Genome-wide linkage analysis of chromogranin B expression in the CEPH pedigrees: implications for exocytotic sympathochromaffin secretion in humans

Tiffany A. Greenwood2,4, Peter E. Cadman1,5, Mats Stridsberg6, Susan Nguyen1,5, Laurent Taupenot1,5, Nicholas J. Schork2,4 and Daniel T. O’Connor1,3,4,5

1 Department of Medicine, University of California at San Diego
2 Department of Psychiatry, University of California at San Diego
3 Center for Molecular Genetics, University of California at San Diego
4 Polymorphism Research Laboratory, University of California at San Diego
5 Veterans Affairs San Diego Healthcare System, San Diego, California 92161
6 Department of Medical Sciences, Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chromogranin B (CgB), a major member of the chromogranin/secretogranin family of catecholamine storage vesicle secretory proteins, plays both intracellular (vesiculogenic) and extracellular (prohormone) roles in the neuroendocrine system, and its biosynthesis and release are under the control of efferent sympathetic nerve traffic ("stimulus-transcription coupling"). To explore the role of heredity in control of CgB, we conducted a genome-wide linkage analysis of CgB release in 12 extended CEPH (Centre d’Etude du Polymorphisme Humain) pedigrees. Region-specific radioimmunoassays were used to measure five CgB fragments in plasma: CgB1–16, CgB312–331, CgB439–451, CgB568–577, and CgB647–657. Substantial heritability, as measured by h2r, was observed for three of the fragment concentrations, CgB312–331, CgB439–451, and CgB568–577, which yielded h2r estimates ranging from 0.378 (P = 0.002) to 0.910 (P < 0.0000001). Variance-component genome-wide linkage analysis with 654 microsatellite markers at ~5 cM spacing identified a major quantitative trait locus for CgB312–331 on chromosome 11q24-q25 with a maximum multipoint LOD score of 5.84. Significant allelic associations between markers in the region and CgB levels were also observed. Although the 2-LOD confidence interval for linkage did not include the CgB locus itself, known trans-activators of the CgB gene promoter, or prohormone cleaving proteases, examination of positional candidate loci within this region yielded novel and plausible physiological candidates for further exploration. Allelic variation in this region may thus influence effects of sympathetic outflow on target organs in humans.

chromogranin B; CHGB; chromaffin; catecholamine


    INTRODUCTION
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE CHROMOGRANINS/SECRETOGRANINS comprise a family of acidic, soluble proteins that are stored in secretory granules with different hormones, transmitters, and neuropeptides throughout the endocrine and nervous system (52). Chromogranin B (CgB) was first uniquely described in 1985 (11, 36), although human CgB may have been observed even earlier (28, 29). CgB is a major chromogranin in humans and perhaps the most abundant chromogranin in human chromaffin cells (38) from which it is released, along with catecholamines, by exocytosis (22).

Hypertension and cardiovascular disease are complex, multifactorial diseases with many genetic and environmental determinants. There are many sets of genes implicated in a wide variety of biochemical pathways that are likely to influence such syndromes (31). Of these pathways, those influencing the release of catecholamines may be some of the most important. Catecholamine release is controlled, in part, by members of the chromogranin/secretogranin family of proteins (52), which makes this family of proteins logical candidates for influence on sympathetic outflow.

CgB has both intracellular and extracellular roles in the neuroendocrine system. Extracellular roles for CgB are dependent on its extensive proteolytic processing within chromaffin granules at dibasic cleavage sites (13) to form biologically active peptides, including GAWK (bovine CgB398–464, human CgB420–493; 6), CCB (human CgB597–656; 6), bovine BAM-1475 (12), PE-11 (rat CgB532–542, human CgB536–545; 19), and the endogenous antimicrobial/bacteriolytic peptides chrombacin (bovine CgB564–626, human CgB597–657; 25) and secretolytin (bovine CgB614–626, human CgB647–657; 49). Such peptides may have a role in the neuroendocrine/sympathoadrenal stress response to systemic infection, perhaps providing innate immunity (52). CgB, or its fragments, may also play an autocrine inhibitory role in the release of co-stored hormones, such as insulin (17).

