Human adrenoceptor polymorphisms: evolving recognition of clinical importance

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Human adrenoceptor polymorphisms: evolving recognition of clinical importance Rainer Büscher, Volker Herrmann and Paul A. Insel

R. Büscher, Postdoctoral Fellow, V. Herrmann, Postdoctoral Fellow, and P. A. Insel, Professor, University of California at San Diego, Department of Pharmacology, La Jolla, CA 92093-0636, USA.

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The central dogma of molecular biology (DNA → RNA → protein) has played a dominant role in biological research in many fields and has recently begun to direct new ways of thinking in pharmacology. The Human Genome Project, with its goal of sequencing the entire genome, provides an opportunity to identify each of the genetic ‘players’ but ignores the ‘teams’ of similar, but different, players. Genetic polymorphisms, which are frequently occurring genetic variants (by contrast with rarer occurring mutations), define such ‘teams’. A rapidly growing literature in the areas of drug metabolism and drug action has begun to identify which genes show substantial variability and, more importantly, the physiological and pharmacological significance of such variations. A working hypothesis is that polymorphisms in neurotransmitter and drug receptors might underline interindividual variability in both pharmacological response and propensity for disease. As key regulators of many organ systems, adrenoceptors provide an attractive system for exploring a possible role between receptor polymorphism, drug response and disease susceptibility or progression1. Adrenoceptors belong to the superfamily of seven transmembrane domain receptors that produce their effects through coupling with G proteins. Nine different subtypes of adrenoceptors have been identified: a1A, a1B, a1D; a2A, a2B, a2C; and b1, b2, b3, based on results of pharmacological and molecular cloning studies. This article provides an overview of recent progress in the assessment of adrenoceptor polymorphisms in human subjects, obtained by use of a variety of techniques (Fig. 1) and by DNA sequencing. Results for

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the b3-adrenoceptor polymorphisms have been discussed in more detail in a recent TiPS review2.

a-Adrenoceptors Although there are three subtypes each of both a1- and a2-adrenoceptors, research on the a-adrenoceptors has, to date, focused on the a1A- (Fig. 2a), a2A- and a2C-subtypes (Table 1, Refs 3–10). As shown in Table 1, the analysis performed on the a1A- and a2C-adrenoceptors has not yet provided definitive evidence for an association of polymorphisms with disease states or drug response. However, the data in those publications cited is of limited validity because only single polymorphic sites were identified by using Restriction Fragment Length Polymorphism (RFLP, Fig. 1). Even so, studies using RFLP to assess the association of the a2C-adrenoceptor with the restriction enzyme DraI have identified a link between the occurrence of a DraI polymorphism and a higher trunk/extremity skinfold ratio7, increased prevalence of thrombotic stroke8 and the development of hypertension in Caucasians9. Identification of the base changes responsible for this (or the other) RFLP, or of alterations in receptor function or pharmacological responses resulting from such changes have not yet been demonstrated. In as-yet unpublished studies we have analysed the entire coding sequence of a1B-adrenoceptor from 51 individuals and have identified three different silent polymorphisms but found no apparent association between these genetic variations and either essential hypertension or variation in response to infused phenylephrine (R. Büscher et al., unpublished observations). We anticipate that data will be forthcoming for the subtypes of a1- and

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a2-adrenoceptors for which information regarding polymorphisms is not yet available.

