Short communication: catechol-O-methyltransferase allelic variation in relation to psychological and hormonal indices of stress in children and adolescents with chromosome 22q11.2 deletion syndrome (22q11.2DS)

Abstract

Chromosome 22q11.2 deletion syndrome (22q11.2DS) is a developmental disorder with high rates of anxiety and psychosis. Catechol-O-methyltransferase (COMT) regulates epinephrine (E), norepinephrine (NE), and dopamine (DA) and is implicated in both anxiety and psychotic disorders. The aim of this study was to determine how COMT variation relates to psychological anxiety and associated stress physiology responsiveness to better understand symptom heterogeneity in people with 22q11.2DS. We examined COMT allelic variation in relation to anxiety and hypothalamic-pituitary-adrenocortical (HPA) and sympathetic-adrenomedullary (SAM) hormonal stress indicators in 30 children and adolescents with 22q11.2DS. Contrary to expectation, individuals with the higher activity COMTval allele had higher anxiety levels versus those with the low activity (COMTmet) allele (p = 0.021; Glass’ Δ = 0.69). Anxiety was not correlated with salivary cortisol (CORT) or alpha-amylase (sAA) in either group. Groups did not differ in CORT levels (p = 0.58), but the COMTmet group had higher sAA (p = 0.026; Glass’ Δ = 0.67, uncorrected) suggesting greater SAM reactivity but not HPA activity. This suggests that COMT allelic variation may contribute to differences in acute SAM but not slower HPA stress reactivity in those with 22q11.2DS.

Introduction

Chromosome 22q11.2 deletion syndrome (22q11.2DS), historically referred to as Velocardiofacial and DiGeorge syndromes, is a multifaceted neurodevelopmental disorder with a reported prevalence ranging from 1:2,000 to 1:6,000 live births (Du et al. 2020). 22q11.2DS results from a hemizygous deletion, ranging in size from 0.5 megabases (Mb) to 3.0 Mb, on the long arm of chromosome 22. It encompasses between 30 and 90 known genes. Symptoms associated with 22q11.2DS include skeletal abnormalities, distinct facial features, genitourinary anomalies, gastrointestinal problems, endocrine abnormalities, heart defects, developmental delays, brain abnormalities, and psychiatric and behavioral disorders (McDonald-McGinn & Sullivan 2011). Anxiety disorders are common, and perhaps most strikingly, 25 to 30% of people with 22q11.2DS develop schizophrenia spectrum disorder in adolescence or emerging adulthood. The origin of this exceptionally high risk for developing a psychotic disorder such as schizophrenia in people with 22q11.2DS is not fully understood but likely arises from a multivariate interaction of factors that contribute to a diathesis that includes complex gene–gene interactions (Karayiorgou et al. 2010; Schneider et al. 2014). Indirect factors such as anxiety and stress that can arise from and exacerbate coping with the effects of the 22q11.2DS may contribute to this risk endophenotype. Allelic variation in genes involved in stress responsivity and psychiatric disorders may provide insight into risk profiles and the schizophrenia endophenotype in people with 22q11.2DS. One such gene found in the deleted region of 22q11.2DS is catechol-O-methyltransferase (COMT).

