High Resolution T2 Association Tests of Complex Diseases Based on Family Data

This paper proposes family based Hotelling's T² tests for high resolution linkage disequilibrium (LD) mapping or association studies of complex diseases. Assume that genotype data of multiple markers or haplotype blocks are available for a sample of nuclear families, in which some offspring are affected. Paired Hotelling's T² test statistics are proposed for a high resolution association study using parents as controls for affected offspring, based on two coding methods: haplotype/allele coding and genotype coding. The paired Hotelling's T² tests take not only the correlation between the haplotype blocks or markers into account, but also take the correlation within each parent‐offspring pair into account. The method extends two sample Hotelling's T² test statistics for population case control association studies, which are not valid for family data due to correlation of genetic data among family members. The validity of the proposed method is justified by rigorous mathematical and statistical proof under the large sample theory. The non‐centrality parameter approximations of the test statistics are calculated for power and sample size calculations. From power comparison and type I error calculations, it is shown that the test statistic based on haplotype/allele coding is advantageous over the test statistic of genotype coding. Analysis using multiple markers may provide higher power than single marker analysis. If only one marker is utilized the power of the test statistic based on haplotype/allele coding is nearly identical to that of 1‐TDT. Moreover, a permutation procedure is provided for data analysis. The method is applied to data from a German asthma family study. The results based on the paired Hotelling's T² statistic tests confirm the previous findings. However, the paired Hotelling's T² tests produce much smaller P‐values than those of the previous study. The permutation tests produce similar results to those of the previous study; moreover, additional marker combinations are shown to be significant by permutation tests. The proposed paired Hotelling's T² statistic tests are potentially powerful in mapping complex diseases. A SAS Macro, Hotel_fam.sas, has been written to implement the method for data analysis.     

Association of RGS2 variants with panic disorder in a Japanese population

Panic disorder (PD) is a severe and chronic psychiatric disorder with significant genetic components underlying its etiology. The gene regulator of G protein signaling 2 (RGS2) has been reported to be associated with anxiety disorders. To confirm the association of RGS2 with PD, we investigated three single nucleotide polymorphisms (SNPs) of RGS2 (rs10801152, rs4606, and rs1819741) in 677 Japanese PD cases and 460 controls. The SNP rs10801152 was suggestive of an association with PD (allele P = 0.045 adjusted using sex and age as confounding factors). The three‐SNP haplotype was significantly associated with PD (global permutation P = 4 × 10^?4). The haplotypes T‐G‐C and T‐C‐T showed significant association and protective effect on PD (T‐G‐C, permutation P = 0.038, OR = 0.80, 95%CI = 0.68–0.95; T‐C‐T, permutation P = 0.004, OR = 0.38, 95%CI = 0.21–0.70). These results provide support for an association of RGS2 with PD in a Japanese population. © 2011 Wiley‐Liss, Inc.     

Rank‐Based Tests for Identifying Multiple Genetic Variants Associated with Quantitative Traits

We consider the analysis of multiple genetic variants within a gene or a region that are expected to confer risks to human complex diseases with quantitative traits, where the trait values do not follow the normal distribution even after some transformations. We rank the phenotypic values, calculate a score to measure the trend effect of a particular allele for each marker, and then construct three statistics based on the quadratic frameworks of methods Hotelling T², the summation of squared univariate statistic and the inverse of the square root weighted statistics to combine the scores for different marker loci. Simulation results show that the above three test statistics can control the type I error rate well and are more robust than standard tests constructed based on linear regression. Application to GAW16 data for rheumatoid arthritis successfully detects the association between the HLA‐DRB1 gene and anticyclic citrullinated protein measure, while the standard methods based on normal assumption cannot detect this association.     

On Sample Size and Power Calculation for Variant Set‐Based Association Tests

Sample size and power calculations are an important part of designing new sequence‐based association studies. The recently developed SEQPower and SPS programs adopted computationally intensive Monte Carlo simulations to empirically estimate power for a series of variant set association (VSA) test methods including the sequence kernel association test (SKAT). It is desirable to develop methods that can quickly and accurately compute power without intensive Monte Carlo simulations. We will show that the computed power for SKAT based on the existing analytical approach could be inflated especially for small significance levels, which are often of primary interest for large‐scale whole genome and exome sequencing projects. We propose a new χ²‐approximation‐based approach to accurately and efficiently compute sample size and power. In addition, we propose and implement a more accurate “exact” method to compute power, which is more efficient than the Monte Carlo approach though generally involves more computations than the χ² approximation method. The exact approach could produce very accurate results and be used to verify alternative approximation approaches. We implement the proposed methods in publicly available R programs that can be readily adapted when planning sequencing projects.     

