Identification of loci associated with schizophrenia by genome-wide association and follow-up.

We carried out a genome-wide association study of schizophrenia (479 cases, 2,937 controls) and tested loci with P < 10(-5) in up to 16,726 additional subjects. Of 12 loci followed up, 3 had strong independent support (P < 5 x 10(-4)), and the overall pattern of replication was unlikely to occur by chance (P = 9 x 10(-8)). Meta-analysis provided strongest evidence for association around ZNF804A (P = 1.61 x 10(-7)) and this strengthened when the affected phenotype included bipolar disorder (P = 9.96 x 10(-9)).

Collaborative genome-wide association analysis supports a role for ANK3 and CACNA1C in bipolar disorder.

To identify susceptibility loci for bipolar disorder, we tested 1.8 million variants in 4,387 cases and 6,209 controls and identified a region of strong association (rs10994336, P = 9.1 x 10(-9)) in ANK3 (ankyrin G). We also found further support for the previously reported CACNA1C (alpha 1C subunit of the L-type voltage-gated calcium channel; combined P = 7.0 x 10(-8), rs1006737). Our results suggest that ion channelopathies may be involved in the pathogenesis of bipolar disorder.

A genome-wide association meta-analysis identifies a novel locus at 17q11.2 associated with sporadic amyotrophic lateral sclerosis.

Identification of mutations at familial loci for amyotrophic lateral sclerosis (ALS) has provided novel insights into the aetiology of this rapidly progressing fatal neurodegenerative disease. However, genome-wide association studies (GWAS) of the more common (∼90%) sporadic form have been less successful with the exception of the replicated locus at 9p21.2. To identify new loci associated with disease susceptibility, we have established the largest association study in ALS to date and undertaken a GWAS meta-analytical study combining 3959 newly genotyped Italian individuals (1982 cases and 1977 controls) collected by SLAGEN (Italian Consortium for the Genetics of ALS) together with samples from Netherlands, USA, UK, Sweden, Belgium, France, Ireland and Italy collected by ALSGEN (the International Consortium on Amyotrophic Lateral Sclerosis Genetics). We analysed a total of 13 225 individuals, 6100 cases and 7125 controls for almost 7 million single-nucleotide polymorphisms (SNPs). We identified a novel locus with genome-wide significance at 17q11.2 (rs34517613 with P = 1.11 × 10(-8); OR 0.82) that was validated when combined with genotype data from a replication cohort (P = 8.62 × 10(-9); OR 0.833) of 4656 individuals. Furthermore, we confirmed the previously reported association at 9p21.2 (rs3849943 with P = 7.69 × 10(-9); OR 1.16). Finally, we estimated the contribution of common variation to heritability of sporadic ALS as ∼12% using a linear mixed model accounting for all SNPs. Our results provide an insight into the genetic structure of sporadic ALS, confirming that common variation contributes to risk and that sufficiently powered studies can identify novel susceptibility loci.

A pathway-based analysis provides additional support for an immune-related genetic susceptibility to Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease affecting 1-2% in people >60 and 3-4% in people >80. Genome-wide association (GWA) studies have now implicated significant evidence for association in at least 18 genomic regions. We have studied a large PD-meta analysis and identified a significant excess of SNPs (P < 1 × 10(-16)) that are associated with PD but fall short of the genome-wide significance threshold. This result was independent of variants at the 18 previously implicated regions and implies the presence of additional polygenic risk alleles. To understand how these loci increase risk of PD, we applied a pathway-based analysis, testing for biological functions that were significantly enriched for genes containing variants associated with PD. Analysing two independent GWA studies, we identified that both had a significant excess in the number of functional categories enriched for PD-associated genes (minimum P = 0.014 and P = 0.006, respectively). Moreover, 58 categories were significantly enriched for associated genes in both GWA studies (P < 0.001), implicating genes involved in the 'regulation of leucocyte/lymphocyte activity' and also 'cytokine-mediated signalling' as conferring an increased susceptibility to PD. These results were unaltered by the exclusion of all 178 genes that were present at the 18 genomic regions previously reported to be strongly associated with PD (including the HLA locus). Our findings, therefore, provide independent support to the strong association signal at the HLA locus and imply that the immune-related genetic susceptibility to PD is likely to be more widespread in the genome than previously appreciated.

Using genome-wide complex trait analysis to quantify 'missing heritability' in Parkinson's disease.

