Journal of Human Hypertension (2003) 17, 319–324 & 2003 Nature Publishing Group All rights reserved 0950-9240/03 $25.00 www.nature.com/jhh ORIGINAL ARTICLE Physical mapping of autonomic/ sympathetic candidate genetic loci for hypertension in the : a somatic cell radiation hybrid library approach

SW Chitbangonsyn, P Mahboubi, D Walker, BK Rana, KL Diggle, DS Timberlake, RJ Parmer and DT O’Connor Department of Medicine and Center for Molecular Genetics, University of California at San Diego, and the V.A. San Diego Healthcare System, San Diego, CA, USA

Allelic variation at multiple genetic loci may contribute segments) to avoid cross-species (hamster) amplifica- to hypertension. Since autonomic/sympathetic dysfunc- tion. Chromosomal positions were assigned in cR tion may play an early, pathogenic, heritable role in (centi-Ray) units (B270 Kbp/cR3000 for GeneBridge 4). A hypertension, we evaluated candidate loci likely to total of 13 loci were newly assigned chromosomal contribute to such dysfunction, including catechola- positions; of particular interest was a cluster of mine biosynthetic enzymes, catecholamine transpor- adrenergic candidate loci on 5q (including ters, neuropeptides, and adrenergic receptors. Since ADRB2, ADRA1A, DRD1, GPRK6, and NPY6R), a region chromosomal locations and physical map positions of harbouring linkage peaks for blood pressure. Such many of these loci had not yet been identified, we used physical map positions will enable more precise selec- the GeneBridge4 human/hamster radiation (somatic cell) tion of polymorphic microsatellite and single nucleotide hybrid library panel (resolution B1toB1.5 Mb), along polymorphism markers at these loci, to aid in linkage with specifically designed oligonucleotide primers and and association studies of autonomic/sympathetic dys- PCR (200–400 bp products) to position these loci in the function in human hypertension. human genome. Primers were designed from sequences Journal of Human Hypertension (2003) 17, 319–324. outside the coding regions (30-flanking or intronic doi:10.1038/sj.jhh.1001550

Keywords: ; sympathetic; autonomic; locus; blood pressure

Introduction Previous allelic association (ie, case/control) studies in hypertension have documented the role Human hypertension is an aetiologically complex 1 of allelic variation (or allele frequency differences) trait, which displays substantial heritability in 14,15 2–5 at adrenergic receptor loci (ADRB2, ), postrecep- family and twin studies. The autonomic nervous tor signal transduction component loci (GNAS116, system is a key regulator of minute-to-minute 17 6 and GNB3 ), and catecholamine biosynthetic en- changes in blood pressure. Autonomic dysfunction 18 6–13 zyme loci (TH ). Genomewide linkage scans for in hypertension seems to be heritable and blood pressure elevation have also implicated alterations in autonomic activity may precede overt several chromosomal regions harbouring hypertension in subjects at genetic risk of the 19 6–13 likely to affect autonomic activity. syndrome. In order to facilitate more comprehensive allelic association studies of autonomic dysfunction in Correspondence: Dr D T O’Connor, Department of Medicine and hypertension, we sought to define chromosomal Center for Molecular Genetics (9111H), University of California at positions for loci encoding crucial for the San Diego and VASDHS, 3350 La Jolla Village Drive, San Diego, operation of the sympathetic neuroeffector junction. CA 92161-9111H, USA. Here we report such localizations, using human/ E-mail: [email protected] hamster cell radiation hybrid libraries as a mapping Internet: http://medicine.ucsd.edu/hypertension; http://elcapitan. 20–22 ucsd.edu/hyper tool. Our results suggest that a cluster of such Support: Department of Veterans Affairs, National Institutes of loci occurs on chromosome 5q, a region linked to 23–24 Health. variation in systolic blood pressure in humans. Autonomic hypertension gene SW Chitbangonsyn et al