b-Adrenoceptors Substantially more data have been obtained for b-adrenoceptors than for a-adrenoceptors, with a large number of recent articles reporting on both b2 and b3 subtypes (Table 2, Refs 9, 11–21, 30). b2-Adrenoceptors are of particular interest because of their ubiquitous expression and their role as drug targets. Reihsaus et al.13 were the first to show that the b2adrenoceptor is highly polymorphic. In this study, which evaluated whether b2-adrenoceptor polymorphisms contribute to the development of asthma, the authors identified nine different polymorphisms: five ‘silent’ ones and four that change the amino acid sequence (Arg16→Gly, Gln27→Glu, Val34→ Met and Thr164→Ile). Subsequent characterization of the variants with coding changes included expression in heterologous cells and revealed important functional changes22 (Fig. 2b): Arg16→Gly leads to enhanced downregulation of the b2-adrenoceptor, Gln27→Glu is characterized by decreased downregulation and Thr164→Ile leads to several functional effects, which include lower binding affinities for agonists and deficient coupling of the receptor to adenylate cyclase. Transgenic mice expressing the Thr164→Ile receptor targeted to the heart show impaired myocardial signalling and function23. The overall frequency of the Arg16→ Gly and Gln27→Glu polymorphisms is much higher than that of the Thr164→Ile (Table 2). Although β2adrenoceptor polymorphisms do not seem to be more common in patients with asthma, the Arg16→Gly occurs more frequently in patients with nocturnal asthma14. Evidence has also shown that b2-adrenoceptor polymorphisms can influence the response to bronchodilator and bronchoconstrictor agents such that polymorphisms predisposed to downregulation lead to greater muscarinic cholinergic receptor-mediated bronchoconstriction and blunted

0165-6147/99/$ – see front matter © 1999 Elsevier Science. All rights reserved.

PII: S0165-6147(99)01322-X

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Technique

Definition

Restriction Fragment Length Polymorphism (RFLP)

variations among individuals in the length of restriction fragments from identical regions of the genome; these variations can create or destroy restriction-enzyme recognition sites

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Mechanism a

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a b mutation cleaving enzyme b

gel electrophoresis

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Advantage

Disadvantage

• easy and relatively inexpensive screening for polymorphic sites in populations

• cannot readily identify polymorphic sites present other than at restriction-enzyme recognition sites

• no limitation in fragment size (100 bp – >10 kb) • radioactive or nonradioactive • discrimination: homozygous/heterozygous

has loss of restriction site b

Single-Stranded Conformation Polymorphism (SSCP)

electrophoretic detection of single base-pair differences that lead to altered conformations

a

b

a

single-base change electrophoretic detection of single a base-pair alterations b from wild-type (WT) sequences using temperature, or another denaturant, which allows detection of the mutant genotype

• only in DNA fragments <500 bp

• discrimination: homozygous/heterozygous

b

Temperature Gradient Gel Electrophoresis (TGGE) or Denaturing Gradient Gel Electrophoresis (DGGE)

• easy and relatively inexpensive screening for polymorphic sites in populations

gel electrophoresis a

single-base change

b

TGGE

• detection of multiple polymorphic sites at once • discrimination: homozygous/ heterozygous • highly sensitive approach generally without false positives

• PCR prducts that are positive for deviations from WT must be sequenced to identify exact mutation • size limitation: <1 kb • labour intensive

PCR Fig. 1. Definition, mechanisms, advantages and disadvantages of different techniques to identify genetic variations. Restriction Fragment Length Polymorphism (RFLP), SingleStranded Conformational Polymorphism (SSCP) and Temperature Gradient Gel Electrophoresis (TGGE) can provide hints regarding polymorphic sites and are techniques that are frequently used for the detection of genetic variations within a population.

response or greater desensitization to the bronchodilating effects of b2adrenoceptor agonists12,24–26. A second condition in which b2adrenoceptor polymorphisms have begun to be examined is essential hypertension. RFLP analysis of a hypertensive African–American study population revealed a linkage of hypertension with the 3.7/3.4 kb BanIRFLP (Ref. 9), but it should be noted that this RFLP was very different from the one obtained by others who used the same restriction enzyme in a different population11 (Table 2). The frequency of the Arg16→Gly b2adrenoceptor polymorphism was significantly higher in African– Caribbean essential hypertensive subjects than in normotensives15, consistent with a hypothesis of blunted vasodilation in hypertension. However, a recent study of offspring from Norwegian hypertensives found