Genome-wide association studies (GWASs) have been successful at identifying single-nucleotide polymorphisms (SNPs) highly associated with common traits; however, a great deal of the heritable variation associated with common traits remains unaccounted for within the genome. Genome-wide complex trait analysis (GCTA) is a statistical method that applies a linear mixed model to estimate phenotypic variance of complex traits explained by genome-wide SNPs, including those not associated with the trait in a GWAS. We applied GCTA to 8 cohorts containing 7096 case and 19 455 control individuals of European ancestry in order to examine the missing heritability present in Parkinson's disease (PD). We meta-analyzed our initial results to produce robust heritability estimates for PD types across cohorts. Our results identify 27% (95% CI 17-38, P = 8.08E - 08) phenotypic variance associated with all types of PD, 15% (95% CI -0.2 to 33, P = 0.09) phenotypic variance associated with early-onset PD and 31% (95% CI 17-44, P = 1.34E - 05) phenotypic variance associated with late-onset PD. This is a substantial increase from the genetic variance identified by top GWAS hits alone (between 3 and 5%) and indicates there are substantially more risk loci to be identified. Our results suggest that although GWASs are a useful tool in identifying the most common variants associated with complex disease, a great deal of common variants of small effect remain to be discovered.

Most genome-wide significant susceptibility loci for schizophrenia and bipolar disorder reported to date cross-traditional diagnostic boundaries.

Recent findings from genetic epidemiology and from genome-wide association studies point strongly to a partial overlap in the genes that contribute susceptibility to schizophrenia and bipolar disorder (BD). Previous data have also directly implicated one of the best supported schizophrenia-associated loci, zinc finger binding protein 804A (ZNF804A), as showing trans-disorder effects, and the same is true for one of the best supported bipolar loci, calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNA1C) which has also been associated with schizophrenia. We have undertaken a cross-phenotype study based upon the remaining variants that show genome-wide evidence for association in large schizophrenia and BD meta-analyses. These comprise in schizophrenia, SNPs in or in the vicinity of transcription factor 4 (TCF4), neurogranin (NRGN) and an extended region covering the MHC locus on chromosome 6. For BD, the strongly supported variants are in the vicinity of ankyrin 3, node of Ranvier (ANK3) and polybromo-1 (PBRM1). Using data sets entirely independent of their original discoveries, we observed strong evidence that the PBRM1 locus is also associated with schizophrenia (P = 0.00015) and nominally significant evidence (P < 0.05) that the NRGN and the extended MHC region are associated with BD. Moreover, considering this highly restricted set of loci as a group, the evidence for trans-disorder effects is compelling (P = 4.7 × 10(-5)). Including earlier reported data for trans-disorder effects for ZNF804A and CACNA1C, six out of eight of the most robustly associated loci for either disorder show trans-disorder effects.

Evaluation of an approximation method for assessment of overall significance of multiple-dependent tests in a genomewide association study.

We describe implementation of a set-based method to assess the significance of findings from genomewide association study data. Our method, implemented in PLINK, is based on theoretical approximation of Fisher's statistics such that the combination of P-vales at a gene or across a pathway is carried out in a manner that accounts for the correlation structure, or linkage disequilibrium, between single nucleotide polymorphisms. We compare our method to a permutation-based product of P-values approach and show a typical correlation in excess of 0.98 for a number of comparisons. The method gives Type I error rates that are less than or equal to the corresponding nominal significance levels, making it robust to the effects of false positives. We show that in broadly similar populations, reference data sets of markers are an appropriate substrate for deriving marker-marker linkage disequilibrium (LD), negating the need to access individual level genotypes, greatly facilitating its generic applicability. We show that the method is thus robust to LD-associated bias and has equivalent performance to permutation-based methods, with a significantly shorter runtime. This is particularly relevant at a time of increasing public availability of significantly larger genetic data sets and should go a long way to assist in the rapid analysis of these data sets.

Genetic differences between five European populations.

AIMS: We sought to examine the magnitude of the differences in SNP allele frequencies between five European populations (Scotland, Ireland, Sweden, Bulgaria and Portugal) and to identify the loci with the greatest differences. METHODS: We performed a population-based genome-wide association analysis with Affymetrix 6.0 and 5.0 arrays. We used a 4 degrees of freedom χ(2) test to determine the magnitude of stratification for each SNP. We then examined the genes within the most stratified regions, using a highly conservative cutoff of p < 10(-45). RESULTS: We found 40,593 SNPs which are genome-wide significantly (p ≤ 10(-8)) stratified between these populations. The largest differences clustered in gene ontology categories for immunity and pigmentation. Some of the top loci span genes that have already been reported as highly stratified: genes for hair color and pigmentation (HERC2, EXOC2, IRF4), the LCT gene, genes involved in NAD metabolism, and in immunity (HLA and the Toll-like receptor genes TLR10, TLR1, TLR6). However, several genes have not previously been reported as stratified within European populations, indicating that they might also have provided selective advantages: several zinc finger genes, two genes involved in glutathione synthesis or function, and most intriguingly, FOXP2, implicated in speech development. CONCLUSION: Our analysis demonstrates that many SNPs show genome-wide significant differences within European populations and the magnitude of the differences correlate with the geographical distance. At least some of these differences are due to the selective advantage of polymorphisms within these loci.