Within chromaffin cells and sympathetic axons, CgB may play a role in the sorting and trafficking of peptide hormone and neuropeptide precursors to secretory granules, as well as an important role in granulogenesis (27). CgB forms heterodimeric complexes with CgA at pH 7.5, with progression to heterotetrameric complexes at the secretory granule interior pH of 5.5 (56). CgB also binds calcium at high capacity (93 mol calcium/mol CgB at pH 5.5) within secretory granules (56). Finally, an amino terminal domain of CgB binds to the secretory vesicle membrane (55). The secretory vesicle binding target for CgB seems to be the inositol 1,4,5-trisphosphate receptor/Ca2+ channel, whose opening by CgB might be of physiological importance in the release of calcium from secretory granules to the cytosolic exocytotic machinery in secretory cells (56).

The human CgB locus (CHGB) has been mapped to chromosome 20p12-pter by in situ hybridization (24). Selective neuroendocrine CgB gene expression has been extensively studied (23), and CgB transcription in chromaffin cells and sympathetic axons is exquisitely sensitive to such efferent preganglionic sympathetic neurotransmitters as acetylcholine and PACAP (pituitary adenylyl cyclase activating peptide), illustrative of the principle of "stimulus-transcription coupling" (24). Characterization of the proximal promoter region of CgB has revealed that a cAMP response element (CRE) and two G/C-rich domains are crucial for both neuroendocrine cell type-specific and preganglionic efferent sympathetic secretagogue-inducible expression (23).

Family studies have indicated that blood pressure is heritable (26, 31), although the genetic loci responsible for variation in blood pressure have thus far remained elusive. One approach to finding possible susceptibility genes for complex diseases or traits, such as hypertension and blood pressure, may be to focus on simpler, monogenic traits associated with the complex trait of interest, so-called "intermediate phenotypes" (31). CgB is overexpressed in catecholaminergic cells in rodent models of genetic (spontaneously hypertensive rat; 30, 39) and acquired (renovascular; 49) hypertension, thus suggesting augmented sympathoadrenal activity in the pathogenesis of these syndromes. Therefore, plasma concentrations of CgB fragments may provide useful intermediate phenotypes for exploration of sympathoadrenal activity in human essential hypertension.

To identify candidate regions of the genome likely harboring loci contributing to hypertension susceptibility, we first assessed the genetic contribution to quantitative CgB expression by measuring the heritability of several circulating CgB fragments. We then performed linkage analysis with genomic DNA markers at an approximate 5-cM density in 12 extended families from the Centre d’Etude du Polymorphisme Humain (CEPH) database, to identify quantitative trait loci (QTLs) for these CgB intermediate phenotypes. Although these families have no history of hypertension, analysis of these pedigrees will allow us to examine the heritable basis of naturally occurring phenotypic variation in CgB expression.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects.
Subjects were recruited through the Utah Genetic Reference Project (UGRP), which is based at the University of Utah in Salt Lake City, and reevaluated by Hall and colleagues (15). Institutional review board approval was obtained, and each subject gave informed consent. These subjects comprise 12 three-generational families from Utah that were originally collected as a subset of the CEPH reference family collection, which has served as an important scientific resource for polymorphism discovery and human genetic map construction (9, 21). The following 12 CEPH pedigrees were analyzed in the present study: 1334, 1345, 1346, 1349, 1350, 1358, 1362, 1377, 1408, 1418, 1421, and 1424. Physical examinations were performed and interviews were conducted for each family on the General Clinical Research Center at the University of Utah Medical Center. Blood pressure data were not available for these analyses. Families were included in this study without regard to disease status. All UGRP study subjects gave informed consent under University of Utah IRB-approved protocol number 6090-96.