COMT is responsible for degrading catecholamines including dopamine, epinephrine, and norepinephrine in the central and peripheral nervous system (Motahari et al. 2019). While this gene harbors multiple single nucleotide polymorphisms (SNPs), this study focuses on the SNP at position 158 (rs4680). This SNP is a missense mutation that causes a change from a guanine to an adenine and results in a change in amino acid from a valine to a methionine. A 40–50% reduction of enzymatic activity is seen in the minor allele (methionine variant) of COMT. This decrease in enzymatic activity should result in longer catecholaminergic activity within the synapse. Epinephrine is part of the rapid sympathetic autonomic stress response, whereas dopamine and norepinephrine are involved in modulating anxiety (Zarrindast & Khakpai 2015). While this is not a forgone conclusion, less efficient catecholamine breakdown could, in part, contribute to individual differences in the perception or physiological response to stress or anxiety. As the deletion is hemizygotic in 22q11.2DS, allelic variation in non-deleted copies of this gene has a strong effect on the enzymatic activity of their products and the effects of any other genes that they interact with in its pathway. The role of this gene in anxiety, the stress and coping response, and risk for psychiatric disorders is well characterized (Motahari et al. 2019) and may contribute to poorer stress-coping responses that heighten the risk of developing psychosis later in life in adolescents and young adults with 22q11.2DS (Beaton & Simon 2011). Previously, Gothelf and colleagues (2014) found associations between the low-activity COMTmet allele and a range of negative outcomes in people with 22q11.2DS, including attention deficit disorder (ADHD), psychosis, cognitive decline, obsessive–compulsive disorder (OCD), and frontal lobe atrophy. They further showed that elevated baseline negative mood (including anxiety), reduced verbal IQ, and elevated psychotic symptoms, along with having the lower-activity COMT genotype, predicted both the onset and severity of adult psychosis.

Along with psychological measures of stress and anxiety, physiological measures of stress in the form of salivary hormones and enzymes can be an important corroborative metric and can also provide insights into the etiopathology of psychopathology. Individuals with 22q11.2DS cope with a variety of stressors from birth through adulthood that include serious medical problems and interventions, behavioral difficulties, and comorbid psychological conditions that can interfere with socioemotional and educational development. Moreover, chronic elevations of glucocorticoids and pro-inflammatory cytokines are associated with degrading neuronal health and function in human and animal studies, including patients with schizophrenia (Armbruster et al. 2012; Walker et al. 2013). The stress response can be characterized into two interrelated systems — the acute sympathetic nervous response that involves the release of activational hormones from the sympathetic-adrenal-medullary (SAM) system including epinephrine and norepinephrine and the slower, but longer responding hypothalamic-pituitary-adrenocortical (HPA) system that releases glucocorticoids such as cortisol. While activation of the SAM system almost instantly mobilizes flight, flee, or freeze behaviors and accompanying physiological arousal and attention, longer, repeated, and/or chronic stressors activate HPA within tens of minutes and can be active over extended periods of time to draw from stored resources in the coping response. Cortisol can be measured in saliva or blood as a proxy of HPA activation, and in most cases, cortisol levels rise in response to a stressor and return to baseline levels after resolution of the stressor. Chronic, repeated, or severe stress is associated with a depletion of coping reserves and increased allostatic load that can lead to anxiety and other psychiatric disorders (McEwen 1998) Increased sympathetic activation in response to a stressor can be measured via salivary alpha-amylase (sAA) concentrations. Moreover, alpha-amylase is a measure of stress not directly controlled by the hypothalamic-pituitary part of the HPA axis. Thus, elevations in sAA can serve as a corroborative measure of stress reactivity, even if the HPA system is dysregulated (Ali & Nater 2020).

The aim of the current study was to measure psychological and physiological indicators of stress in the form of parent-reported anxiety about their child, indicators of the acute SAM stress response, and the slower HPA stress response in the context of COMT allelic variation in children and adolescents with 22q11.2DS. We hypothesized that individuals with 22q11.2DS and the less active methionine (the minor allele) enzyme variant of the COMT gene (COMTmet) would have higher reported trait anxiety on parental assessments, greater HPA activity as measured by salivary cortisol, and greater SAM activation as measured by sAA compared to those with the more active valine variant (the major allele) of the COMT gene (COMTval) in response to a mild stressor consisting of completing a series of computer-based cognitive tasks in a laboratory setting.

Materials and methods

Participants

Participants were 30 children and adolescents (13 females) ages 7.8 years to 18.1 years (M = 12.4 years, SD = 2.5 years) and their parents/guardians as part of a larger ongoing study. The sample included participants who were identified by their parents as Caucasian (73.4%), Hispanic (10%), African American (3.3%), or not specified (13.3%). Medical records provided by parents included a fluorescence in situ hybridization (FISH) confirmation of a 22q11.2DS diagnosis. Participants with a history of head injuries, central nervous system infections, or other neurologic abnormalities were excluded. This research was approved by the Institutional Review Board at the University of New Orleans (IRB#: 06-Aug-12).