Association between TCF4 and schizophrenia does not exert its effect by common nonsynonymous variation or by influencing cis-acting regulation of mRNA expression in adult human brain.

Large collaborative Genome-wide Association studies of schizophrenia have identified genes and genomic regions that are associated with the disorder at highly stringent levels of statistical significance. Among these, transcription factor 4 (TCF4) is one of the best supported although the associated SNP (rs9960767) is located within intron 3 and has no obvious function. Seeking the mechanism at TCF responsible for the association, we examined TCF4 for coding variants, and for cis regulated variation in TCF4 gene expression correlated with the associated SNP using an assay to detect differential allelic expression. Using data from the 1000 genomes project, we were unable to identify any nonsynonymous coding variants at the locus. Allele specific expression analysis using human post mortem brain samples revealed no evidence for cis-regulated mRNA expression related to genotype at the schizophrenia associated SNP. We conclude that association between schizophrenia and TCF4 is not mediated by a relatively common non-synonymous variant, or by a variant that alters mRNA expression as measured in adult human brain. It remains possible that the risk allele at this locus exerts effects on expression exclusively in a developmental context, in cell types or brain regions not adequately represented in our analysis, or through post-transcriptional effects, for example in the abundance of the protein or its sub-cellular distribution.

No evidence that extended tracts of homozygosity are associated with Alzheimer's disease.

We sought to investigate the contribution of extended runs of homozygosity in a genome-wide association dataset of 1,955 Alzheimer's disease cases and 955 elderly screened controls genotyped for 529,205 autosomal single nucleotide polymorphisms. Tracts of homozygosity may mark regions inherited from a common ancestor and could reflect disease loci if observed more frequently in cases than controls. We found no excess of homozygous tracts in Alzheimer's disease cases compared to controls and no individual run of homozygosity showed association to Alzheimer's disease.

Strong evidence that GNB1L is associated with schizophrenia.

Evidence that a gene or genes on chromosome 22 is involved in susceptibility to schizophrenia comes from two sources: the increased incidence of schizophrenia in individuals with 22q11 deletion syndrome (22q11DS) and genetic linkage studies. In mice, hemizygous deletion of either Tbx1 or Gnb1l can cause deficits in pre-pulse inhibition, a sensory motor gating defect which is associated with schizophrenia. We tested the hypothesis that variation at this locus confers risk of schizophrenia and related disorders in a series of case-control association studies. First, we found evidence for a male-specific genotypic association (P = 0.00017) TBX1/GNB1L in 662 schizophrenia cases and 1416 controls from the UK. Moreover, we replicated this finding in two independent case-control samples (additional 746 cases and 1330 controls) (meta analysis P = 1.8 x 10(-5)) and also observed significant evidence for genotypic association in an independent sample of 480 schizophrenia parent-proband trios from Bulgaria with markers at this locus, which was again strongest in the male probands (P = 0.004). Genotyping the most significant SNPs in a sample of 83 subjects with 22q11DS with and without psychosis again revealed a significant allelic association with psychosis in males with 22q11DS (P = 0.01). Finally, using allele specific expression analysis, we have shown that the markers associated with psychosis are also correlated with alterations in GNB1L expression, raising the hypothesis that the risk to develop psychosis at this locus could be mediated in a dose sensitive manner via gene expression. However, other explanations are possible, and further analyses will be required to clarify the correct functional mechanism.

Variation at the GABAA receptor gene, Rho 1 (GABRR1) associated with susceptibility to bipolar schizoaffective disorder.

We have previously reported evidence that variation at GABA(A) receptor genes is associated with susceptibility to bipolar disorder with schizophrenia-like psychotic features (Research Diagnostic Criteria (RDC) schizoaffective disorder, bipolar type) with gene-wide significance at GABRB1, GABRA4, GABRB3, GABRA5, and GABRR3. Here we provide suggestive evidence implicating a sixth member of the gene family, GABRR1 (gene-wide P = 0.0058; experiment-wide corrected significance P = 0.052).