Biochemical phenotyping.
Heparinized plasma was obtained from each subject and stored at –70°C prior to assay. CgB region-specific radioimmunoassays (for CgB1–16, CgB312–331, CgB439–451, CgB568–577, and CgB647–657) were based on synthetic peptides, as previously described (48). 125I radiolabeling of each peptide was enabled by either an endogenous or adventitious (terminal) tyrosine residue. Polyclonal rabbit antisera were developed to the synthetic CgB regions (33). Several of the CgB region-specific radioimmunoassays have been described in detail previously (4648). Table 1 describes the synthetic peptides used in these assays, and Fig. 1 details the structure of CgB, as well as its derived peptides and radioimmunoassay epitopes.


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Table 1. hCgB regions targeted by the synthetic peptides in the radioimmunoassays

 


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Fig. 1. Diagram of the structural/functional domains of human chromogranin B (CgB), indicating the peptide regions accessed by the synthetic peptide epitopes used in the radioimmunoassays. *Multibasic proteolytic processing sites.

 
Catecholamine release in vitro.
Catecholamine release from PC12 pheochromocytoma cells in vitro was studied as previously described (51). Vesicular catecholamine stores were prelabeled with L-[3H]norepinephrine, after which exocytosis was triggered by K+ channel blockade with extracellular Ba2+ (2 mM, in the absence of extracellular calcium; 3). Released and residual cellular norepinephrine were quantified by liquid scintillation counting, and results were expressed as means ± SE percent of cellular stores released (n = 3 replicates/condition).

Genotype information.
Genotype data for the 12 CEPH families was obtained from the CEPH Genotype Database (http://www.cephb.fr/cephdb). A total of 654 autosomal STR (short tandem repeat; microsatellite) polymorphisms that had been typed on all or most of the pedigrees in our study were selected for analysis at ~5 cM spacing. Marker positions were assigned according to the newly published deCODE sex-averaged genetic map (18), which is based on more meioses and corrects several inaccuracies of the widely used Marshfield map (7). For those markers not available on the deCODE map, sex-averaged genetic locations were interpolated based on physical location according to the UCSC November 2002 freeze (http://www.genome.ucsc.edu; C. M. Nievergelt, personal communication).

Statistical analyses.
Heritability (h2r) estimates were obtained via the variance component methodology implemented in the SOLAR ("sequential oligogenic linkage analysis routines") package (5). This maximum likelihood method assumes a multivariate normal distribution of phenotypes in a pedigree and can accommodate a defined set of covariates (41, 42). The null hypothesis of no heritability (h2r = 0) is tested by comparing a "full" model, which assumes that some fraction of the phenotypic variation is explained by genetic factors, to a "reduced" model, which assumes that no variation is explained by genes, using likelihood ratio tests. Covariates (sex and age) that were significant at P < 0.05 in the heritability analysis were retained and considered in the linkage-analyses.

To showcase the power of our approach, we computed the power of our 12 CEPH pedigrees to identify a QTL and compared it to the power for identifying a QTL associated with a comparable number of sibling pairs using standard variance component power formulas (http://statgen.iop.kcl.ac.uk/gpc/; Refs. 34, 42).

Two-point and multipoint quantitative trait linkage analyses of the DNA marker data were conducted using the SOLAR package, which employs a variance-components-based algorithm. In this approach, missing genotypes are imputed and assessed probabilistically by conditioning on all other linked marker data and pedigree structure, and the proportion of marker alleles shared identical-by-descent (IBD) among all relative pairs is estimated independently for all autosomal markers. Linkage is assessed by fitting a polygenic model that does not incorporate genotype information provided by marker loci and comparing it with models that incorporate genotype data either with a specific marker (two-point analysis) or with multiple markers (multipoint). The log (base 10) of the ratio of the likelihoods of the marker-specific and polygenic models is the log-of-the-odds (LOD) score, a traditional measure of genetic linkage. As there are many different methodologies that can be used to assess linkage for quantitative traits, we also took advantage of the regression-based linkage statistics in the software package MERLIN (2).