Biological sample collection

Blood collection

Blood was collected using ethylenediaminetetraacetic acid (EDTA) and/or serum separator tubes (SST). Samples were centrifuged to remove the plasma/serum and aliquoted into multiple microcentrifuge tubes and stored at − 80 °C until DNA extraction was performed.

Saliva collection

All saliva samples were collected in the afternoon as part of another larger study (Sanders et al. 2017). Each participant completed a series of mildly stressful computer-based cognitive tasks that included a countback task and a progressively challenging mathematics task. Five saliva samples were collected over the course of the tasks via passive drool into 2-ml microcentrifuge tubes and stored at − 80 °C until assayed for CORT and sAA.

DNA extraction

DNA extraction

The Qiagen DNA Mini and Blood Mini kits were used to isolate DNA from 200 μl of whole blood cells. After Buffer AW2 was centrifuged, the sample was placed in a new collection tube and centrifuged again. Buffer AE was incubated on the column at room temperature for 5 min. For participants for whom there was no blood sample, DNA was extracted from saliva using the Qiagen DNA Mini and Blood Mini kits. Adjustments included using 100 versus 200 μl of Buffer AE to yield more DNA. Sample concentrations were determined using a Qubit fluorometer.

Polymerase chain reaction amplification

COMT allele and DNA sequencing

A 20-mM primer solution (in TE) was made for each of the forward and reverse primers. In the Premixed Simpleseq sequencing tubes, 5 μl of the DNA sample and 5 μl of the primer solution were added to the samples (blood and saliva). Sequencing data from Eurofins was analyzed on UGENE with each sample compared to the reference to determine which nucleotide was present at the location of the SNP.

Anxiety measure

Participants’ trait anxiety levels were assessed using the parent-report version of the Behavioral Assessment System for Children (BASC) 2nd Edition. The anxiety subscale is comprised of 14 true/false and Likert format items that quantitatively assess the presence and severity of anxiety-related symptoms in children and adolescents including both somatic and cognitive dimensions of anxiety (Kamphaus et al. 2014).

Salivary hormone measures

Saliva samples were assayed for CORT and sAA using enzyme-linked immunosorbent assay (ELISA) kits in duplicate. CORTand sAA were measured in μg/dL, and the total levels from the five measures of CORT and sAA were used for analyses.

Results

Statistical analyses

All variables were screened for missing data and outliers. Uncorrected results are reported with effect sizes, given the small sample size conferred by a relatively rare special population of children and adolescents and the smaller number of minor allele (n = 9) versus the larger group with the major allele (n = 21). The frequency of the minor allele in these participants was 0.3, which is close to the frequency in the general population as reported in the National Center for Biotechnology Information (approximately 0.4).

Anxiety results

As shown in Fig. 1, an independent t-test indicated that the COMTval allele group had higher reported anxiety compared to individuals in the COMTmet allele group (t(25) = 2.36, p = 0.021; Glass’s delta effect size = 0.69).

Fig. 1.

Mean BASC-2 anxiety subscale T-scores in the COMTval and COMTmet groups

Salivary hormone results

An independent t-test indicated that the COMT groups did not statistically differ in total CORT (t(13) = 0.568. p = 0.58; Glass’ Δ effect size = 0.24). This is shown in Fig. 2A. However, as illustrated in Fig. 2B, the COMTmet group had a higher total sAA compared to the COMTval group (t(26) = − 2.366. p = 0.026 uncorrected; Glass’ Δ effect size = 0.67).

Fig. 2.

