Fish oil supplementation does not lower C-reactive protein or interleukin-6 levels in healthy adults.

BACKGROUND: The n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may prevent a range of chronic conditions through anti-inflammatory actions. However, as clinical trials using these fatty acids for primary prevention are yet unavailable, their putative role in disease prevention rests, in part, on evidence of anti-inflammatory actions in healthy individuals. OBJECTIVE: To investigate in a double-blind, placebo-controlled clinical trial whether supplementation with a moderate dose of EPA+DHA reduces common biomarkers of chronic, systemic inflammation in healthy individuals. METHODS: A total of 261 healthy individuals aged 30-54 years who were free of inflammatory conditions and consumed ≤ 300 mg per day EPA+DHA were included in the study. Participants were randomly assigned to 18 weeks of either fish oil supplementation providing 1400 mg per day EPA+DHA or matching placebo. Outcome measures were serum levels of C-reactive protein (CRP) and interleukin (IL)-6. In a substudy, ex vivo cytokine production was measured. Missing data for CRP and IL-6 were estimated using regression imputation. Data analyses conformed to intention-to-treat principles. RESULTS: Participant blinding was verified. Red blood cell EPA+DHA increased by 64% in the active treatment group, but serum CRP and IL-6 were not affected by supplementation (P ≥ 0.20). Findings were consistent with and without imputed values and across subgroups. Similarly, EPA+DHA supplementation did not alter ex vivo production of four pro-inflammatory cytokines (P ≥ 0.20). CONCLUSIONS: Supplementation with 1400 mg EPA+DHA did not reduce common markers of systemic inflammation in healthy adults. Whether this or a higher dose affects other measures of inflammation, oxidative stress or immune function warrants examination.

Association between amygdala reactivity and a dopamine transporter gene polymorphism.

Essential for detection of relevant external stimuli and for fear processing, the amygdala is under modulatory influence of dopamine (DA). The DA transporter (DAT) is of fundamental importance for the regulation of DA transmission by mediating reuptake inactivation of extracellular DA. This study examined if a common functional variable number tandem repeat polymorphism in the 3' untranslated region of the DAT gene (SLC6A3) influences amygdala function during the processing of aversive emotional stimuli. Amygdala reactivity was examined by comparing regional cerebral blood flow, measured with positron emission tomography and [(15)O]water, during exposure to angry and neutral faces, respectively, in a Swedish sample comprising 32 patients with social anxiety disorder and 17 healthy volunteers. In a separate US sample, comprising 85 healthy volunteers studied with blood oxygen level-dependent functional magnetic resonance imaging, amygdala reactivity was assessed by comparing the activity during exposure to threatening faces and neutral geometric shapes, respectively. In both the Swedish and the US sample, 9-repeat carriers displayed higher amygdala reactivity than 10-repeat homozygotes. The results suggest that this polymorphism contributes to individual variability in amygdala reactivity.

PPARα gene polymorphisms modulate the association between physical activity and cardiometabolic risk.

BACKGROUND AND AIMS: Habitual physical activity is understood to help prevent type 2 diabetes and atherosclerotic cardiovascular disease via beneficial effects on both metabolism and the vascular system. However, individuals do not have uniform cardiometabolic responses to physical activity. Here we explore the extent to which variation in the proliferator-activated receptor-alpha (PPARα) gene, which modulates carbohydrate and lipid metabolism, vascular function, and inflammation, predicts the overall cardiometabolic risk (CMR) profile of individuals engaging in various levels of physical activity. METHODS AND RESULTS: 917 unrelated, community volunteers (52% female, of Non-Hispanic European ancestry) aged 30-54 years, participated in the cross-sectional study. Subjects were genotyped for 5 single nucleotide polymorphisms in the PPARα gene, from which common haplotypes were defined. A continuous measure of CMR was calculated as an aggregate of 5 traditional risk factors: waist circumference, resting blood pressure, fasting serum triglycerides, HDL-cholesterol and glucose. Regression models were used to examine the main and interactive effects of physical activity and genetic variation on CMR. One common PPARα haplotype (H-23) was associated with a higher CMR. This association was moderated by daily physical activity (B = -0.11, SE = 0.053, t = -2.05, P = 0.04). Increased physical activity was associated with a steeper reduction of CMR in persons carrying the otherwise detrimental H-23 haplotype. CONCLUSIONS: Variations in the PPARα gene appear to magnify the cardiometabolic benefits of habitual physical activity.