that the occurrence of the Arg16 allele was more frequent in the hypertensive offspring16. A number of studies are in progress to extend these observations to other hypertensive cohorts and to assess the b2-adrenoceptor in other cardiovascular and pulmonary disease settings. For the b3-adrenoceptor, a leading question has been whether a Trp64→Arg polymorphism is associated with an earlier onset of noninsulin-dependent diabetes (NIDDM), is a marker for morbid obesity, or is without clinical significance2 (Table 2, Fig. 2c). Additional studies with larger study populations, as well as detailed functional studies, will be necessary to define the role of this polymorphism. Surprisingly, no data regarding b1-adrenoceptor polymorphic sites have thus far been reported, even

though the high level of expression of this receptor subtype in heart and adipose tissue implies that b1adrenoceptor polymorphisms could have important effects on cardiovascular and metabolic homeostasis.

Where do we go from here? In spite of the growing literature on adrenoceptor polymorphisms, the database remains incomplete. Data for certain types and subtypes of adrenoceptor are either insufficient or absent, but, in addition, some of the available information is rather limited in scope. For example, very few studies have examined the precise functional consequence of receptor polymorphisms using both in vitro and in vivo studies, especially human studies. Data from such studies will be crucial both to place disease associations in a rational context and, from a pharmacological

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α1A-adrenoceptor

a

NH2 extracellular

TM7

TM1

intracellular 347

Arg347 – wild-type Cys347 – apparently similar pharmacological characteristics

COOH

β2-adrenoceptor

b

Gln27 – wild-type Glu27 – minimal downregulation

Arg16 – wild-type Gly16 – enhanced downregulation 16

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NH2 extracellular

TM1

TM7

164

intracellular Thr164 – wild-type Ile164 – decreased binding

COOH

β3-adrenoceptor

c

NH2 extracellular

TM7

TM1

intracellular 64

Trp64 – wild-type Arg64 – decreased cAMP production

COOH

Fig. 2. Polymorphisms of human adrenoceptors. Each figure depicts the seven transmembrane domain receptor with an extracellular amino (NH2) and an intracellular carboxy (COOH) terminus. a: a1A-Adrenoceptor. The circle indicates the position of a most commonly occurring polymorphism that results in changes in the amino acid sequence and its functional significance in this receptor subtype. b: b2-Adrenoceptor. The three circles indicate the positions of the most commonly occurring polymorphisms and their functional significance. c: b3-Adrenoceptor. For this receptor subtype a controversial question has been whether a Trp64→Arg polymorphism is associated with an earlier onset of non-insulin-dependent diabetes, is a marker for morbid obesity, or is without clinical significance.

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perspective, to provide mechanismbased rationales for predicting efficacy and toxicity. The overriding hypothesis is that adrenoceptor polymorphisms might predispose an individual to the onset of a disease, alter the clinical course of a disease (prognosis) or the response to treatment, or both. Pharmacogenomic classification of individual patients and heterogeneous groups of patients into distinct subpopulations based on adrenoceptor genotyping and identification of polymorphisms is thus an attractive, but elusive, goal. Moreover, there are several issues that need to be considered. Most studies published thus far have been based on the analysis of RFLPs. Unfortunately, because RFLPs depend on having appropriate restriction-enzyme cleavage sites, RFLP analysis cannot readily identify polymorphic sites present elsewhere in a sequence (Fig. 1). Other screening methods, such as singlestranded conformational polymorphism (SSCP), temperature or denaturing gradient gel electrophoresis (TGGE, DGGE) and related techniques can provide hints regarding polymorphic sites but definitive identification requires DNA sequence analysis (Fig. 1). Sequencing of an entire coding block might be necessary to define the full range of polymorphic sites27. Analysis of 59 and 39 non-coding sequences could also prove to be crucial. Recent data suggesting evidence for a polymorphism in a 59 cistron open reading frame upstream of the start site of the b2-adrenoceptor coding sequence is particularly intriguing because such a polymorphism might be involved in regulating receptor expression16. Thus, as a starting point, it seems essential to sequence each of the adrenoceptor genes in a reasonable number of subjects (~50) to define the existence of frequent polymorphic sites. Such pilot studies can then be followed by analysis of samples from larger numbers of patients, perhaps selected on the basis of a particular phenotype (e.g. disease or drug response). Follow-up studies could be facilitated by the use of