Influence of population stratification on population‐based marker‐disease association analysis

Population‐based genetic association analysis may suffer from the failure to control for confounders such as population stratification (PS). There has been extensive study on the influence of PS on candidate gene‐disease association analysis, but much less attention has been paid to its influence on marker‐disease association analysis. In this paper, we focus on the Pearson χ² test and the trend test for marker‐disease association analysis. The mean and variance of the test statistics are derived under presence of PS, so that the power and inflated type I error rate can be evaluated. It is shown that the bias and the variance distortion are not zero in the presence of both PS and penetrance heterogeneity (PH). Unlike candidate gene‐disease association analysis, when PS is present, the bias is not zero no matter whether PH is present or not. This work generalises the published results, where only the fully recessive penetrance model is considered and only the bias is calculated. It is shown that candidate gene‐disease association analysis can be treated as a special case of marker‐disease association analysis. Consequently, our results extend previous studies on candidate gene‐disease association analysis. A simulation study confirms the theoretical findings.     

Novel sequences and epitopes of diagnostic value derived from the Anisakis simplex Ani s 7 major allergen

Background: Anisakis simplex allergens may cause severe allergic reactions in infected patients. Human anisakiasis can be specifically diagnosed by detection of immunoglobulin E (IgE) antibodies against O‐deglycosylated nAni s 7 allergen captured by monoclonal antibody (mAb) UA3 (UA3‐ELISA), although the nature of this important allergen is unknown. The aim of this study was to clone and characterize the Ani s 7 major allergen, and to obtain a recombinant fragment suitable for serodiagnosis. Methods: An Anisakis cDNA library was screened with mAb UA3 and a cDNA clone (rAni s 7) encoding a 1096‐amino acid fragment of Ani s 7 (GenBank: EF158010 ) was identified. Bioinformatic tools and immunological and biochemical techniques were used to characterize the allergen obtained. Results: The rAni s 7 fragment comprised 19 repeats of a novel CX_17?25CX_9?22CX₈CX₆ tandem repeat motif not seen in any previously reported protein sequence. An internal ⁴³⁵Met–⁷¹³Arg fragment of the rAni s 7 (t‐Ani s 7) was expressed in Escherichia coli and evaluated for serodiagnostic utility. Indirect enzyme‐linked immunosorbent assay (ELISA) with t‐Ani s 7 identified as positive the same 60 sera as UA3‐ELISA. The sequence MCQCVQKYGTEFCKKRLA from rAni s 7 was identified as the epitope recognized by mAb UA3, and is the target for over 60% of human IgE antibodies that react with O‐deglycosylated nAni s 7. Conclusions: In addition to their clear value for serodiagnosis of human anisakiasis, the nature of the novel sequences and epitopes identified in the Ani s 7 allergen are of interest for a better understanding of the mechanisms operating in Anisakis‐induced allergy.     

A Likelihood Ratio Test for Genome‐Wide Association under Genetic Heterogeneity

Most existing association tests for genome‐wide association studies (GWASs) fail to account for genetic heterogeneity. Zhou and Pan proposed a binomial‐mixture‐model‐based association test to account for the possible genetic heterogeneity in case‐control studies. The idea is elegant, however, the proposed test requires an expectation‐maximization (EM)‐type iterative algorithm to identify the penalised maximum likelihood estimates and a permutation method to assess p‐values. The intensive computational burden induced by the EM‐algorithm and the permutation becomes prohibitive for direct applications to GWASs. This paper develops a likelihood ratio test (LRT) for GWASs under genetic heterogeneity based on a more general alternative mixture model. In particular, a closed‐form formula for the LRT statistic is derived to avoid the EM‐type iterative numerical evaluation. Moreover, an explicit asymptotic null distribution is also obtained, which avoids using the permutation to obtain p‐values. Thus, the proposed LRT is easy to implement for GWASs. Furthermore, numerical studies demonstrate that the LRT has power advantages over the commonly used Armitage trend test and other existing association tests under genetic heterogeneity. A breast cancer GWAS dataset is used to illustrate the newly proposed LRT.     