320 Materials and methods dried template genomic DNA (50 ng in 10 ml; ie, 5 ng/ ml). The DNA templates for optimization were PCR primer design human genomic DNA, hamster genomic DNA, and Primer pairs were designed for human candidate a negative control (no DNA). The mixture for an loci using human nucleotide information from individual PCR reaction (10 ml) consisted of the genomic reference clones in the National Center following final concentrations: 400 nM forward/ for Biotechnology Information owww.ncbi.nlm. reverse primers, 0.2 mM dNTPs (Genset), 0.5 U nih.gov4 and online Mendelian inheritance in AmpliTaqGold thermostable DNA polymerase man (OMIM) at the same internet address. Complete (Applied Biosystems), 1 mM MgCl2, in TM buffer intronic coding regions (in FASTA format) were (1 Â concentration ¼ 34 mM Tris pH ¼ 8, 25 mM KCl, chosen as targets for polymerase chain reaction 2.5 mM MgCl2, 8.3 mM (NH4)2SO4, 0.85 mg/ml (PCR) amplification. Noncoding regions were used bovine serum albumin), supplemented with as template targets, either intronic or 3’-untranslated 18 MO deionized/distilled water (Sigma), to achieve regions (eg, ADMR) to avoid amplification of the final desired total volume (10 ml/amplifica- conserved sequences across species. Once the desired tion). ‘Touch Down Profile’ programs (MJ Research, regions were identified, MacVector (Oxford Molecu- Watertown, MA, USA) were used, which began lar) was used to design primer pairs, typically 21–24- at annealing temperatures of 661C and ran down mers spanning 200–400 bp target regions (amplicons), to 501Cat11C/ cycle for the first 16 PCR and avoiding primer dimer formation. Primer pairs cycles, followed by a uniform three-step amplifica- with the lowest practical percent G/C content tion profile (941C denaturing step for 30 s, 1 1 (typically o60% G/C) were chosen for annealing 50 C annealing step for 30 s, 72 C extension step temperatures between 45 and 651C. for 30 s) for another 24 cycles, then finally holding at 101C. After PCR, the products were visualized using 2% agarose gels and ethi- PCR primer optimization dium bromide stain. Primers that amplified only human (but not hamster) DNA targets were Optimization of primer pairs for PCR was performed further applied to the radiation hybrid (RH) on plates containing duplicate wells of evaporation- panels.

Figure 1 Pre- and postsynaptic locations, within the sympathetic neuroeffector junction, of proteins encoded by loci examined in this study. The schematic neuron is illustrative of the functions of an efferent postganglionic sympathetic axon, innervating such cardiovascular targets as vascular smooth myocytes or cardiac myocytes. Note that the biosynthesis and vesicular uptake of catecholamines (although not soluble peptides) can take place throughout the neuron, even in the terminus. By contrast, vesicular soluble peptides (eg, chromogranin A and neuropeptide Y) can only enter the vesicle as it buds off the Golgi apparatus in the cell soma.

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321 RH mapping A total of 42 loci encoding key components of the

14,15 sympathetic neuroeffector junction have been tabu- The GeneBridge4 RH library (G4) RH mapping lated in Table 1. Of these, 13 were newly mapped panel (Research Genetics, Huntsville, AL, USA) is a here: 11 using the G4 RH panel, and two with the G3 human/hamster somatic cell hybrid panel, in which panel. The remaining 30 loci were previously donor human fibroblasts were X-irradiated at mapped, with information provided by GeneMap 3000 rad, fused to hamster A23tk-cells, and 93 clones were selected in HAT (hypoxanthine/ami- nopterin/thymine) media.15 The mapping resolution of this RH library is approximately B1 to 1.5 Mb. The approximate physical distance conversion scale for this 3000 rad G4 RH library is 1 cR3000 (centi- Ray3000)E270 kbp. Two loci (VMAT1 and VMAT2) were mapped using the Stanford G3 RH library,14 which has n ¼ 83 clones derived from a 10 000 rad irradiation of human genetic material. To convert G4 cR3000 to G3 (Stanford G3 RH panel) cR10 000. the approximation E is: 0.1 cR3000 1.0 cR10 000. G4 RH panel genomic DNAs were diluted 1 : 5 (to achieve 50 ng in 10 ml; ie, 5 ngml), and then under- went the same amplification procedure as for optimization (see above). Each locus (primer pair) was RH-mapped twice.

Informatics G4 library amplification results (n ¼ 93 clones) were transmitted to the Radiation Hybrid Map- ping server at the Whitehead Institute ohttp:// www-genome.wi.mit.edu/cgi-bin/contig/rhmapper4.