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Table 1. Association between occurrence of polymorphisms of human a-adrenoceptor genes and disease states: an overview Population

No.

Enzyme Allelic frequency used

Clinical association

Refs

a1A-Adrenoceptor (chromosome 8p21) American, 83 Arg492Cys RFLP Caucasian

PstI

0.34 (allele 1) 0.66 (allele 2)

First description of a polymorphic site

3

Japanese

PstI

WT: 0.90 : 0.10 BPH: 0.87 : 0.13

Similar radioligand binding, Ins(1,4,5)P3 formation and Ca2+ signalling; no association between BPH and a1A-adrenoceptor gene polymorphism

4

Bsu361, PstI

Hypertensive (HT): No difference in frequency of polymorphic site in 0.52 (12 kb), 0.48 (5.8 kb); hypertensive and normotensive subjects Normotensive (NT): 0.45 (12 kb), 0.55 (5.8 kb)

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SSCP

HhaI

–

No association between phenotypes and RFLP genotypes

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a2C-Adrenoceptor (chromosome 10q24–26) French 280 – RFLP Canadian

DraI

–

Women (not men), with the 6.3 kb allele DraI RFLP had a significantly higher trunk to extremity skinfold ratio

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DraI

Caucasians: 0.20 African–American. 0.30 (6.3 kb fragment)

Increased prevalence of thrombotic stroke, augmented baroreceptor sensitivity, diminished sodium excretion in hypertensive individuals

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267

Mutation

Arg492Cys

Assay

RFLP

a2A-Adrenoceptor (chromosome 4p16.3) American, 107 – RFLP ethnicity unspecified Catalan, Caucasian

55

–

American, 81 Caucasians and African– American

–

RFLP

American, 175 Caucasians and African– American HT and NT

–

RFLP, Dra I Southern blot

Afr. Am. HT: 0.52 (6.7/ No association between hypertension and 9 6.7 kb), 0.44 (6.7/6.3 kb), genotype in the group as a whole or in African– 0.04 (6.3/6.3 kb); Americans, but association of 6.7 kb and Afr. Am. NT: 0.49 (6.7/ absence of 6.3 kb in Caucasians with hypertension 6.7 kb), 0.45 (6.7/6.3 kb), 0.06 (6.3/6.3 kb): Caucasian HT: 0.82 (6.7/ 6.7 kb), 0.18 (6.7/6.3 kb), 0.00 (6.3/6.3 kb); Caucasian NT: 0.62 (6.7/ 6.7 kb), 0.30 (6.7/6.3 kb), 0.08 (6.3/6.3 kb)

American, 227 Caucasians and African– American, HT and NT

–

RFLP

Afr. Am. HT: 0.16 (6.3/6.3 kb); Afr. Am. NT: 0.03 (6.3/6.3 kb); Caucasian HT: 0.00 (6.3/6.3 kb); Caucasian NT: 0.015 (6.3/6.3 kb)

DraI

Possible association of 6.3 kb RFLP with hypertension in African–Americans

10

Afr. Am., African–American; BPH, benign prostata hypertrophy; Enzyme used, restriction enzyme used for Restriction Fragment Length Polymorphism (RFLP); HT, hypertensive; Ins(1,4,5)P3, inositol(1,4,5)trisphosphate; No., number of individuals tested; NT, normotensive; WT, wild-type.

strategies that focus on smaller DNA fragments containing polymorphisms and for which RFLPs or allelicspecific oligonucleotide assays might be used to assess polymerase chain reaction (PCR)-isolated products. This general approach, although more expensive and time consuming, could provide the most definitive information regarding expression of polymorphisms in adrenoceptors (or other types of molecules).