Genetic variants affecting the neural processing of human facial expressions: evidence using a genome-wide functional imaging approach.

Human faces present crucial visual information for social interaction. Specialized brain regions are involved in the perception of faces, with the fusiform face area (FFA) a key neuronal substrate. Face processing is genetically controlled, but by which specific genes is unknown. A genome-wide approach identified common single nucleotide polymorphisms (SNPs) associated with areas of increased brain activity in response to affective facial expressions, measured with functional magnetic resonance imaging. SNPs in 20 genetic regions were linked with neural responses to negative facial expressions in a Norwegian sample (n=246), which included patients with mental illness. Three genetic regions were linked with FFA activation in a further discovery experiment using positive facial expressions and involving many of the same individuals (n=284). Two of these three regions showed significant association with right FFA activation to negative facial expressions in an independent North American replication sample of healthy Caucasians (n=85, 3q26.31, P=0.004; 20p12.3, P=0.045). The activation patterns were particularly striking for the SNP in 3q26.31, which lies in a gene TMEM212; only the FFA was activated. The specialized function of this brain region suggests that TMEM212 could contribute to the innate architecture of face processing.

Impairment of ovarian function and associated health-related abnormalities are attributable to low social status in premenopausal monkeys and not mitigated by a high-isoflavone soy diet.

BACKGROUND: Psychological stress may impair premenopausal ovarian function and contribute to risk for chronic disease. Soy isoflavones may also influence ovarian function and affect health. Here, we report the effects of a psychological stressor (subordinate social status) and dietary soy on reproductive function and related health indices in female monkeys. We hypothesized that reproductive compromise and adverse health outcomes would be induced in subordinate when compared with dominant monkeys and be mitigated by exposure to soy. METHODS: Subjects were 95 adult cynomolgus monkeys (Macaca fascicularis) housed in social groups of five or six. Animals consumed a soy-free, animal protein-based diet during an 8-month Baseline phase and then, during a 32-month Treatment phase, consumed either the baseline diet or an identical diet that substituted high-isoflavone soy protein for animal protein. RESULTS: Across more than 1200 menstrual cycles, subordinate monkeys consistently exhibited ovarian impairment [increased cycle length (P < 0.02) and variability (P < 0.02) and reduced levels of progesterone (P < 0.04) and estradiol (P < 0.04)]. Subordinate status was confirmed behaviorally and was associated with elevated cortisol (P < 0.04) and relative osteopenia (P < 0.05). Consumption of the soy diet had no significant effects. CONCLUSIONS: (i) Psychological stress adversely affects ovarian function and related health indices in a well-accepted animal model of women's health; (ii) Similar effects may extend to women experiencing reproductive impairment of psychogenic origin; (iii) soy protein and isoflavones neither exacerbate nor mitigate the effects of an adverse psychosocial environment; and (iv) this study was limited by an inability to investigate the genetic and developmental determinants of social status.

Genetic variation in components of dopamine neurotransmission impacts ventral striatal reactivity associated with impulsivity.

Individual differences in traits such as impulsivity involve high reward sensitivity and are associated with risk for substance use disorders. The ventral striatum (VS) has been widely implicated in reward processing, and individual differences in its function are linked to these disorders. Dopamine (DA) plays a critical role in reward processing and is a potent neuromodulator of VS reactivity. Moreover, altered DA signaling has been associated with normal and pathological reward-related behaviors. Functional polymorphisms in DA-related genes represent an important source of variability in DA function that may subsequently impact VS reactivity and associated reward-related behaviors. Using an imaging genetics approach, we examined the modulatory effects of common, putatively functional DA-related polymorphisms on reward-related VS reactivity associated with self-reported impulsivity. Genetic variants associated with relatively increased striatal DA release (DRD2 -141C deletion) and availability (DAT1 9-repeat), as well as diminished inhibitory postsynaptic DA effects (DRD2 -141C deletion and DRD4 7-repeat), predicted 9-12% of the interindividual variability in reward-related VS reactivity. In contrast, genetic variation directly affecting DA signaling only in the prefrontal cortex (COMT Val158Met) was not associated with variability in VS reactivity. Our results highlight an important role for genetic polymorphisms affecting striatal DA neurotransmission in mediating interindividual differences in reward-related VS reactivity. They further suggest that altered VS reactivity may represent a key neurobiological pathway through which these polymorphisms contribute to variability in behavioral impulsivity and related risk for substance use disorders.

