus, and frontal cortex. This dynamic system is mediated through the secretion of adrenal glucocorticoid hormones, and is involved in the regulation of physiological and behavioral responses to stress. Activity of the HPA axis is a component of normal coping (Gunnar, 1987, de Kloet, Oitzl and Joels, 1999). The HPA response to stress is adaptive in that it prepares the individual for dealing with the source of the stress, however chronic elevations or disruptions in the typical diurnal rhythm of cortisol can lead to medical problems associated with immune suppression (McEwen et al., 1997) and adverse effects on the brain that interfere with learning and memory (Sapolsky, 2000). In addition, while it has long been known that the experience of stress can cause an HPA response, recent evidence suggests that a genetic-biological predisposition to neuroendocrine reactivity can lead to abnormal behavioral responses to stressful stimuli (Bakshi and Kalin, 2000).
Neuroendocrine studies in fragile X syndrome implicate abnormalities in the hypothalamic-pituitary system. For example, precocious puberty and elevated gonadotrophin levels have been described in individuals with fragile X (Butler and Najjar, 1988, Moore, Chudley and Winter, 1990). In an evaluation of hypothalamic-pituitary-thyroid (HPT) function in 12 males with fragile X, Bregman et al. (1990) reported normal levels of thyroid stimulating hormone (TSH) but a blunted TSH response to thyrotropin releasing hormone (TRH). Further, Loesch and colleagues (Loesch et al., 1995) found that despite a high rate of physical growth in the preadolescent period, individuals with fragile X show less pubertal growth compared to normal relatives. These investigators hypothesized that premature activation of the hypothalamic-pituitary-gonadal axis may be cause of growth impairment in individuals with fragile X. While these studies do not directly show hypothalamic-pituitary-adrenal dysfunction in fragile X, they demonstrate that disruption of hormonal processes mediated by the hypothalamus and pituitary may be affected.
Children with fragile X often have abnormally strong physiological and behavioral responses to physical and social stimuli, thereby increasing their levels of arousal and possibly stress. For example, Miller et al. (1999) used a laboratory paradigm to study electrodermal responses to auditory, visual, touch, vestibular, and olfactory stimuli to assess sympathetic nervous system activity in children and adults with fragile X. In this study, increased electrodermal response (EDR) to stimulation and lower rates of habituation to stimulation were found in fragile X as compared to age and gender matched control subjects. Boccia and Roberts (2000), utilizing spectral analysis of heart beat intervals, found that boys with fragile X had increased heart rate and lower parasympathetic activity during experimental challenge. Thus, the anxiety, behavioral distress, and gaze avoidance typically observed in individuals with fragile X may be due to sympathetic-adrenomedullary over-reactivity.
To date, no study has comprehensively examined HPA function in individuals with fragile X syndrome. We conducted a pilot assessment of salivary cortisol levels in 15 children with fragile X in comparison to a group of normally developing children (Wisbeck et al., 2000). These results provided preliminary evidence for increased adrenocortical activity in fragile X; however the small sample sizes and the comparison group limited the generalizability of these findings. In the current study, we assessed adrenocortical activity and its relation to behavior via measurement of salivary cortisol in a large group of children with the fragile X full mutation in comparison to their unaffected siblings. For each child, cortisol data were obtained at 4 sample times on two typical weekend days and at 6 times during a home visit that included cognitive and social challenges. By choosing a sibling comparison group, we were able to examine the effect of fragile X syndrome on adrenocortical activity by comparing two groups of children who share similar environments and inheritance of HPA function (Wust et al., 2000), but who differ primarily by virtue of their fragile X status.
Based on the behavioral phenotype, as well as evidence of autonomic and behavioral over-reactivity and other hormonal abnormalities, we hypothesized that children with fragile X would have higher levels of cortisol in comparison to their unaffected siblings. We hypothesized that this difference would be present on typical days and during the home visit, but more pronounced during the cognitive and social challenges. We also predicted that males with fragile X would demonstrate higher levels of cortisol than females with fragile X, given the known gender differences in FMR1 protein expression and phenotype severity (Tassone et al., 1999). Finally, we predicted that, after controlling for factors that significantly influence behavior in fragile X such as FMRP, intelligence, the effectiveness of educational and therapeutic services and parental psychopathology, increased adrenocortical activity would further contribute to the severity of behavior problems in these children.
Section snippets
Subjects
Subjects were 109 children with the fragile X full mutation (39 girls and 70 boys) and their unaffected siblings (58 girls and 51 boys). In families having more than one child with fragile X and/or more than one unaffected child, matched pairs were chosen based on age and gender when possible. Children were between 6 and 17 years of age (fragile X: M = 10.82, SD = 2.83; unaffected siblings: M = 11.26, SD = 3.16). The sample of children was 91.7% Caucasian, 2.5% Hispanic, 2.5% African American,
Random effects: individual and familial variation in salivary cortisol
To test for familial variation, we fitted models using subject as the only random effect, and compared them to models that also included the family effect using the likelihood ratio test. The addition of the family effect significantly improved the fit of the models on the evaluation day (LR = 42.83, p < 0.0001) and on the typical days (LR = 51.08, p < 0.0001). In fact, as can be seen by the estimates of the random effects (Table 1, Table 2), a greater proportion of the variance in cortisol was
Discussion
Fragile X syndrome is a single gene disorder characterized by increased risk for a behavioral phenotype that includes social anxiety, gaze aversion, social withdrawal, and autistic behavior. In the current study, we found significant elevations in cortisol, a hormone of the HPA axis associated with stress, in children with fragile X in comparison to their unaffected biological siblings. The increases in cortisol were more pronounced in males, especially during conditions associated with
Acknowledgements
The authors wish to thank the families participating in this study, as well as the following individuals who made substantial contributions: Rita Gabriela Barajas, Donna Mumme, Cindy Johnston, Kate Ritter, and Jacob Wisbeck. This work was supported by NIH Grants: MHO1142 and MH50047. Further support was received from the Packard Foundation and the Lynda and Scott Canel Fund for Fragile X Research.
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