Dynamic intravoxel incoherent motion imaging of skeletal muscle at rest and after exercise

The purpose of this work was to demonstrate the feasibility of intravoxel incoherent motion imaging (IVIM) for non‐invasive quantification of perfusion and diffusion effects in skeletal muscle at rest and following exercise. After IRB approval, eight healthy volunteers underwent diffusion‐weighted MRI of the forearm at 3 T and eight different b values between 0 and 500 s/mm² with a temporal resolution of 57 s per dataset. Dynamic images were acquired before and after a standardized handgrip exercise. Diffusion (D) and pseudodiffusion (D*) coefficients as well as the perfusion fraction (F_P) were measured in regions of interest in the flexor digitorum superficialis and profundus (FDS/FDP), brachioradialis, and extensor carpi radialis longus and brevis muscles by using a multi‐step bi‐exponential analysis in MATLAB. Parametrical maps were calculated voxel‐wise. Differences in D, D*, and F_P between muscle groups and between time points were calculated using a repeated measures analysis of variance with post hoc Bonferroni tests. Mean values and standard deviations at rest were the following: D*, 28.5 ± 11.4 × 10^?3 mm²/s; F_P, 0.03 ± 0.01; D, 1.45 ± 0.09 × 10^?3 mm²/s. Changes of IVIM parameters were clearly visible on the parametrical maps. In the FDS/FDP, D* increased by 289 ± 236% (p < 0.029), F_P by 138 ± 58% (p < 0.01), and D by 17 ± 9% (p < 0.01). A significant increase of IVIM parameters could also be detected in the brachioradialis muscle, which however was significantly lower than in the FDS/FDP. After 20 min, all parameters were still significantly elevated in the FDS/FDP but not in the brachioradialis muscle compared with the resting state. The IVIM approach allows simultaneous quantification of muscle perfusion and diffusion effects at rest and following exercise. It may thus provide a useful alternative to other non‐invasive methods such as arterial spin labeling. Possible fields of interest for this technique include perfusion‐related muscle diseases, such as peripheral arterial occlusive disease. Copyright © 2014 John Wiley & Sons, Ltd.     

A Comparison of the Likelihood Ratio Test and the Variance‐Stabilising Transformation‐Based Tests for Detecting Association of Rare Variants

In a recent paper in this journal, the use of variance‐stabilising transformation techniques was proposed to overcome the problem of inadequacy in normality approximation when testing association for a low‐frequency variant in a case‐control study. It was shown that tests based on the variance‐stabilising transformations are more powerful than Fisher's exact test while controlling for type I error rate. Earlier in the journal, another study had shown that the likelihood ratio test (LRT) is superior to Fisher's exact test, Wald's test, and Pearson's χ² test in testing association for low‐frequency variants. Thus, it is of interest to make a direct comparison between the LRT and the tests based on the variance‐stabilising transformations. In this commentary, we show that the LRT and the variance‐stabilising transformation‐based tests have comparable power greater than Fisher's exact test, Wald's test, and Pearson's χ² test.     

A Comparison of Case-Control and Family-Based Association Methods: The Example of Sickle-Cell and Malaria

There has been much debate about the relative merits of population‐ and family‐based strategies for testing genetic association, yet there is little empirical data that directly compare the two approaches. Here we compare case‐control and transmission/disequilibrium test (TDT) study designs using a well‐established genetic association, the protective effect of the sickle‐cell trait against severe malaria. We find that the two methods give similar estimates of the level of protection (case‐control odds ratio = 0.10, 95% confidence interval 0.03–0.23; family‐based estimate of the odds ratio = 0.11, 95% confidence interval 0.04–0.25) and similar statistical significance of the result (case‐control: χ²= 41.26, p= 10^?10 , TDT: χ²= 39.06, p= 10^?10 ) when 315 TDT cases are compared to 583 controls. We propose a family plus population control study design, which allows both case‐control and TDT analysis of the cases. This combination is robust against the respective weaknesses of the case‐control and TDT study designs, namely population structure and segregation distortion. The combined study design is especially cost‐effective when cases are difficult to ascertain and, when the case‐control and TDT results agree, offers greater confidence in the result.     