The APC Variant p.Glu1317Gln predisposes to colorectal adenomas by a novel mechanism of relaxing the target for tumorigenic somatic APC mutations.

Multiple rare nonsynonymous variants in APC predispose to colorectal adenomas. The mechanisms through which such variants act have been unclear, but it has been proposed that a specific ("just-right") level of beta-catenin signaling is required for colorectal tumorigenesis. This appears to be mediated by selection for APC genotypes that retain one, or rarely two, 20 amino acid beta-catenin downregulating repeats (20AARs). We investigated the mechanism through which the variant p.Glu1317Gln (c.3949G>C) contributes to colorectal tumorigenesis. We compared the patterns of somatic APC mutations in tumors from patients with attenuated familial adenomatous polyposis (AFAP) who did, or did not, coinherit p.Glu1317Gln with their AFAP-causing APC mutations. Only 8.2% (4/49) of tumors carrying p.Glu1317Gln had somatic mutations predicted to result in mutant polypeptides retaining a single 20AAR, compared to 62.1% (36/58) of those which did not carry this variant (P=5.64 x 10(-9)). Furthermore, tumors with p.Glu1317Gln often carried somatic mutations that were unusually early or late (downstream of the second 20AAR) in the APC open reading frame. These data support a novel mechanism in which p.Glu1317Gln in combination with other weak mutant APC alleles (generating polypepetides with zero, two, or three 20AARs) can provide the necessary growth advantage for colorectal tumorigenesis.

On multiple-testing correction in genome-wide association studies.

The interpretation of the results of large association studies encompassing much or all of the human genome faces the fundamental statistical problem that a correspondingly large number of single nucleotide polymorphisms markers will be spuriously flagged as significant. A common method of dealing with these false positives is to raise the significance level for the individual tests for association of each marker. Any such adjustment for multiple testing is ultimately based on a more or less precise estimate for the actual overall type I error probability. We estimate this probability for association tests for correlated markers and show that it depends in a nonlinear way on the significance level for the individual tests. This dependence of the effective number of tests is not taken into account by existing multiple-testing corrections, leading to widely overestimated results. We demonstrate a simple correction for multiple testing, which can easily be calculated from the pairwise correlation and gives far more realistic estimates for the effective number of tests than previous formulae. The calculation is considerably faster than with other methods and hence applicable on a genome-wide scale. The efficacy of our method is shown on a constructed example with highly correlated markers as well as on real data sets, including a full genome scan where a conservative estimate only 8% above the permutation estimate is obtained in about 1% of computation time. As the calculation is based on pairwise correlations between markers, it can be performed at the stage of study design using public databases.

Sex differences in symptom patterns of recurrent major depression in siblings.

The objectives of this study were to examine sex differences in depressive symptom patterns in 475 sib pairs with well-defined recurrent major depression and to test the hypotheses that (a) symptom patterns show higher intraclass correlations within same sex sib pairs versus mixed sex sib pairs; and (b) symptoms more associated with women, e.g. atypical depressive and anxiety symptoms, account for differences between male and female siblings within the same family. A total of 878 individuals, with a past history of at least two depressive episodes, were interviewed using the Schedules for Clinical Assessment in Neuropsychiatry interview and diagnosed according to DSM-IV using a computerized scoring program (CATEGO5). Intraclass correlations were compared between mixed and same sex sibs, and a conditional regression analysis in mixed sex sib pairs was performed to test whether specific symptoms account for differences between male and female siblings within the same family. Women showed a significantly earlier onset of depression compared with men (23.0 years, SD=10.6 versus 25.5, SD=12.5 years, P=0.0004), and a significantly greater frequency of several aspects of depressed mood was found in women compared with men, including atypical depressive features of fatiguability, appetite gain, weight gain and hypersomnia. Discordant sib-pair data analyses revealed five symptoms that accounted for the sex differences between siblings (P=.000035): phobia (exp(B)=2.04, P=0.017), hypersomnia (exp(B)=1.37, P=0.055), appetite loss (exp(B)=1.38, P=0.004) and appetite gain (exp(B)=2.19, P<0.001). Sex significantly modifies clinical features of depression and an earlier onset of depression and atypical depressive symptoms occur more frequently in women.

Association analysis of AKT1 and schizophrenia in a UK case control sample.