Selected Log10 of the odds ratio for assignment to a particular chromosomal position (LOD) thresholds were always LOD 47, and were incremented to as high as LOD 417 in order to achieve mapping to a single chromosome. Results are reported as the LOD value in comparison with the next best model for marker order. In order to compare our G4 mapping results to marker positions previously determined by other laboratories in screening G3 (Stanford G3 RH panel, irradiated at 10 000 rads) RH maps, we used this E approximation: 0.1cR3000 1.0 cR10000 When marker order could be assigned for only a single nearby marker, rather than interpolated between two mar- kers, the results are reported as the distance from Figure 2 Chromosomal clusters of adrenergic candidate loci 7 observed during RH mapping. The map units are cR3000, where that single nearby marker, in cR3000 units. B 1cR3000 corresponds to 270 kbp. Abbreviations: ADM, adreno- medullin; ADRA1B, alpha-1 B-adrenergic receptor; ADRA2A, alpha-2A-adrenergic receptor; ADRB1, beta-1–adrenergic recep- Results and discussion tor; ADRB2, beta-2-adrenergic receptor; DRD1, dopamine receptor isoform 1; DRD2, dopamine receptor isoform 2; DRD4, dopamine Human hypertension is a complex trait6 with receptor isoform 4; GPRK6, G--coupled receptor kinase substantial heritability.2–5 The autonomic nervous isoform 6; NPY6R, neuropeptide Y receptor isoform 6; TH, tyrosine hydroxylase; VMAT2, vesicular monoamine transporter, system plays a central role in minute-to-minute isoform 2. (a) Chromosome 5q, in a region linked to systolic blood control of blood pressure, and heritable alterations pressure. The microsatellite marker linked to systolic blood in autonomic function are associated with hyperten- pressure variation in sibling pairs is D5S1471,23,24 while the sion.6 Therefore, we sought to assign chromosomal microsatellite associated with systolic blood pressure variation is 23,24 positions for loci encoding crucial components of D5S2093. A neuroeffector candidate locus newly mapped by the RH technique to chromosome 5q is ADRA1B. Other locus the sympathetic neuroeffector junction (Figure 1), in positions are already in the public domain ohttp:// order to facilitate studies of the genetic under- www.ncbi.nlm.nih.gov/genome/guide/human. b) Chromosome pinnings of autonomic alterations in hypertension. 10q. (c) Chromosome 11p.

Journal of Human Hypertension ora fHmnHypertension Human of Journal 322

Table 1 Physical map assignments of loci encoding proteins crucial for the sympathetic neuroeffector junction

Region of the Category Name Symbol OMIM # Previously PCR primers RH LOD Human cR3000 neuroeffector RH-mapped scorea chromosome from junction p-ter Reference Upstream Downstream clone

Position Sequence Position Sequence

Presynaptic Catecholamine Vesicular monoamine VMAT1 193 002 New AC025853 22411–22431 AAAGTGCTGGGAT 22687–22666 GGTGGTGATTCCATT >3.0 8p21.3 83.76 7 2.25 uptake or transporter 1 TACAGGCG TGGCTTC gene hypertension Autonomic storage

Vesicular monoamine VMAT2 193 001 New AC022283 39023–39043 GCTTGAGTCGGACA 39278– GCAACTACTGAGGTC >3.0 10q25 516.07 7 2.3 Chitbangonsyn SW transporter 2 GGAGCAC 39257 GGTTTGG Norepinephrine NET 163 970 Yes R34127 CCCAAAGTCTAGG GGGCTGTTAGCTC 0.7 I6q122 354.26 transporter (uptake-1) CAAATGT AGAGGA Solute carrier family SLC22A3 604 842 New AL355506 11955–11979 TGACGGATACTTTG 12176–12198 ATGGCATTTGGAGC 2.75 6q27 621.94 22 member 3 OCT3 GAAGCATTCTG ATTGTAACG (uptake-2 catecholamine