A novel bayesian approach with conditional autoregressive specification for intravoxel incoherent motion diffusion‐weighted MRI

The Intra‐Voxel Incoherent Motion (IVIM) model is largely adopted to estimate slow and fast diffusion coefficients of water molecules in biological tissues, which are used in cancer applications. The most reported fitting approach is a voxel‐wise segmented non‐linear least square, whereas Bayesian approaches with a direct fit, also considering spatial regularization, were proposed too. In this work a novel segmented Bayesian method was proposed, also in combination with a spatial regularization through a Conditional Autoregressive (CAR) prior specification. The two segmented Bayesian approaches, with and without CAR specification, were compared with two standard least‐square and a direct Bayesian fitting methods. All approaches were tested on simulated images and real data of patients with head‐and‐neck and rectal cancer. Estimation accuracy and maps noisiness were quantified on simulated images, whereas the coefficient of variation and the goodness of fit were evaluated for real data. Both versions of the segmented Bayesian approach outperformed the standard methods on simulated images for pseudo‐diffusion (D^?) and perfusion fraction (f), whilst the segmented least‐square fitting remained the less biased for the diffusion coefficient (D). On real data, Bayesian approaches provided the less noisy maps, and the two Bayesian methods without CAR generally estimated lower values for f and D^? coefficients with respect to the other approaches. The proposed segmented Bayesian approaches were superior, in terms of estimation accuracy and maps quality, to the direct Bayesian model and the least‐square fittings. The CAR method improved the estimation accuracy, especially for D^?.     

Multilocus analysis reveals three candidate genes for Chinese migraine susceptibility

Several genome‐wide association studies (GWASs) in Caucasian populations have identified 12 loci that are significantly associated with migraine. More evidence suggests that serotonin receptors are also involved in migraine pathophysiology. In the present study, a case–control study was conducted in a cohort of 581 migraine cases and 533 ethnically matched controls among a Chinese population. Eighteen polymorphisms from serotonin receptors and GWASs were selected, and genotyping was performed using a Sequenom MALDI‐TOF mass spectrometry iPLEX platform. The genotypic and allelic distributions of MEF2D rs2274316 and ASTN2 rs6478241 were significantly different between migraine patients and controls. Univariate and multivariate analysis revealed significant associations of polymorphisms in the MEF2D and ASTN2 genes with migraine susceptibility. MEF2D, PRDM16 and ASTN2 were also found to be associated with migraine without aura (MO) and migraine with family history. And, MEF2D and ASTN2 also served as genetic risk factors for the migraine without family history. The generalized multifactor dimensionality reduction analysis identified that MEF2D and HTR2E constituted the two‐factor interaction model. Our study suggests that the MEF2D, PRDM16 and ASTN2 genes from GWAS are associated with migraine susceptibility, especially MO, among Chinese patients. It appears that there is no association with serotonin receptor related genes.     

The mean measure of divergence: Its utility in model‐free and model‐bound analyses relative to the Mahalanobis D2 distance for nonmetric traits

The mean measure of divergence (MMD) distance statistic has been used by researchers for nearly 50 years to assess inter‐sample phenetic affinity. Its widespread and often successful use is well documented, especially in the study of cranial and dental nonmetric traits. However, the statistic has accumulated some undesired mathematical baggage through the years from various workers in their attempts to improve or alter its performance. Others may not fully understand how to apply the MMD or interpret its output, whereas some described a number of perceived shortcomings. As a result, the statistic and its sometimes flawed application(s) have taken several well‐aimed hits; a few researchers even argued that it should no longer be utilized or, at least, that its use be reevaluated. The objective of this report is to support the MMD, and in the process: (1) provide a brief history of the statistic, (2) review its attributes and applicability relative to the often‐used Mahalanobis D² statistic for nonmetric traits, (3) compare results from MMD and D² model‐free analyses of previously‐recorded sub‐Saharan African dental samples, and (4) investigate its utility for model‐bound analyses. In the latter instance, the ability of the D² and other squared Euclidean‐based statistics to approximate a genetic relationship matrix and Sewall Wright's fixation index using phenotypic data, and the inability of the MMD to do so, is addressed. Three methods for obtaining such results with nonlinear MMD distances, as well as an assessment of the fit of the isolation‐by‐distance model, are presented. Am. J. Hum. Biol., 2010. © 2009 Wiley‐Liss, Inc.     