A regulatory variant of the human tryptophan hydroxylase-2 gene biases amygdala reactivity.

Recent studies have indicated that a newly identified second isoform of the tryptophan hydroxylase gene (TPH2) is preferentially involved in the rate-limiting synthesis of neuronal serotonin. Genetic variation in the human TPH2 gene (hTPH2) has been associated with altered in vitro enzyme activity as well as increased risk for mood disorders. Here, we provide the first in vivo evidence that a relatively frequent regulatory variant (G(-844)T) of hTPH2 biases the reactivity of the amygdala, a neural structure critical in the generation and regulation of emotional behaviors.

Influence of estradiol on cortisol secretion in ovariectomized cynomolgus macaques (Macaca fascicularis).

In an investigation of cortisol secretion in fully mature, ovariectomized cynomolgus monkeys (Macaca fascicularis), we compared monkeys that were given either placebo (OVX, n = 26) or 17beta estradiol (E(2 )) (EST, n = 26) in a daily oral dose. Serum cortisol concentrations were measured prior to the experimental manipulation and 3, 6, 9, and 12 months following initiation of treatment. Pretreatment cortisol values did not differ between groups. Assessment of the treatment period values revealed that cortisol concentrations were significantly higher ( approximately 10%) in the EST than in the OVX monkeys. Cortisol also varied significantly across periods of sampling. This time-dependent variation was attributable to elevations in months 6 and 9 (when daylight was generally long), relative to months 3 and 12 (when daylight was relatively short). The modest stimulatory effect of estrogen on corticosteroid production observed in this study is consistent with what has been seen in women, and contrasts with the more robust effects observed in New World monkeys. The possible relationship between season and cortisol secretion observed here has not been previously described in monkeys.

Premenopausal social status and hormone exposure predict postmenopausal atherosclerosis in female monkeys.

OBJECTIVE: To determine whether premenopausal social subordination in female monkeys predicts postmenopausal atherosclerosis, and whether any such effect is altered by chronic exposure to contraceptive steroids or postmenopausal hormone replacement. METHODS: One hundred seventy-seven (177) premenopausal cynomolgus monkeys (Macaca fascicularis) housed in social groups of five or six were fed an atherogenic diet that, for half of the animals, also contained an oral contraceptive (OC). Individuals were judged socially dominant or subordinate based on behavioral observations. After 26 months animals were oophorectomized, biopsied for iliac atherosclerosis, and for the next 36 months were fed one of three atherogenic diets containing soy protein: 1) phytoestrogen-free; 2) phytoestrogens intact; and 3) phytoestrogen-free plus conjugated equine estrogens. Plasma lipids and menstrual cyclicity were also assessed. Finally, all animals were necropsied and the extent of atherosclerosis measured in the coronary and iliac arteries. RESULTS: The interaction of premenopausal social status and OC exposure predicted postmenopausal coronary artery atherosclerosis (P =.02). Subordinate animals not receiving OCs developed twice the coronary atherosclerosis of similarly untreated dominants (P <.01), an outcome mitigated by premenopausal OC exposure (P <.01). These effects occurred across postmenopausal treatment groups and independent of variation in plasma lipids. The same associations were observed in the iliac arteries, and, to a similar extent, both pre- and post-menopausally. Hormone data suggest that untreated premenopausal subordinates may have been estrogen deficient. CONCLUSION: Premenopausal social subordination exacerbates postmenopausal atherosclerosis, an effect possibly mediated by estrogen deficiency and shown here to be prevented by premenopausal OC exposure. These results occur irrespective of postmenopausal treatment.

The nature of the association between diet and serum lipids in the community: a twin study.