transporter) al et Catecholamine Phenylalanine hydroxylase PAH 241 600 Yes K03020 GTCCGAAAAGC TCTTGATGAAA >3 12q24.1 399.61 biosynthetic AAACCTTA TGCGACA enzymes Tyrosine hydroxylase TH 191 290 Yes D00269 CCTCCCCAGGGCGG CACTTACTTAC 0.1 11p15.5 22.72 CCCGGGGCCCA CCTTGGGGT GGGGG GTP cyclohydrolase 1 GCH1 600 225 Yes S44049 CGATCGTACTG CAGTTCCCTCTC 0 14q22.1– 166.29 CCAGTAGCA ATTCCCAA q22.2 GTP cyclohydrolase GCHFR 602 437 Yes U78190 AGGAGTGACCTT CACTGCTTGAC 0.59 15 126.94 1 feedback CTCATGCTGATTT GCAGGGTTT regulatory protein Aromatic L-amino acid DDC/AADC 107 930 Yes M76180 AAGCCATCCA GCGTGAACATT >3 7p11 229.31 decarboxylase GAGGGTTG GATTGCC Dopamine b-hydroxylase DBH 223 360 New AL357623 93957–93978 TGCTTCAGCCCCA 94255–94234 TTTCTG TTGCTTC 1.39 9q34 404.72 TCATCTTAC CTTCGTTGG Phenylethanolamine PNMT 171 190 New J032801 1218–1237 GAAAGGGCCTAATT 1341–1363 CACAATCTTTAGACC 2.61 17q21–q22 296.29 N-methyltransferase CCCCAG CCCTCACC Catecholamine Catechol-O COMT 116 790 Yes R41599 CAATCCAGTGTTG GTGCCCAGACGTG >3 22q112 1.50 catabolic enzymes -methyltransferase CAGTTCAG CTCCT Monoamine oxidase A MAOA 309 850 Yes AA035648 TGCAGAGGTAA CTCCCTGCTTAG 0.14 Xp11.23 116.53 CCATGTTGAAG CTCTGTGG Monoamine oxidase B MAOB 309 860 Yes R37282 CCCAAAGTGTGT GAACACAAATCCC 0.37 Xp11.23 129.54 ACTGTCCTTTG AACGCTT Sulfotransferase family SULT1A3 600 641 New NT_010584 114747–114778 GGCTCACTGTC 114984–115004 TTAGGCTCCTCC >3.0 16p11.2 297.96 1A phenol AACCCTGTTCG CCTGCGAC preferring member 3 (catecholamine sulfation) Chromogranins Chromogranin A CHGA 118 910 Yes U03749 CATCCTTTGC AGTCAGGAGTTCTC 0.02 14q32 246.79 AGGGCAG AGCTTTCAC Chromogranin B CHGB 118 920 Yes R44737 CCAACTGAGACAAAC TGTCAGAATGCTA >3 20pter-p12 43.14 ATTTTTCC TTGAAAATGTG Secretogranin II SCG2 118 930 Yes M25756 CACTGGGGA AACAATGCCACA 2.41 2q35–q36 697.35 GTCTGCTTCA GCCATTAG Sympathetic Neuropeptide Y NPY 162 640 Yes M14298 GGAAATGAGACTTG TGAGGAGGATACACT 1.39 7p15.1 103.44 neuropeptides CTCTCTGG TTTGAATGC Pituitary adenylate PACAP 102 980 Yes S83513 ATGGACTGGTTT GCATGGACAGC 0.01 18p11 14.82 cyclase activating GAGATTGC ACTGGAG polypeptide Adrenomedullin ADM 103 275 Yes D14874 GCGCAAGCCTC GCGGCGAACAA 0.9 11p15.4 50.28 ACTATTAC CTTTAC Table 1

Region of the Category Name Symbol OMIM # Previously PCR primers RH LOD Human cR3000 neuroeffector RH-mapped scorea chromosome from junction p-ter Reference Upstream Downstream clone