The QTDT, an extended version of the transmission/disequilibrium test designed for quantitative traits and extended pedigrees, was used to evaluate association between CgB312–331 and microsatellite markers on chromosome 11 (1). We used the orthogonal model, incorporating environmental, polygenic, and additive major locus effects.

Although somewhat skewed toward larger values, most of the distributions of CgB values among the individuals in our sample were not grossly non-Gaussian. We did not further assess departures from multivariate normality of the phenotypes within our 12 families.


    RESULTS
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Phenotype distributions and heritability.
A total of 125 individuals from 12 UGRP/CEPH multigenerational pedigrees were analyzed in the current study. Table 2 shows the distributions of values, including the number of people phenotyped, means, standard deviations, and minimum and maximum values, for the plasma concentrations of the five CgB fragments studied. Correlations were observed between several of the phenotypes studied, and Pearson product-moment as well as Spearman rank correlations are summarized in Table 3.


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Table 2. Summary statistics of the five CgB variables measured in the 12 UGRP/CEPH families used for linkage analysis

 

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Table 3. Correlations among the five CgB variables

 
The heritability estimates for CgB expression, as well as the corresponding number of parent-offspring and sibling pairs, are indicated in Table 4. Plasma concentrations of CgB fragments CgB312–331, CgB439–451, and CgB568–577 were found to be substantially heritable, with heritability estimates ranging from h2r = 0.378 (P = 0.002) to h2r = 0.910 (P < 0.0000001). However, CgB647–657 was not found to be heritable in this sample (h2r = 0) and was thus excluded from further analyses.


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Table 4. Heritability estimates for the five CgB variables measured in the 12 UGRP/CEPH families used for linkage analysis

 
Statistical power of the study.
We assessed the utility of the CEPH pedigrees for linkage analyses by evaluating the power of our 12 pedigrees with an average of 8 siblings per family. We also compared the power of these 12 pedigrees with 48 sibling pairs, which gives the same total number of siblings (i.e., 96) and 336 sibling pairs, which equals the total number of sibling pairs that can be extracted from 12 families with 8 siblings each [number of sibling pairs per family is n(n – 1)/2, where n is the number of siblings]. Figure 2 provides the results and clearly emphasizes the power of our sample.



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Fig. 2. Power of these UGRP/CEPH families to detect linkage, compared with the equivalent number of individuals and sibling pairs. The solid line represents the 12 CEPH families with an average of 8 siblings each. The dashed line represents the equivalent number of sib pairs (336). The dotted line represents the equivalent number of individuals in sib pair families (48 sib pairs). CEPH, Centre d’Etude du Polymorphisme Humain; UGRP, Utah Genetic Reference Project.

 
Quantitative trait linkage analysis.
Table 5 summarizes the highest two-point and multipoint LOD scores from SOLAR for the genome-wide scan of the five CgB fragment plasma concentrations in the 12 UGRP/CEPH pedigrees. Two-point LOD scores >3.0 were observed for CgB312–331 on chromosomes 6 and 11. Additionally, two-point LOD scores >1.5 were observed for CgB312–331 on chromosomes 2, 3, 5, 7, 10, and 22; CgB439–451 (with age as a covariate) on chromosomes 5, 12, and 13; and CgB568–577 on chromosome 6. Multipoint analyses revealed a significant linkage for CgB312–331 on chromosome 11, as well as suggestive evidence for linkage of CgB312–331 at 84 cM on chromosome 6. All other regions of suggestive linkage as implicated by the two-point analyses were not observed to be either significant or suggestive of linkage with the multipoint analyses.