Diet is commonly thought to be an environmental influence on serum lipid concentrations. This study evaluated whether total caloric and fat intake predict total cholesterol (TC), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), and triglyceride (TRIG) concentrations for environmental, as compared with genetic, reasons among 137 monozygotic and 67 dizygotic young adult twins. When genetic influences were controlled by correlating differences between monozygotic co-twins, a significant association remained between diet and TC, LDL, and HDL, suggesting that these dietary and serum lipid measures correlate for environmental reasons. Twin structural equation modeling confirmed these results. Overall, these results provide additional support for the hypothesis that diet is an environmental influence on TC, LDL, and HDL.

Associations between stress, trait negative affect, acute immune reactivity, and antibody response to hepatitis B injection in healthy young adults.

Eighty-four healthy graduate participants were administered the standard course of 3 hepatitis B vaccinations. Five months after the first dose (shortly after the second injection), each participant completed psychosocial measures, and a blood sample was drawn for determination of hepatitis B surface antibody titer. After completion of the vaccination series, participants performed an acute stress protocol, consisting of a 30-min adaptation period and a 5-min evaluative speech task. Blood was drawn at the end of the resting and task periods for assessment of cellular immune measures. Lower antibody response, as assessed after the second hepatitis B injection, was predicted independently by (a) high trait negative affect and (b) diminished T-cell proliferation in response to PHA. These data provide evidence that trait negative affect and the magnitude of stress-induced suppression of immune function may have clinical significance.

Genetic and environmental influences on schizotypy: a community-based twin study.

This study investigated the factor structure and etiology of four self-report schizotypy questionnaires during young adulthood (age 18-27) in 98 monozygotic and 59 same-sex dizygotic twin pairs from the community. A single phenotypic factor was identified that was primarily associated with Perceptual Aberration, Magical Ideation, and the Rust Inventory of Schizotypal Cognitions scales, and less so with Social Anhedonia. Univariate etiologic models suggested that in addition to nonshared environmental influences, Perceptual Aberration and Social Anhedonia were significantly influenced by either genes or shared family environment, whereas Magical Ideation and the Rust Inventory were influenced by shared family environment, but not genes. Multivariate twin analyses detected a common schizotypy factor, primarily defined by Perceptual Aberration, Magical Ideation, and the Rust Inventory scales, that was influenced by genes or shared environment as well as nonshared environment. Contrary to expectations, these results suggest that, at least in community-based samples, these "positive" schizotypy questionnaires are not strongly genetically influenced.

Cholesterol reduction and non-illness mortality: meta-analysis of randomised clinical trials.

OBJECTIVE: To investigate the association between cholesterol lowering interventions and risk of death from suicide, accident, or trauma (non-illness mortality). DESIGN: Meta-analysis of the non-illness mortality outcomes of large, randomised clinical trials of cholesterol lowering treatments. STUDIES REVIEWED: 19 out of 21 eligible trials that had data available on non-illness mortality. INTERVENTIONS REVIEWED: Dietary modification, drug treatment, or partial ileal bypass surgery for 1-10 years. MAIN OUTCOME MEASURE: Deaths from suicides, accidents, and violence in treatment groups compared with control groups. RESULTS: Across all trials, the odds ratio of non-illness mortality in the treated groups, relative to control groups, was 1.18 (95% confidence interval 0.91 to 1.52; P=0.20). The odds ratios were 1.28 (0.94 to 1.74; P=0.12) for primary prevention trials and 1.00 (0.65 to 1.55; P=0.98) for secondary prevention trials. Randomised clinical trials using statins did not show a treatment related rise in non-illness mortality (0.84, 0.50 to 1.41; P=0.50), whereas a trend toward increased deaths from suicide and violence was observed in trials of dietary interventions and non-statin drugs (1.32, 0.98 to 1.77; P=0.06). No relation was found between the magnitude of cholesterol reduction and non-illness mortality (P=0.23). CONCLUSION: Currently available evidence does not indicate that non-illness mortality is increased significantly by cholesterol lowering treatments. A modest increase may occur with dietary interventions and non-statin drugs.

Memory performance and the apolipoprotein E polymorphism in a community sample of middle-aged adults.

