Katie R. Kryski

Assessing stress reactivity indexed via salivary cortisol in preschool-aged children

Identifying a stressor paradigm that elicits mean increases in salivary cortisol in young children has proven elusive, possibly due to characteristics of the paradigms used and how and when cortisol is sampled. We therefore examined the validity of a standardized task (adapted from Lewis and Ramsay, 2002) and procedures developed to assess cortisol reactivity in 215 preschool-aged children. Children participated in a standardized stress task during a home visit, which was videorecorded for future coding. Salivary cortisol samples were obtained at baseline and 10, 20, 30, 40, and 50 min post-stress. In support of the validity of the task, significant increases in cortisol levels from baseline were found, followed by a significant decline, and a quadratic function provided a good fit to the data. Children also showed a significant increase in negative emotions and a decrease in positive emotions over the course of the stress task. Results indicate that the task successfully elicited the hypothesized cortisol response in 3-year-old children.

Corticotropin-releasing hormone system polymorphisms are associated with children's cortisol reactivity.

The hypothalamic-pituitary-adrenal (HPA) axis underlies both adaptive and maladaptive responses to stress and may be an important marker of childhood vulnerability to psychopathology, although little is known about genetic variants that influence cortisol reactivity. We therefore examined associations between corticotrophin-releasing hormone (CRH) system gene (CRH, CRHR1 and CRHBP) variants and cortisol reactivity in preschoolers. A community sample of 409 three-year-old children completed a standardized stress task to elicit HPA axis activation. Salivary samples were obtained at the baseline and at 10-min intervals post-stress for a total of six samples. Salivary cortisol was measured using standard ELISA (enzyme-linked immunosorbent assay) protocols and cortisol reactivity was operationalized by calculating cortisol change scores ([baseline]-[peak cortisol post-stressor]). A single nucleotide polymorphism (SNP) marker panel containing 18 SNPs was used to tag the full-length CRH (4 SNPs), CRHR1 (7 SNPs) and CRHBP (7 SNPs) genes. Significant main effects on childrens cortisol reactivity (all ps<0.05) were found for loci on CRHR1 and CRHBP. Haplotypes of the CRHR1 linkage region were also associated with cortisol reactivity (all ps<0.01). Additionally, we found multiple interactions between tag-SNPs in all three gene-coding regions predicting cortisol reactivity (all ps<0.05). Individual differences in childrens cortisol reactivity are related to genetic variation in CRH system gene-coding regions. Our results have important implications for future research on the role of HPA axis function in the development of disorders such as anxiety and depression.

Genotyping the BDNF rs6265 (val66met) polymorphism by one- step amplified refractory mutation system PCR

Background The brain derived neutrophic factor (BDNF), a 27 kD polypeptide, is one of the most widely expressed neurotrophins in the brain, regulating neural development and plasticity. The BDNF gene contains a functional single-nucleotide polymorphism (rs6265), which results in a valine to methionine substitution (val66met), leading to reduced mature BDNF expression. This polymorphism has been widely implicated in a host of psychiatric disorders and is a focus of many ongoing psychiatric genetic studies. Objective To develop an efficient and rapid method to detect the val66met polymorphism in a one-step PCR reaction. Method and results We have designed four PCR primers that amplify the BDNF gene region containing rs6265. The specificity of the four primers in a single PCR reaction amplifies two allele-specific amplicons (253 and 201 bp) and the entire region (401 bp) as an internal control, which are easily distinguished on a polyacrylamide gel. The effectiveness and efficiency of the results are validated by traditional NlaIII restriction enzyme digestion, sequencing of resulting bands and confirmation on 308 genomic DNA samples. Conclusion This new method describes a rapid, sensitive, cost effective and high throughput genotyping of the BDNF val66met polymorphism, ideal for large-scale genotyping studies.

Hair cortisol concentrations in higher- and lower-stress mother–daughter dyads: A pilot study of associations and moderators

Hair cortisol concentrations (HCC) are receiving increased attention as a novel biomarker of psychophysiological responses to chronic stress, with potential relevance for psychopathology risk research. We examined the validity of HCC as a marker of psychosocial stress in mother (M(age)  = 37.87 years)-daughter (M(age)  = 7.62 years) dyads characterized by higher (n = 30) or lower (n = 30) maternal chronic stress. Additionally, we examined whether early care moderated similarity of HCC levels within dyads. Higher-stress mothers had significantly lower HCC compared to lower-stress mothers, consistent with other research showing that chronic stress leads to blunted HPA axis activity over time. Further, HCC in daughters were significantly and positively associated with previously assessed salivary cortisol stress reactivity. Finally, mother-daughter HCC associations were significantly moderated by negative parenting styles, such that associations became stronger as quality of parenting decreased. Findings overall indicate that HCC may be a useful marker of cortisol responses to chronic stress.

Catechol-O-methyltransferase gene val158met polymorphism and depressive symptoms during early childhood.

Catechol‐O‐Methyltransferase (COMT) is a critical regulator of catecholamine levels in the brain. A functional polymorphism of the COMT gene, val158met, has been linked to internalizing symptoms (i.e., depression and anxiety) in adolescents and adults. We extended this research by investigating whether the val158met polymorphism was associated with childhood symptoms of depression and anxiety in two independent samples of young children (Ns = 476 and 409). In both samples, preschool‐aged children were genotyped for the COMT val158met polymorphism. Symptoms of psychopathology were assessed via parent interviews and primary caregiver reports. In both samples, children homozygous for the val allele had higher levels of depressive symptoms compared to children with at least one copy of the met allele. Our findings extend previous research in older participants by showing links between the COMT val158met polymorphism and internalizing symptoms in early childhood.