A Mean salivary cortisol levels in the COMTval and COMTmet groups; B mean salivary alpha-amylase in the COMTval and COMTmet groups

Discussion

Chromosome 22q11.2DS is a complex neurodevelopmental disorder with a heterogeneous symptom presentation and uncertain developmental path that includes a significant risk of psychiatric illness. To the best of our knowledge, this is the first study to investigate the acute SAM and chronic HPA stress responses in people with 22q11.2DS in the context of COMT allelic variation and anxiety. Contrary to our hypothesis that the COMTmet allele would be associated with higher levels of anxiety, this group had lower anxiety compared to the COMTval group. This was unexpected given prior findings that COMTmet was associated with increasing anxiety over time in patients with 22q11.2DS with psychosis (Gothelf et al. 2014). Moreover, Bauman and colleagues (2013) found that children who experience early adverse life events and the COMTmet allele also had higher levels of anxiety sensitivity. There could also be interactions with sex that would exacerbate the limitations of our small sample size, although BASC-2 anxiety T-scores are standardized by both age and sex. Nevertheless, the association between higher anxiety symptoms and the COMTval major allele in the current study reflects similar prior findings showing that the COMTval polymorphism is associated with increased neuroticism, harm avoidance, negative emotionality, and phobic anxiety (for a review and meta-analysis see Lee & Prescott 2014). One possible explanation may involve dopamine (DA) depletion, which is associated with elevated anxiety. Lower DA and NE levels resulting from higher enzymatic activity of the COMTval gene could also influence HPA axis activity in response to stress (Feldman & Weidenfeld 2004).

We found no group differences in the total salivary CORT levels. However, the COMTval group did show lower acute stress SAM activity as measured with sAA. This reflects previous studies indicating greater emotional control and efficiency in emotion processing in a variety of population samples possessing the COMTval polymorphism (Lee & Prescott 2014). Our findings indicated that the low-activity COMTmet group had increased sympathetic adrenal medullary activity in response to the mildly stressful cognitive tasks. Others have found similar HPA versus SAM activity patterns in response to stress with COMTmet associated with elevated sAA levels in otherwise healthy adults in a pain induction task with no group differences in CORT responsivity (Serrano et al. 2019). Increased bioavailability of NE in the COMTmet group may drive the increased SAM reactivity independently from the slower hypothalamic-mediated CORT response. Dysregulation of monoamine neurotransmitters (DA, NE, serotonin) is associated with mood and anxiety disorders. Increased activity of the central NE system from the locus coeruleus is seen in humans and animal models of anxiety, stress, NE, and fear responses via actions on limbic structures including the amygdala (Montoya et al. 2016). We suggest that the elevated anxiety reported in the COMTval group could be driven by increased levels of NE, a consequence of lower enzymatic breakdown of NE. Moreover, greater enzymatic action in COMTval may lead to lower DA levels in the brains of those individuals leading to lower stress resilience and elevated anxiety (Šanjug et al. 2023).

While this is the first study to examine SAM and HPA activity in children and adolescents with 22q11.2DS, it is limited due to the small sample size, expanded age range including children and adolescents, the inherent heterogeneity of the 22q11.2DS syndrome, a complex medical and pharmaceutical profile for each child, and a focus on a single gene polymorphism. The differences in development, morphology, and symptoms are the result of many gene–gene interactions, copy number repeats, and allelic variation of many other genes along with epigenetic changes over time. Psychological anxiety was also determined via parent reports using a standardized psychological tool. Including self-reports and/or teacher-reports of anxiety-related behavior along with semi-structured interviews may be a better approach. While parents/guardians are front-line observers, they may not always recognize anxiety in their children, and this may be even more difficult in children with developmental disorders.

Nevertheless, the strengths of the study include novel measurements in an important special population of children and adolescents. Lower anxiety with elevated sAA in the COMTmet group was unexpected. However, this pattern suggests a synergistic interaction between the low-activity COMTmet gene and adrenal hormones to increase catecholamine availability leading to increased SAM stress reactivity in a subset of children and adolescents with 22q11.2DS. Based on the results of this study, we suggest that further investigation via replication and extension with a larger sample of children and adolescents with 22q11.2DS is the next step. Coping with chronic, intense, and unpredictable stressors contributes to the risk of psychopathology and psychosis, making it a critical point for further investigation.

Data availability

Study data is available upon request.