Age-dependence of the lateral diffusion coefficient of Concanavalin-A receptors in the plasma membrane of ex vivo prepared brain cortical nerve cells of BN/BiRijHsd rats

A new method has been developed for ex vivo preparation of brain cortical cells of BN/BiRijHsd rats to make them suitable for the measurement of the lateral diffusion coefficient of the membrane components by means of fluorescence recovery after photobleaching (FRAP). The method involves chopping the brain cortex into pieces of less than 1 mm. These parts are stained with a fluorescent label (e.g., concanavalin-A-fluorescein, Con-A-FL conjugate) and then gently pressed onto a microscope slide using the coverslip. In the resulting specimen, the largest cells of the cortex can be recognized in phase-contrast image, sufficiently stained by the label and ready for the FRAP measurement. The lateral diffusion coefficient of Con-A-receptor proteins (D _p) was measured in such brain cell preparations of 15 female rats in four age groups (5.6–31.8 months) and 11 males in three age groups (13.8–31.8 months). Highly significant negative, linear age correlation of D _p (R=−0.9958 in females, and −0.9956 in males) were found, the regression equations being D _p,=(8.8311–0.1425 X)⁻¹⁰ and D _p█=(9.3240−0.1630 X)⁻¹⁰ cm²/s, respectively, where X is age in months. The data confirm that the lateral mobility of plasma membrane proteins represents an important biomarker of cellular aging in the brain cortical cells of BN/BiRijHsd rats. Received: 22 June 1998 / Accepted: 24 September 1998     

The Scaling Law between Molecular Mass and Diffusivity and its Influence on the Molecular Weight Distribution as Derived from a Stretched Exponential PGSTE NMR Response Curve

The molecular weight distribution characteristics derived from an inverse Laplace transformation (ILT) of a stretched exponential PGSTE NMR response function (SEF) is discussed with reference to the scaling law between diffusivity (D) and molecular mass (M) (D = KM^?α). Two general equations are presented, enabling the polydispersity index (PDI) and the average molecular weights (M _w, M _n, and M_med) to be determined from α and the two characteristic parameters β and D₀ of the (normalized) SEF (= exp [(?D₀t)^β]). A detailed discussion regarding the derivation of the two equations is presented.     

Lipopolysaccharide induces proinflammatory cytokines and chemokines in experimental otitis media through the prostaglandin D2 receptor (DP)-dependent pathway

Otitis media is one of the most common and intractable ear diseases, and is the major cause of hearing loss, especially in children. Multiple factors affect the onset or development of otitis media. Prostaglandin D₂ is the major prostanoid involved in infection and allergy. However, the role of prostaglandin D₂ and prostaglandin D2 receptors on the pathogenesis of otitis media remains to be determined. Recent studies show that D prostanoid receptor (DP) and chemoattractant receptor‐homologous molecule expressed on T helper type 2 (Th2) cells (CRTH2) are major prostaglandin D₂ receptors. In this study, homozygous DP single gene‐deficient (DP^–/–) mice, CRTH2 single gene‐deficient (CRTH2^–/–) mice and DP/CRTH2 double gene‐deficient (DP^–/– CRTH2^–/–) mice were used to investigate the role of prostaglandin D₂ and its receptors in otitis media. We demonstrate that prostaglandin D₂ is induced by lipopolysaccharide (LPS), a major component of Gram‐negative bacteria, and that transtympanic injection of prostaglandin D₂ up‐regulates macrophage inflammatory protein 2 (MIP‐2), interleukin (IL)‐1β and IL‐6 in the middle ear. We also show that middle ear inflammatory reactions, including infiltration of inflammatory cells and expression of MIP‐2, IL‐1β and IL‐6 induced by LPS, are reduced significantly in DP^–/– mice and DP^–/– CRTH2^–/– mice. CRTH2^–/– mice display inflammatory reactions similar to wild‐type mice. These findings indicate that prostaglandin D₂ may play significant roles in LPS‐induced experimental otitis media via DP.     

A polymorphism in the promoter region of the CD86 (B7.2) gene is associated with systemic sclerosis