In the future, it is likely that ‘gene chip’ technology will provide a means for rapid screening and precise identification of adrenoceptor polymorphisms in large numbers of patients and healthy controls. However, thus far, chip technology is suitable only for known mutations. This technology might be particularly well suited for linkage studies in which family members are analysed or association studies in which unrelated individuals

are assessed. Studies that involve sibling-pair analysis and examination of large numbers of families and family members will be needed to increase statistical power for genetic linkage analysis. Studies on adrenoceptor polymorphisms have thus far focused primarily on genotyping and identification of genetic variations, but relatively few physiological and pharmacological studies have been

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Table 2. Association between occurrence of polymorphisms of human b-adrenoceptor genes and disease states: an overview Population

No.

Mutation

Assay

b2-Adrenoceptor (chromosome 5q31–32) Japanese 58 – RFLP, Southern blot

Enzyme used

Allelic frequency

Clinical association

Refs

BanI

Asthmatics: 2.3/2.3 kb + 2.3/2.1 kb (0.12), 2.1/2.1 kb (0.44); Non-asthmatics: 2.3/2.3 kb + 2.3/2.1 kb (0.88), 2.1/2.1 kb (0.56)

Association between BanI RFLP with airway 11 responses to β2-adrenoceptor agonists and incidence of asthma. Trend towards reduced cAMP. Responses of peripheral mononuclear cells in subjects without 2.3 kb allele compared with those with this allele

American, 175 Caucasians and African– American HT and NT

–

RFLP, Southern blot

BanI

Total study population: Association of hypertension and black race with 0.46 (3.7/3.7 kb), increased frequency of 3.7/3.4 kb and decreased 0.73 (3.7/3.4 kb), frequency of 3.4/3.4 kb 0.54 (3.4/3.4 kb); Afr. Am. HT: 0.17 (3.7/3.7 kb), 0.81 (3.7/3.4 kb), 0.02 (3.4/3.4 kb); Afr. Am. NT: 0.06 (3.7/3.7 kb), 0.76 (3.7/3.4 kb), 0.18 (3.4/3.4 kb); Caucasian HT: 0.00 (3.7/3.7 kb), 0.84 (3.7/3.4 kb), 0.16 (3.4/3.4 kb); Caucasian NT: 0.13 (3.7/ 3.7 kb), 0.49 (3.7/3.4 kb), 0.38 (3.4/3.4 kb)

9

African– American sibling pairs

–

RFLP, Southern blot

BanI

Not reported

Diastolic blood pressure response to sodium loading/volume depletion linked to the β2-adrenoceptor RFLP

30

American, 496 Caucasian and Hispanic children, with/without bronchospasm

Arg16→Gly Gln27→Glu

RFLP of PCR products

NcoI, BbvI

Caucasian Gly16: 0.62, mixed Gly16: 0.61, Hispanic Gly16 0.59; Caucasian Glu27: 0.39, mixed Glu27: 0.34, Hispanic Glu27: 0.27

Children with and without a history of wheezing with Arg16 were more likely to show bronchodilator response to the β2-adrenoceptor agonist albuterol

12

American, 107 ethnicity unspecified, asthmatics/ non-asthmatics

Arg16→Gly TGGE, – Gln27→Glu direct Met34→Val sequencing Thr164→Ile

Occurrence of homozygous Asthmatics have similar allelic frequencies, but the polymorphism: Arg16→Gly polymorphism was associated with Asthmatics: Gly16Gly (0.53), increased use of corticosteroids Glu27Glu (0.24); Healthy controls: Gly16Gly (0.59), Glu27Glu (0.28)