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Table 5. Summary of the results of the whole-genome scan of CgB fragment phenotypes using SOLAR

 
Figure 3 shows a plot of the SOLAR multipoint linkage scan of chromosome 11 for CgB312–331 performed at 1-cM intervals. A peak LOD score of 5.84 was observed at 147 cM on 11q24-q25, between markers AFM154YH6 (146.24 cM, 133.24 Mb) and MFD161 (147.52 cM, 133.62 Mb). A second peak with a maximum LOD of 3.13 was observed at 43 cM on 11p. Figure 3 also offers the linkage results for CgB312–331 using the MERLIN regression-based analysis, which identified a LOD peak of 3.57 at 148 cM.



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Fig. 3. Results of the multipoint linkage analysis of CgB312–331 on chromosome 11, using SOLAR (solid line) and MERLIN (dashed line). A peak LOD score of 5.84 was observed at 147 cM with SOLAR, and a peak LOD of 3.57 was observed at 148 cM with MERLIN. Microsatellite markers used for analysis are indicated according to their genetic locations at the top.

 
Additionally, evidence for linkage was evaluated for log-transformed CgB312–331 to assess the robustness of our finding, as it is known that variance component procedures may be influenced by large departures of a trait’s distribution from normality (4). However, it also known that transformations of data to normality can actually decrease evidence for a QTL (see, e.g., 40, 42). Reanalysis of log-transformed CgB312–331 still provided evidence for linkage, albeit somewhat reduced (SOLAR maximum multipoint LOD = 2.9; data not presented).

Table 6 lists the confirmed and predicted genes within a two-LOD interval of the SOLAR peak multipoint LOD for CgB312–331 on chromosome 11q24–25 as obtained from a search of the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). This region encompasses ~12 cM, which corresponds to about 6 Mb, flanked by AFM157XH6 (137.93 cM, 130.14 Mb) on the left and extending to the q-terminal end of the chromosome.


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Table 6. List of genes identified within a 2-LOD interval of the SOLAR multipoint peak LOD on chromosome 11q24-q25

 
Allelic association analyses.
We used the program QTDT to assess the degree of association between CgB312–331 and the chromosome 11 microsatellites. Significant associations were observed for two nearby markers: MFD251 (P = 0.002) and AFM248WF5 (P = 0.037). These markers are located at 132.30 cM (127.34 Mb) and 133.07 cM (127.59 Mb), respectively. These results, along with the results of the multipoint linkage analysis are summarized in Fig. 4 with the locations of the two most interesting candidate genes in the region.



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Fig. 4. Diagram of the chromosome 11q24–25 region indicating the location of the SOLAR multipoint LOD peak, individual two-point LOD scores, associated microsatellites as indicated by QTDT, and two potential candidate genes: the potassium channels KCNJ1 (ROMK) and KCNJ5 (GIRK4, KATP1). All physical locations (Mb) are according to NCBI build 31. QTDT, transmission/disequilibrium test designed for quantitative traits.

 
K+ channel blockade and catecholamine release in vitro.
To explore whether K+ channel alterations might affect exocytosis, we studied catecholamine release from chromaffin cells in the presence or absence of the K+ channel blocker Ba2+ (Fig. 5). Extracellular Ba2+ triggered substantial catecholamine release from these cells by ~17-fold (from 4.67 ± 0.24% to 79.6 ± 0.37% of cell stores).



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Fig. 5. Effect of K+ channel blockade on catecholamine secretion from chromaffin cells. PC12 chromaffin cell catecholamine vesicular catecholamine stores were prelabeled with L-[3H]norepinephrine, and release was triggered by the nonselective K+ channel blocker Ba2+ (2 mM extracellular Ba2+, in the absence of extracellular calcium), vs. mock stimulation (Ba2+-free secretion buffer). Results are plotted as % secretion of norepinephrine (means ± 1SE).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Since CgB biosynthesis and release are highly responsive to efferent sympathetic stimulation (23), and CgB expression is augmented in experimental hypertension (30, 39, 50), we sought to determine the degree to which genes influence the expression of CgB in humans, with the goal of furthering insight into genetic control of autonomic activity in man (31). Here we found that the expression of several CgB fragments was highly heritable in the CEPH pedigrees; in addition, a major locus on chromosome 11q24-q25 cosegregated (LOD = 5.84) with the plasma concentration of CgB312–331, suggesting that a previously unsuspected gene in this chromosomal region might exert a major influence on exocytotic sympathochromaffin secretion in humans.