The apolipoprotein E genotype (APOE) is an established risk factor for Alzheimer disease, with the age-at-onset occurring earlier in individuals having at least one APOE epsilon 4 allele, relative to the APOE epsilon 3 or APOE epsilon 2 isoforms. Moreover, nondemented older adults with the APOE epsilon 4 allele also show diminished cognitive performance, particularly on tests of learning and memory, and an accelerated decline in memory performance with increasing age. The current investigation extends the study of the APOE epsilon 4 allele and cognitive performance to healthy, middle-aged adults. A community sample of 220 non-Hispanic Caucasian men and women, aged 24-60 (average age = 46), were genotyped for the APOE polymorphism and completed a battery of neuropsychological tests. Multivariate analyses were conducted on measures of verbal learning and memory (e. g., learning a list of words and recalling them 30 min later), visual memory (e.g., reproducing a previously copied figure from memory), and attention span (e.g., repeating long lists of digits), after adjustments for age, and estimated IQ. Results indicated that performance on learning and memory tasks was significantly poorer in adults having any APOE epsilon 4 allele, relative to adults with APOE epsilon 2 and epsilon 3 genotypes (P <.01). Attention span did not differ by genotype. These findings, the first in a sample of middle-aged adults, suggest that the APOE polymorphism is a marker for age-related decline in memory (detectable prior to overt, clinical manifestations of memory loss), and/or a marker for individual differences in memory ability across the life span. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:707-711, 2000.

Anticipatory blood pressure responses to exercise are associated with left ventricular mass in Finnish men: Kuopio Ischemic Heart Disease Risk Factor Study.

BACKGROUND: Exaggerated cardiovascular reactivity to psychological demands may contribute to the development of left ventricular (LV) hypertrophy. We examined the cross-sectional association between anticipatory blood pressure (BP) responses to bicycle exercise and LV mass in the Kuopio Ischemic Heart Disease Risk Factor Study, a population-based epidemiological sample. METHODS AND RESULTS: Among 876 men from 4 age cohorts (ages 42, 48, 58, and 64 years), we collected echocardiographic assessments of LV mass along with measures of BP response taken before bicycle ergometry testing. Anticipatory BP responses were positively associated with LV mass, with significant associations only among younger (age <50 years) subjects with elevated resting pressures (3-way interactions for anticipatory BP x age x resting pressure for systolic and diastolic BP, all P:<0.05; for younger subjects with elevated systolic BP, P:<0. 01; and for younger subjects with elevated diastolic BP, P:<0.001). Among these subgroups, exaggerated anticipatory BP responses (top quartile) were associated with an incremental increase in LV mass of 10% or greater, corrected for body surface area. Results remained significant after adjusting for age, education, salt consumption, and resting BP, and the pattern of findings was maintained among men with no previous history of cardiovascular disease. CONCLUSIONS: The tendency to show exaggerated pressor responses to psychological demands may be a significant independent correlate of LV mass, especially among young men with high resting pressures. This is the first study to examine such associations in a middle-aged population sample.

Enhancing the laboratory-to-life generalizability of cardiovascular reactivity using multiple occasions of measurement.

Studies examining the association between laboratory measures of stress-related cardiovascular (CV) reactivity and responses to psychological challenges in the natural environment have yielded mixed results. Frequently, single laboratory tasks have been used to predict responses to natural stressors on a single occasion of measurement. Because aggregation broadens the range of stimuli sampled and reduces measurement error, laboratory-to-life generalizability should be more easily detected when multiple predictor and criterion observations are used. Sixty students in a public speaking course were administered multitask assessments of CV reactivity during two laboratory sessions and during two in-class public speech assignments. CV responses to the classroom speeches were greater among those who showed larger responses in the laboratory, and these associations became stronger as measures were aggregated across multiple samples of behavior. These results support the generalizability of CV reactivity as a measure of individual difference, and they help to shed light on previous inconsistent findings in this area.

A regulatory polymorphism of the monoamine oxidase-A gene may be associated with variability in aggression, impulsivity, and central nervous system serotonergic responsivity.