13

American, 55 ethnicity unspecified, nocturnal asthmatics/ asthmatics/ non-asthmatics

Arg16→Gly Direct – Gln27→Glu sequencing Thr164→Ile

Nocturnal asthma: Gly16 (0.80), Glu27 (0.50), Ile164 (0.00); non-nocturnal asthma: Gly16 (0.52), Glu27 (0.52), Ile164 (0.05);

Arg16→Gly was overrepresented in nocturnal asthma and may be a genetic factor in the expression of this asthmatic phenotype

14

African Caribbeans

217

Arg16→Gly

Direct – sequencing

Occurrence of homozygous polymorphism: HT: Gly16Gly (0.74), Arg16Arg (0.05); NT: Gly16Gly (0.52), Arg16Arg (0.18)

Essential hypertension in African Caribbeans was associated with an increased frequency of the Gly16 allele

15

Norwegian Caucasians

34

Arg16→Gly

Direct sequencing

Occurrence of homozygous polymorphism: HT: Gly16Gly (0.22), Arg16Arg (0.39); NT: Gly16Gly (0.5), Arg16Arg (0.06)

Frequency of Arg16 allele was significantly higher in offspring from hypertensives than in offspring from normotensives

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Cont British, ethnicity unspecified

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No.

Mutation

Assay

419

Arg16→Gly Gln27→Glu

Allelic – specific oligonucleotides

b3-Adrenoceptor (chromosome 8p11.1–12) French 185 Trp64→Arg RFLP

T

Enzyme used

A

W

A

R

E

N

E

S

S

Allelic frequency

Clinical association

Refs

Arg16Arg (0.09), Arg16Gly (0.48), Gly16Gly (0.43); Gln27Gln (0.22), Gln27Glu (0.30), Glu27Glu (0.16)

Gln27 (homozygous or heterozygous), but not Gly16 was associated with asthma

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BstNI

Trp64→Arg: heterozygote (0.076)

Heterozygote patients with Trp64→Arg have an increased capacity to gain weight

18

BstNI

Trp64→Arg: heterozygote (0.0025)

No association or linkage with BMI, insulin or glucose levels, or diabetes

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American Caucasians

491

Trp64→Arg

RFLP

Pima Indians

642

Trp64→Arg

Direct – sequencing, SSCP

Arg64Arg: 0.11

72% of Pima Indians with Trp64→Arg develop earlier onset of NIDDM vs. 60% of WT

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Finnish

254

Trp64→Arg

Solid – phase minisequencing

Trp64Trp: 0.81, Arg64Arg: 0.016 (morbid obesity); Trp64Trp: 0.83, Arg64Arg: 0.007 (lean subjects)

Data do not support a significant role for the codon 64 polymorphism as a marker of morbid obesity

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BMI, Body Mass Index (i.e. ratio of weight over square of height in cm); enzyme used, restriction enzyme used for Restriction Fragment Length Polymorphism (RFLP); HT, hypertensive; NIDDM, non insulin-dependent diabetes mellitus; No., number of individuals tested; NT, normotensive; WT, wild-type.

conducted. It is essential, we believe, to relate in vitro and in vivo functional data to adrenoceptor polymorphisms that are found in different human population or patient groups. The large number of agonists and antagonists available to study adrenoceptors and the numerous physiological and biochemical assays that can be conducted makes this approach highly feasible. Moreover, measurement of adrenoceptor drug responses, such as vasoconstriction, can provide a straightforward means to define the impact of different genotypes. The recent report by Lerman et al.28, who identified an association between right ventricular outflow tract tachycardia and a somatic cell mutation in a G protein subunit emphasizes that studies of genomic DNA to identify polymorphisms will not provide information about tissue-specific expression of altered adrenoceptor that might occur in peripheral tissues. Thus, a definitive assessment of the role of adrenoceptor polymorphisms (and mutations) in disease might require analysis of target tissue DNA or RNA in addition to analysis of genomic DNA.