Heritability.
The plasma concentrations of three of the five CgB regions assayed seemed to be under substantial genetic control (Table 4), with heritability values ranging from h2r = 0.378 for CgB568–577 (P = 0.0007), to h2r = 0.448 for CgB312–331 (P = 0.002), to h2r = 0.910 for CgB439–451 (P < 0.0000001). Insignificant heritability estimates for CgB fragments CgB1–16 and CgB647–657 suggest that nongenetic factors such as interindividual variability in fragment processing, distribution, or excretion (16) are important in determining the steady-state concentrations of these two peptides. CgB312–331 is of particular interest for metabolic and cardiovascular disease, in that "subtraction" of CgB (by an antibody directed against the peptide CgB312–331) alters glucose-stimulated insulin release from pancreatic islet ß-cells (17).

Candidate genetic loci.
Genome-wide linkage analysis is used to detect previously unsuspected regions of the genome likely to harbor loci that influence complex traits and diseases (20). We applied this method to CgB expression using the CEPH pedigrees. Linkage analysis of complex traits using CEPH families has been pursued before (8, 15, 32, 37, 43, 53). The CHGB gene itself is located on chromosome 20p12-pter (24). CgB expression is dependent upon a proximal promoter CRE (23), but the CRE-binding factor CREB1 is encoded by chromosome 2q32.3-q34, while its coactivator/binding protein CBP (CREBBP) is on chromosome 16p13.3. Further upstream in the CHGB promoter, two G/C-rich regions are crucial; yet the typical factors that trans-activate G/C-rich promoter regions are also located in genomic regions other than chromosome 11q24-q25 [e.g., SP1 on chromosome 12q13.1; AP2-{alpha} (TFAP2A) on chromosome 6p24; EGR1 on chromosome 5q31.1].

Interindividual variation in several of the CgB phenotypes studied was found to be positively correlated (Table 3; e.g., CgB312–331 with CgB439–451, R = 0.262, P = 0.003), whereas other correlations were actually inverse (e.g., CgB312–331 with CgB568–577, R = –0.231, P = 0.010). Thus interindividual variation in CgB fragment concentrations cannot be a simple monotonic function of biosynthesis and release of the prohormone, raising the possibility of interindividual differences in proteolytic cleavage of CgB or its removal from the bloodstream. The chromogranins may be physiologically processed by several proteases (10, 13, 52); however, none of the previously implicated proteases maps to chromosome 11q24-q25 [e.g., furin (FUR) on chromosome 15q25-q26, prohormone convertase 1 (PCSK1) on chromosome 15q15-q21, prohormone convertase 2 (PCSK2) on chromosome 20p11.1, or cathepsin L (CTSL) on chromosome 9q21-q22]. Thus other heritable mechanisms must contribute to the linkage peak on chromosome 11q24-q25.

Linkage to chromosome 11q24-q25: positional candidate loci.
Since no linkage was observed to the chromosomal regions expected on the basis of the CHGB locus itself, its functional promoter motifs, or its processing enzymes, we investigated other loci beneath the LOD = 5.84 peak on chromosome 11q24-q25. In particular, given the exquisite sensitivity of CgB to efferent sympathetic stimuli ("stimulus-transcription coupling"; 23), we wondered whether the implicated region on 11q might harbor a locus at which allelic variation at which influences sympathetic outflow.