Systemic sclerosis (SSc) is a connective tissue disease of unknown aetiology characterized by fibrosis of the skin and internal organs, vascular abnormalities and humoral autoimmunity. Strong T‐cell‐dependent autoantibody and HLA associations are found in SSc subsets. The co‐stimulatory molecule, CD86, expressed by antigen‐presenting cells, plays a crucial role in priming naïve lymphocytes. We hypothesized that SSc, or one of the disease subsets, could be associated with single‐nucleotide polymorphisms of the CD86 gene. Using sequence specific primer‐polymerase chain reaction (SSP‐PCR) methodology, we assessed four CD86 polymorphisms in 221 patients with SSc and 227 healthy control subjects from the UK. Haplotypes were constructed by inference and confirmed using PHASE algorithm. We found a strong association between SSc and a specific haplotype (haplotype 5), which was more prevalent in patients than in controls (29% vs 15%, OR = 2.3, χ² = 12, P = 0.0005). This association could be attributed to the novel ?3479 promoter polymorphism; a significant difference was observed in the distribution of the CD86 ?3479 G allele in patients with SSc compared to controls (43.7% vs. 32.4%, OR = 1.7, χ² = 12.1, P = 0.0005). TRANSFAC analyses suggest that the CD86‐3479T allele contains putative GATA and TBP sites, whereas G allele does not. We assessed the relative DNA protein‐binding activity of the ?3479 polymorphism in vitro using electromobility gel shift assays (EMSA), which showed that the ?3479G allele has less binding affinity compared to the T allele for nuclear proteins. These findings highlight the importance of co‐stimulatory pathways in SSc pathogenesis.     

Statistical methods for assessing linkage disequilibrium at the HLA-A, B, C loci

A number of standard and new statistics for measuring linkage disequilibrium are introduced applicable to any multilocus system. Included are the usual pairwise gametic disequilibrium function, D, the Lewontin disequilibrium index, D_L, a Euclidean disequilibrium distance, D_E, a hierarchy of stratified linkage disequilibrium functions (e.g. a pairwise disequilibrium value conditioned on the value of a third locus) and an averaged conditional disequilibrium expression, D_W. Various global second order measures of disequilibrium are proposed partly based on contingency table statistics and weighted pairwise disequilibrium quantities. A non-parametric (stochastic) comparison assessment for global linkage disequilibrium is also developed. These measures are compared and contrasted at the HLA-A, B, C gene complex for a sample of 2000 haplotypes from a healthy Austrian population. Several results from applying these methods include: (1) Of the various pairwise disequilibrium measures examined, D, D_E and D_W correlated very closely with each other but differ from D_L. (2) The ‘third-order interaction’ between two loci conditional on an allele (or group of alleles) at a third locus indicated that HLA-AB maintains the classical disequilibrium pairings only for conditioning on C_X (the blank allele at locus C), and they mostly disappear for conditioning on C₁ to C₅. (3) Subpopulations involving C₃ or C₄ alleles exhibit the significant new combinations A₃₃B₁₇ and A₂B₁₅. (4) The B, C loci have a higher total linkage disequilibrium than A, B and A, C; more than expected by mapping distance relationships. (5) The total linkage disequilibrium was significantly larger for conditioning on C_X compared to C_X (non-C_X), but smaller for the population conditioned by {C₃, C₄}. A number of interpretations of the results with respect to heterogeneity classifications of populations are discussed.     

The collecting duct,dopamine and vasopressin‐dependent hypertension

AVP not only increases osmotic water permeability (P_f) in the rat cortical collecting duct (CCD), but also acts synergistically with aldosterone to augment sodium reabsorption (J_Na). These effects are inhibited by catecholamines via α₂ adrenergic receptors, and by dopamine. We review here studies designed to determine the mechanism and receptor involved in dopamine action. The inhibitory effect of dopamine on Na⁺ and water transport was found to be reversible, and was not produced by agonists specific to D_1A and D_1B receptors. D₂‐type (D₂, D₃ or D₄) receptors and activation of the GTP‐binding protein G_i were implicated by the observation that dopamine had no inhibitory effect when J_Na and P_f were stimulated by a cyclic AMP analogue plus isobutylmethylxanthine. The only dopaminergic antagonist that reversed the inhibitory effect of dopamine was clozapine, which is relatively D₄‐specific. We also found that dopamine or D₁‐specific agonists by themselves had no effect on cAMP production. However, dopamine inhibited the high rate of AVP‐dependent cAMP production, and this effect of dopamine was reversed by clozapine but not other antagonists or by inhibitors of protein kinase C. The D₄ receptor was observed in western blots of renal cortical proteins, and it was localized to the collecting duct by RT‐PCR and immuno‐histochemistry using a D₄‐specific antibody. These results show that at least a portion of the natriuretic effect of dopamine can be attributed to inhibition of AVP‐dependent Na⁺ reabsorption by the CCD, and they introduce another signalling system as a candidate in the aetiology of low‐renin, salt‐dependent hypertension.