This study presents preliminary evidence of an association between polymorphic variation in the gene for monoamine oxidase-A (MAOA) and interindividual variability in aggressiveness, impulsivity and central nervous system (CNS) serotonergic responsivity. An apparently functional 30-bp VNTR in the promoter region of the X-chromosomal MAOA gene (MAOA-uVNTR), as well as a dinucleotide repeat in intron 2 (MAOA-CAn), was genotyped in a community sample of 110 men. All participants had completed standard interview and questionnaire measures of impulsivity, hostility and lifetime aggression history; in a majority of subjects (n=75), central serotonergic activity was also assessed by neuropsychopharmacologic challenge (prolactin response to fenfluramine hydrochloride). The four repeat variants of the MAOA-uVNTR polymorphism were grouped for analysis (alleles '1+ 4' vs. '2+3') based on prior evidence of enhanced transcriptional activity in MAOA promoter constructs with alleles 2 and 3 (repeats of intermediate length). Men in the 1/4 allele group scored significantly lower on a composite measure of dispositional aggressiveness and impulsivity (P<0.015) and showed more pronounced CNS serotonergic responsivity (P<0.02) than men in the 2/3 allele group. These associations were also significant on comparison of the more prevalent one and three alleles alone (encompassing 93% of subjects). Although in linkage disequilibrium with the MAOA-uVNTR polymorphism, MAOA-CAn repeat length variation did not vary significantly with respect to behavior or fenflluramine challenge in this sample. We conclude that the MAOA-uVNTR regulatory polymorphism may contribute, in part, to individual differences in both CNS serotonergic responsivity and personality traits germane to impulse control and antagonistic behavior.

White-coat hypertension and carotid artery atherosclerosis: a matching study.

BACKGROUND: Blood pressure (BP) measurements obtained in the clinic have long served as the basis for determining risk of hypertensive vascular disease, yet many patients with high BP in the physician's office are normotensive elsewhere. It remains unclear whether such patients with "white coat" hypertension elude the risk of atherosclerosis. METHODS: Community residents 40 to 70 years of age and not receiving any cardiovascular medications were recruited to participate in a study of cardiovascular risk factors. On the basis of clinic and daytime ambulatory BP and a threshold criterion of 140/90 mm Hg, subjects were classified as having persistent hypertension, white-coat hypertension, or persistent normotension. One-to-one matching was conducted in male participants on the basis of race and BP. Subjects with persistent hypertension and white-coat hypertension were matched on clinic BP, and those with white-coat hypertension and normotension were matched on daytime ambulatory BP. RESULTS: The 3 matched groups of men (n=40 in each group) were similar in age, smoking status, and fasting glucose and lipid levels. Compared with the normotensive subjects, subjects with either persistent or white-coat hypertension had greater mean body mass index, waist-hip ratio, and fasting insulin concentration. On the basis of standardized duplex ultrasound examination of the carotid arteries, mean maximal intimal-medial thickness and plaque index in subjects with white-coat hypertension were greater than among normotensive subjects and equal to that of the subjects with persistent hypertension. CONCLUSION: When compared with unmedicated individuals with comparable elevations in clinic BP, individuals with white-coat hypertension appear not to be protected from the atherosclerotic sequelae of hypertension.

Effects of lovastatin on cognitive function and psychological well-being.

PURPOSE: Animal research and cross-sectional studies suggest that serum lipid concentrations may influence cognitive function, mood, and behavior, but few clinical trials have studied these effects. SUBJECTS AND METHODS: In this double-blind investigation, 209 generally healthy adults with a serum low-density-lipoprotein (LDL) cholesterol level of 160 mg/dL or higher were randomly assigned to 6-month treatment with lovastatin (20 mg) or placebo. Assessments of neuropsychological performance, depression, hostility, and quality of life were conducted at baseline and at the end of the treatment period. Summary effect sizes were estimated as z scores on a standard deviation (SD) scale. RESULTS: Placebo-treated subjects improved between baseline and posttreatment periods on neuropsychological tests in all five performance domains, consistent with the effects of practice on test performance (all P <0.04), whereas those treated with lovastatin improved only on tests of memory recall (P = 0.03). Comparisons of the changes in performance between placebo- and lovastatin-treated subjects revealed small, but statistically significant, differences for tests of attention (z score = 0.18; 95% confidence interval (CI), 0.06 to 0.31; P = 0.005) and psychomotor speed (z score = 0.17; 95% CI, 0.05 to 0.28; P = 0. 004) that were consistent with greater improvement in the placebo group. Psychological well-being, as measured several ways, was not affected by lovastatin. CONCLUSION: Treatment of hypercholesterolemia with lovastatin did not cause psychological distress or substantially alter cognitive function. Treatment did result in small performance decrements on neuropsychological tests of attention and psychomotor speed, the clinical importance of which is uncertain.