AKT1 (V-akt murine thyoma viral oncogene homolog 1) is involved in intracellular signalling pathways postulated as of aetiological importance in schizophrenia. Markers in the AKT1 gene have also recently been associated with schizophrenia in two samples of European origin and in Japanese and Iranian samples. Aiming to replicate these findings, we examined ten SNPs spanning AKT1 in a UK case-control sample (schizophrenia cases n=673, controls n=716). These included all SNPs previously reported to be associated in European, Japanese and Iranian samples, alone or in haplotypes, as well as additional markers defined by the Haploview Tagger program (pair-wise tagging, minimum r(2)=0.8, minor allele frequency=0.02). We found no association with single markers (min p=0.17). We found weak evidence for association (p=0.04) with a four marker haplotype reported as significant in the original positive European sample of Emamian et al. [Emamian, E.S., Hall, D., Birnbaum, M.J., Karayiorgou, M., Gogos, J.A., 2004. Convergent evidence for impaired AKT1-GSK3beta signaling in schizophrenia. Nat. Genet. 36, 131-137] and also an overlapping three marker haplotype (p=0.016) that had previously been reported as significant in a Japanese sample. Nominal p-values for these haplotypes did not survive correction for multiple testing. Our study provides at best weak support for the hypothesis that AKT1 is a susceptibility gene for schizophrenia. Examination of our own data and those of other groups leads us to conclude that overall, the evidence for association of AKT1 as a susceptibility gene for schizophrenia is weakly positive, but not yet convincing.

Convergent evidence for 2',3'-cyclic nucleotide 3'-phosphodiesterase as a possible susceptibility gene for schizophrenia.

CONTEXT: Convergent data make 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) a candidate gene for schizophrenia. Reduced expression has been reported in the schizophrenic brain. The CNP gene maps to a region to which we have reported linkage to schizophrenia. Mice in which the CNP gene has been knocked out display central nervous system pathological characteristics reminiscent of some features observed in schizophrenia. 2',3'-Cyclic nucleotide 3'-phosphodiesterase is used as a marker of myelin-forming cells and is detectable in cells of oligodendrocyte lineage throughout life. Because CNP is thought to be important for oligodendrocyte function, altered expression has been interpreted as supportive of the hypothesis that altered oligodendrocyte function may be an etiological factor in schizophrenia. However, it is unclear whether the observed changes in the schizophrenic brain are primary or secondary. OBJECTIVES: To determine if CNP expression is influenced by DNA polymorphisms and to verify if these polymorphisms are associated with schizophrenia. DESIGN: Allele-specific messenger RNA expression assay and genetic association studies. SETTING: Unrelated subjects were ascertained from secondary psychiatric inpatient and outpatient services. PARTICIPANTS: We used brain tissue from 60 anonymous individuals with no known psychiatric disorder; a case-control sample of 708 white individuals from the United Kingdom meeting DSM-IV criteria for schizophrenia matched for age, sex, and ethnicity to 711 blood donor controls; and a pedigree with DNA from 6 affected siblings and 1 parent, showing evidence for linkage to CNP. MAIN OUTCOME MEASURES: Association between allele and gene expression. Association between allele and schizophrenia. RESULTS: The exonic single nucleotide polymorphism rs2070106 was associated with CNP expression (P<.001). Compatible with underexpression of CNP messenger RNA in schizophrenia, the lower-expressing A allele was significantly associated with schizophrenia (P = .04) in the case-control sample. All affected individuals in the linked pedigree were homozygous for the lower-expression allele, providing independent support for the association (P = .03). CONCLUSIONS: Our data support the hypothesis that reduced CNP expression in the schizophrenic brain is relevant to disease etiology and therefore provide support for the general hypothesis that altered oligodendrocyte function is an etiological factor in schizophrenia.

Assessment of the relationship between pre-chip and post-chip quality measures for Affymetrix GeneChip expression data.