The Role of Parenting and Dopamine D4 Receptor Gene Polymorphisms in Children's Inhibitory Control.

Temperamental effortful control has important implications for childrens development. Although genetic factors and parenting may influence effortful control, few studies have examined interplay between the two in predicting its development. The current study investigated associations between parenting and a facet of childrens effortful control, inhibitory control (IC), and whether these associations were moderated by whether children had a 7-repeat variant of the DRD4 exon III VNTR. A community sample of 409 3-year-olds completed behavioural tasks to assess IC, and observational measures of parenting were also collected. Negative parenting was associated with lower child IC. The association between childrens IC and positive parenting was moderated by childrens DRD4 7-repeat status, such that children with at least one 7-repeat allele displayed lower IC than children without this allele when positive parenting was lower. These effects appeared to be primarily influenced by parent support and engagement. Results extend recent findings suggesting that some genetic polymorphisms may increase vulnerability to contextual influences.

Higher- and lower-order factor analyses of the Children's Behavior Questionnaire in early and middle childhood.

The Childrens Behavior Questionnaire (CBQ; Rothbart, Ahadi, & Hershey, 1994), a 195-item parent-report questionnaire, is one of the most widely used measures of child temperament, with previous analyses of its scales suggesting that 3 broad factors account for the overarching structure of child temperament (Rothbart, Ahadi, Hershey, & Fisher, 2001). However, there are no published item-level factor analyses of the CBQ, meaning that it is currently unclear whether items clearly load onto CBQ scales as proposed by its developers. Additionally, although the CBQ is intended to cover a broad window of development (i.e., ages 3-7), little is known about whether the structure of the CBQ differs depending on child age. The present study used a bottom-up approach to examine the lower- and higher-order structure of the CBQ in a large community sample of children at ages 3 (N = 944) and 5/6 (N = 853). Item-level exploratory factor analyses (EFAs) identified 88 items at age 3 and 87 items at age 5/6 suitable (i.e., with loadings ≥.40) for constructing lower-order factors. Of the lower-order factors derived at ages 3 and 5/6, fewer than half resembled original CBQ scales (Rothbart et al., 1994, 2001). Higher-order EFAs of the lower-order factors suggested that a 4-factor structure was the best fit at both ages 3 and 5/6. Thus, results indicate that a substantial number of CBQ items do not load well on a lower-order factor and that more than 3 factors are needed to account for its higher-order structure.

Catechol-O-Methyltransferase gene (val158met) polymorphisms and anxious symptoms in early childhood: The roles of hypothalamus-pituitary-adrenal axis reactivity and life stress

Individual differences in hypothalamus-pituitary-adrenal (HPA) axis reactivity to stress (measured via salivary cortisol) have been widely implicated in the etiology of internalizing problems such as depression and anxiety. Literature suggests that stress during early childhood is an important source of contextual risk although its effects may be moderated by polymorphisms of neurotransmitter genes. The COMT val158met is one such polymorphism, and literature documents its link to internalizing problems. To extend these findings, and to better understand the role of this polymorphism in developmental risk, we investigated links between the val158met polymorphism and early-age cortisol response. Additionally, we investigated whether cortisol reactivity mediated the link between COMT and emerging internalizing symptoms. The study was conducted in a community sample of 409 preschoolers. Saliva samples were collected pre-stress task (baseline) and every 10min post-stress task for one-hour to asses cortisol response. Child anxious and depressive symptoms were tabulated based on parent-reports. Markers of early childhood stress included marital discord, socio-economic status and the UCLA Life Stress Interview. Findings indicated that the val158met polymorphism is associated with childhood cortisol response (p<0.05). A gene-environment interaction between val158met and life stress also predicted child anxiety symptoms (p<0.01). Finally, cortisol response mediated the main-effect of val158met on child anxiety symptoms (pathway ps<0.05). Analyses suggest that COMT val158met moderates the influence of early life stress on preschool-age symptoms of anxiety. Additionally, cortisol reactivity acts as a mechanistic mediator of the main-effect of COMT genotype on child anxious symptoms.

The serotonin transporter promoter polymorphism moderates the continuity of behavioral inhibition in early childhood

Persistently elevated behavioral inhibition (BI) in children is a marker of vulnerability to psychopathology. However, little research has considered the joint influences of caregiver and child factors that may moderate the continuity of BI in early childhood, particularly genetic variants that may serve as markers of biological plasticity, such as the serotonin transporter linked polymorphic region (5-HTTLPR). We explored this issue in 371 preschoolers and their caregivers, examining whether parent characteristics (i.e., overinvolvement or anxiety disorder) and child 5-HTTLPR influenced the continuity of BI between ages 3 and 5. Measures were observational ratings of child BI, observational and questionnaire measures of parenting, and parent interviews for anxiety disorder history, and children were genotyped for the 5-HTTLPR. Parent factors did not moderate the association between age 3 and age 5 BI; however, child BI at age 3 interacted with childrens 5-HTTLPR variants to predict age 5 BI, such that children with at least one copy of the short allele exhibited less continuity of BI over time relative to children without this putative plasticity variant. Findings are consistent with previous work indicating the 5-HTTLPR short variant increases plasticity to contextual influences, thereby serving to decrease the continuity of BI in early childhood.