Of the genes located in the region under the multipoint LOD peak, two loci pose an intriguing rationale as possible candidate genes: KCNJ1 (ROMK), an inward rectifier potassium channel, and KCNJ5 (GIRK4, KATP1), an ATP-sensitive, inward rectifier potassium channel. These two genes are ~50 kb apart and only about 3 Mb from the peak LOD, within the 2-LOD interval, and midway on the physical map of 11q24-q25 between the LOD peak and the QTDT-associated microsatellite loci (Fig. 4). A previous family study by Sharma and colleagues (44) assessing genetic linkage of essential hypertension in an affected sibling pair design also revealed a locus on chromosome 11q near the marker D11S934 (AFM248WF5 in our study) for which we also observed a modest association with CgB312–331 (P = 0.037). This marker is located at 133.07 cM (127.59 Mb) on 11q, very near these two potassium channels. Of note, KCNJ1 (ROMK) inactivating mutations cause "Bartter syndrome," an autosomal recessive electrolyte disturbance that may be associated with low blood pressure (45). Also, "subtraction" of CgB (by an antibody directed against the peptide CgB312–331) alters glucose-stimulated insulin release from pancreatic islet ß-cells (17); intriguingly, insulin release is regulated by an ATP-sensitive K+ channel, and KCNJ5, a K+ channel beneath the CHGB LOD peak (Fig. 4), is ATP sensitive (also known as KATP1).

Cell surface potassium channels play a dominant role in controlling membrane potential in excitable cells such as neurons and chromaffin cells, both in the basal state and during the hyperpolarization phase just after membrane depolarization; indeed, blockade of outward K+ currents in chromaffin cell potassium channels results in membrane depolarization and consequent catecholamine release (14). Here (Fig. 5), we found that K+ channel blockade by extracellular Ba2+ provoked an ~17-fold increment in catecholamine secretion from chromaffin cells. Thus either qualitative or quantitative variability in such channels might influence exocytotic sympathoadrenal activity, plausibly perturbing both CgB release and blood pressure. We will pursue further studies to probe the involvement of such positional candidate loci in autonomic phenotypic variation. A recent linkage study by Xu et al. (54) examining blood pressure regulation suggested that another locus on chromosome 11 (maximum multipoint LOD = 2.07, single-point LOD = 2.57 at D11S2019, ~90 cM centromeric to our peak) might influence systolic blood pressure.

We did not detect an effect of the CgB gene itself on plasma CgB312–331. Such a negative result could have resulted from the relatively low statistical power of linkage (vs. association) analysis (35), complex interactions of the gene with other genes, and/or complex interactions involving environmental factors. It may also be the case that allelic variation at CHGB does not substantially influence variance of plasma CgB. Further studies involving additional linkage and association analyses, in vitro studies, and animal models may be required to elucidate the genetic basis of CgB expression and its role in hypertension.


    GRANTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We appreciate the assistance of the National Institutes of Health (NIH)/DRR-supported General Clinical Research Center at UCSD (Grant M01-RR-00827). This investigation was supported by National Center for Research Resources Grant M01-RR-00064 to the Huntsman General Clinical Research Center at the University of Utah; by generous gifts from the W. M. Keck Foundation and from the George S. and Delores Doré Eccles Foundation; and by the Department of Veterans Affairs, by NIH Grants HL-58120, HL-69758, DK-48645, HL-54998-07, HL-64777-04, and General Clinical Research Center Grant RR-00827; and by the Swedish Cancer Society.


    ACKNOWLEDGMENTS
 
We thank all family members who participated in the Utah Genetic Reference Project. We also thank Dr. Andreas P. Peiffer (UGRP Medical Director) and Melissa M. Dixon (UGRP Study Coordinator).


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: D. T. O’Connor, Dept. of Medicine and Center for Molecular Genetics, Univ. of California at San Diego and VASDHS (9111H), 3350 La Jolla Village Drive, San Diego, CA 92161 (E-mail: doconnor{at}ucsd.edu; URL: http://medicine.ucsd.edu/hypertension).


    REFERENCES
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 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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