BACKGROUND: Gene expression microarray experiments are expensive to conduct and guidelines for acceptable quality control at intermediate steps before and after the samples are hybridised to chips are vague. We conducted an experiment hybridising RNA from human brain to 117 U133A Affymetrix GeneChips and used these data to explore the relationship between 4 pre-chip variables and 22 post-chip outcomes and quality control measures. RESULTS: We found that the pre-chip variables were significantly correlated with each other but that this correlation was strongest between measures of RNA quality and cRNA yield. Post-mortem interval was negatively correlated with these variables. Four principal components, reflecting array outliers, array adjustment, hybridisation noise and RNA integrity, explain about 75% of the total post-chip measure variability. Two significant canonical correlations existed between the pre-chip and post-chip variables, derived from MAS 5.0, dChip and the Bioconductor packages affy and affyPLM. The strongest (CANCOR 0.838, p < 0.0001) correlated RNA integrity and yield with post chip quality control (QC) measures indexing 3'/5' RNA ratios, bias or scaling of the chip and scaling of the variability of the signal across the chip. Post-mortem interval was relatively unimportant. We also found that the RNA integrity number (RIN) could be moderately well predicted by post-chip measures B_ACTIN35, GAPDH35 and SF. CONCLUSION: We have found that the post-chip variables having the strongest association with quantities measurable before hybridisation are those reflecting RNA integrity. Other aspects of quality, such as noise measures (reflecting the execution of the assay) or measures reflecting data quality (outlier status and array adjustment variables) are not well predicted by the variables we were able to determine ahead of time. There could be other variables measurable pre-hybridisation which may be better associated with expression data quality measures. Uncovering such connections could create savings on costly microarray experiments by eliminating poor samples before hybridisation.

Pooled DNA genotyping on Affymetrix SNP genotyping arrays.

BACKGROUND: Genotyping technology has advanced such that genome-wide association studies of complex diseases based upon dense marker maps are now technically feasible. However, the cost of such projects remains high. Pooled DNA genotyping offers the possibility of applying the same technologies at a fraction of the cost, and there is some evidence that certain ultra-high throughput platforms also perform with an acceptable accuracy. However, thus far, this conclusion is based upon published data concerning only a small number of SNPs. RESULTS: In the current study we prepared DNA pools from the parents and from the offspring of 30 parent-child trios that have been extensively genotyped by the HapMap project. We analysed the two pools with Affymetrix 10 K Xba 142 2.0 Arrays. The availability of the HapMap data allowed us to validate the performance of 6843 SNPs for which we had both complete individual and pooled genotyping data. Pooled analyses averaged over 5-6 microarrays resulted in highly reproducible results. Moreover, the accuracy of estimating differences in allele frequency between pools using this ultra-high throughput system was comparable with previous reports of pooling based upon lower throughput platforms, with an average error for the predicted allelic frequencies differences between the two pools of 1.37% and with 95% of SNPs showing an error of < 3.2%. CONCLUSION: Genotyping thousands of SNPs with DNA pooling using Affymetrix microarrays produces highly accurate results and can be used for genome-wide association studies.

Inhibition of cortisol production with metyrapone prevents mental stress-induced endothelial dysfunction and baroreflex impairment.

OBJECTIVES: This study was designed to investigate the role of cortisol in stress-induced endothelial dysfunction and impaired baroreflex sensitivity (BRS) by blocking cortisol production with metyrapone before subjecting healthy volunteers to mental stress. BACKGROUND: Mental stress raises cortisol levels and is associated with increased coronary heart disease (CHD) morbidity and mortality, especially from sudden cardiac death. It also causes endothelial dysfunction and impaired BRS. METHODS: We measured brachial artery flow-mediated dilation (FMD), a measure of endothelial function, and BRS in 36 subjects without CHD risk factors who were then randomized in a double-blind fashion to oral metyrapone 750 mg x 2 or placebo. Five hours later we subjected subjects to mental stress and then remeasured endothelial function and BRS. RESULTS: Prestress cortisol levels were significantly higher in the placebo group at 270.5 (30.9) nmol/l versus 89.1 (11.8) nmol/l (p = 0.01), and the increase with stress was higher at 57.9 (17.9) nmol/l versus 11.2 (2.2) nmol/l (p < 0.001). In the placebo group, compared to baseline, FMD and BRS fell significantly from 4.5% (0.7%) to 1.4% (1.1%) (p = 0.02) and 21.4 (2.3) ms/mmHg to 16.3 (1.5) ms/mmHg (p = 0.04), respectively. In the metyrapone group, FMD and BRS were unchanged from baseline: 4.3% (0.9%) versus 5.1% (0.8%) (p = 0.48) and 26.4 (2.9) ms/mmHg versus 24.9 (2.6) ms/mmHg (p = 0.62), respectively. Analysis of covariation showed a significant effect of metyrapone on change in both FMD (p = 0.009) and BRS (p = 0.024). CONCLUSIONS: Stress-related endothelial dysfunction and BRS impairment can be prevented by blocking cortisol production with metyrapone, demonstrating a direct or facilitative role for cortisol in these phenomena and suggesting mechanisms by which stress contributes to CHD and sudden cardiac death.