Position Sequence Position Sequence

Presynaptic Adrenergic Alpha-2A-adrenergic ADRA2A 104 210 New M23533 82–102 TGCTGAGCCCTT 261–238 GCCAACAGAGCCTTC 0.87 10q24–q26 594.68 receptors receptor CCTGATGTG TCCTATCTC Alpha-2B-adrenergic ADRA2B 104 260 New AF005900 750–770 TATTCCAGACATT 957–936 ACTCCACATCACC >30 2p13-q13 342.83 receptor GCCGAACG AAGCACCAG Alpha-2C-adrenergic ADRA2C 104 250 Yes J03853 AGCCTGTAC CCTGGTTGAAG >3 4p16.1 31.36 receptor GGCATCTG ACCGTG Postsynaptic Adrenergic Alpha-1A-adrenergic ADRA1A 104 219 Yes M76446 CCACCTGCCAGG GGGATGTCA 1.37 20p13 12.09 receptors receptor CCTACGA CAGAGCAGCT Alpha-1B-adreoergic ADRA1B 104 220 New ACO11343 80628–80651 GGACCACCAAACA 80861–80883 TTTACAGCCACACA 0.01 5q33 595.83 receptor AAAAGAAGACG GCCCCAAC Beta-1-adrenergic ADRB1 109 630 Yes J03019 AGCAGGTGA AGCAGGTGAAC 76 10q24–q26 519.76 receptor ACTCGAAGCC TCGAAGCC Beta-2-adrenergic ADRB2 109 960 Yes M15169 TCAGTTCCTC ACCCCGTGTGAGC 0 5q32–q34 571.96 receptor TTTGCATGGA AAATAAG Beta-3-adrenergic ADRB3 109 691 Yes X70811 AGGCACAAA GATTTTTGTAGC 0 8p12–p11.2 156.44 receptor GCATTGCTTG TGGGGAAGG Dopamine Dopamine receptor D1 DRD1 126 449 Yes X58987 CTTAGGATTTAC ACTTTGCTGGG 0.01 5q35.1 636.91 receptors CAAATAGGGC AACAGTG Dopamine receptor D2 DRD2 126 450 Yes M3065 GTATGATGATGAT ACTGCGAGGC 11 11q23 373.28 CTGGAGAGGC TGACGATC Dopamine receptor D3 DRD3 126 451 Yes U25441 GGCTGCCCTT CAGCAAGACAGG >3 3q13.3 412.11 CTTCTTGAC ATCTTGAGG Dopamine receptor D4 DRD4 126 452 Yes L12398 TGTCTTCAAC TTAACGTACAAA 0.20 11p15.5 18.88 GCCGAGTTC AGCGCCCT Dopamine receptor D5 DRD5 126 453 New U21164 163–186 TTCCCTCACCTC 409–386 TCCTTTCTCCCTC 2.47 4p16.1– 69.06 TTTCACACAATC TGCTCTATCTG p15.3 Sympathetic Neuropeptide NPY1R 162 641 Yes G06447 AACTACTTATAGCC CATAAATTCACA 1.32 4q31.3–q32 650.95 Chitbangonsyn SW gene hypertension Autonomic neuropeptide Y receptor Y1 TATGGTCCCG AAAAGCACTTCA G-protein coupled receptors Neuropeptide Y NPY2R 162 642 Yes G29898 GAATCTGGTTG TCTGCTTTTACTC 0 4q31 695.0 receptor Y2 ATGGCGG ATAATGCAACC Neuropeptide Y NPY6R 601770 New AC073206 140627–140651 AGAAGAGGCAAA 140896–140874 AGTCAGGAGGATA 2.21 5q31 522.46 receptor Y6 CTCACTTAGGGTC GGGATTGTGC al et (truncated receptor) Adrenomedullin ADMR 103 275 New NT_009485 68182–68202 TGAGGCTAAAGA 68481–68503 TGGAGATACCA 0.93 12p 261.28 receptor GGTGCTCGG TCCTGCTGACC G-protein coupled G protein coupled GPRK6 600 869 Yes U00686 ACTCAAGTCG TAAGAATGCTCT 0.2 5q35 636.91 receptor kinase receptor kinase 6 TGGCCTG GGTCGC Heterotrimeric G Gs-alpha GNAS1 139 320 Yes T72916 GCAAGCTGAA GAAGGGAAC 0.47 20q13.2 336.90 protein subunits GGGAAAAGG CCCCAAATTTA Gi-beta isoform 3 GNB3 139 130 Yes H92898 GAAAGCATGAATA CAGGCCCTAGG 1.00 12p13 43.58 AGAAGAGGGC ATTCCTCC Adenylyl cyclase Adenylyl cyclase 6 ADCY6 600 299 New NM_015270 1062–1082 TGCAGG 1430–1450 GAGCGTGTAGGC 1.21 12q12–q13 226.32 isoforms TGTTCC GATGTAGAC AGTCGAAGC

a All LOD scores for a chromosomal position ranged from 7 to 17; LOD scores shown are the comparison with the next best model for marker position. cR3000 : centi-Ray 3000 map units distance ora fHmnHypertension Human of Journal derived from the G4 library, whose human chromosomal segments were irradiated at 3000 rad. For results of existing chromosomal assignments already in the public domain, consult GeneMap ‘99 at ohttp://www.ncbi.nlm.nih.gov/genome/guide/human>. 323 Autonomic hypertension gene SW Chitbangonsyn et al