Lastly, it is important to emphasize that polymorphisms in adrenoceptors might represent only one type of genetic variation in adrenoceptor signalling that could impact on the expression of a disease phenotype or altered pharmacological response. Data showing polymorphisms of a G protein b3 subunit with higher frequency in hypertensives29 indicates that G protein subunits can also be polymorphic. Time will tell whether other G protein subunits, G proteinregulated effectors and more distal components will also show polymorphisms of clinical and pharmacological importance. References 1 Insel, P. A. (1996) New Engl. J. Med. 334, 580–585 2 Strosberg, A. D. (1997) Trends Pharmacol. Sci. 18, 449–454 3 Hoehe, M. R., Berretini, W. H., Schwinn, D. A. and Hsieh, W-T. (1992) Hum. Mol. Genet. 1, 349 4 Shibata, K. et al. (1996) Br. J. Pharmacol. 118, 1403–1408 5 Sun, L., Schulte, N., Pettinger, P., Regan, J. W. and Pettinger, W. A. (1992) J. Hypertens. 10, 1011–1015 6 Bono, M. et al. (1996) Gene Geogr. 10, 151–159 7 Oppert, J. M. et al. (1995) Obes. Res. 3, 249–255 8 Freeman, K. et al. (1995) Am. J. Hypertens. 8, 863–869 9 Svetkey, L. P. et al. (1996) Hypertension 27, 1210–1215

10 Lockette, W. et al. (1995) Am. J. Hypertens. 8, 390–394 11 Ohe, M. et al. (1995) Thorax 50, 353–359 12 Martinez, F. D., Graves, P. E., Baldini, M., Solomon, S. and Erickson, R. (1997 J. Clin. Invest. 100, 3184–3188 13 Reihsaus, E., Innis, M., MacIntyre, N. and Liggett, S. B. (1993) Am. J. Respir. Cell. Mol. Biol. 8, 334–339 14 Turki, J. et al. (1995) J. Clin. Invest. 95, 1635–1641 15 Kotanko, P. et al. (1997) Hypertension 30, 773–776 16 Timmermann, B. et al. (1998) Kidney Int. 53, 1455–1460 17 Hopes, E. et al. (1998) Br. Med. J. 316, 664 18 Clement, K. et al. (1995) New Engl. J. Med. 333, 352–354 19 Elbein, S. C. et al. (1996) J. Clin. Endocrinol. Metab. 81, 4422–4427 20 Walston, J. et al. (1995) New Engl. J. Med. 333, 343–347 21 Oksanen, L. et al. (1996) Int. J. Obes. 20, 1055–1061 22 Liggett, S. B. (1995) News Physiol. Sci. 10, 265–273 23 Turki, J. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10483–10488 24 Hall, I. P., Wheatley, A., Wilding, P. and Liggett, S. B. (1995) Lancet 345, 1213–1214 25 Tan, S., Hall, I. P., Dewar, J., Dow, E. and Lipworth, B. (1997) Lancet 350, 995–999 26 Hall, I. P. (1996) Monogr. Allergy 33, 153–167 27 Büscher, R., Herrmann, V. and Insel, P. A. (1998) Mol. Genet. Metab. 64, 266–270 28 Lerman, B. B. et al. (1998) J. Clin. Invest. 101, 2862–2868 29 Siffert, W. et al. (1998) Nat. Genet. 18, 45–48 30 Svetkey, L. P., Chen, Y. T., McKeown, S. P., Preis, L. and Wilson, A. F. (1997) Hypertension 29, 918–922

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Acknowledgements The authors‘ research was supported by research grants from the National Institutes of Health (NIH) GM 40781, GM 31987, HL 53773 and by postdoctoral fellowships from Deutsche Forschungsgemeinschaft (DFG, RB) and Falk Foundation (VH).

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