324 ‘99 at ohttp://www.ncbi.nlm.nih.gov/genome/guide/ 8 Takiyyuddin MA et al. Chromogranin A in human human4. Target loci have been grouped into two hypertension. Influence of heredity. Hypertension principal anatomic categories (pre- and postsynaptic 1995; 26: 213–220. targets), as well as particular subcategories within 9 Parmer RJ, Cervenka JH, Stone RA. Baroreflex sensi- these domains (eg, catecholamine uptake/storage, tivity and heredity in essential hypertension. Circula- tion 1992; 85: 497–503. adrenergic receptors, dopamine receptors, catecho- 10 Kailasam MT, O’Connor DT, Parmer RJ. Hereditary lamine biosynthetic enzymes, catecholamine cata- intermediate phenotypes in African American hyper- bolic enzymes, neuropeptide Y (NPY) and its recep- tension. Ethn Health 1996; 1: 117–128. tors, the chromogranins/secretogranins, G-protein 11 Bachmann AW, Ballantine DM, Gordon RD. Effect of coupled receptor kinases, and other neuropeptides, positive family history of hypertension on the blood specifically pituitary adenylate cyclase activating pressure and catecholamine responses to a 6 hour polypeptide (PACAP) and adrenomedullin (ADM). adrenaline infusion. Clin Exp Pharmacol Physiol 1993; Chromosomal positions are uniformly expressed as 20: 395–398. distance from the chromosome’s p-terminus, in 12 De Lima JJ et al. Pressor response to norepinephrine in cR units, where each cR representsB270 kbp. essential hypertension. A study in families. Hyperten- 3000 3000 sion 1990; 15 (Suppl): 1137–1139. Of the newly mapped loci, two (NPY6R and 13 Ferrier C, Cox H, Esler M. Elevated total body ADRA1B) were found to be located near a region noradrenaline spillover in normotensive members of on chromosome 5q previously found to be linked to hypertensive families. Clin Sci 1993; 84: 225–230. (D5S1471) and associated (D5S2093) with systolic 14 Bray MS, et al. Positional genomic analysis identifies

blood pressure in a genome scan of discordant the b2 adrenergic receptor gene as a susceptibility sibling pairs23,24. Of note, this region (Figure 2a) locus for human hypertension. Circulation 2000; 101: includes not only several adrenergic receptors 2877–2882. (ADRB2, ADRA1B, and DRD1) but also a G-protein- 15 Kotannko P et al. Essential hypertension in African coupled receptor kinase (GPRK6), and an NPY Caribbeans associates with a variant of the beta2- receptor gene encoding a truncated protein adrenoceptor. Hypertension 1997; 30: 773–776. 25 16 Jia H et al. Association of the G(s) alpha gene with (NPY6R). essential hypertension and response to beta-blockade. Two other chromosomal regions (on Hypertension 1999; 34: 8–14. 10q (Figure 2b) and 11p (Figure 2c) also possessed 17 Siffert W et al. Association of a human G-protein beta3 clusters of adrenergic candidate loci. subunit variant with hypertension. Nat Genet 1998; 18: More precise chromosomal positioning of these 45–48. neuroeffector loci should facilitate their analysis in 18 Sharma P et al. Positive association of tyrosine genetic linkage and allelic association studies of hydroxylase microsatellite marker to essential hyper- autonomic dysfunction in human hypertension.6 tension. Hypertension 1998; 32: 676–682. 19 Timberlake DS, O’Connor DT, Parmer RJ. Molecular genetics of essential hypertension: recent results and emerging strategies. Curr Opin Nephrol Hypertens References 2001; 10: 71–79. 20 Dracopoli N et al. Somatic cell hydrids. In: Boyle A 1 Lander E, Schork N. Genetic dissection of complex (ed). Human Genetics. John Wiley & Sons Inc: New traits. Science 1994; 265: 2037–2048. York, 1995, pp 3.0.1–3.4.12. 2 Lifton RP. Molecular genetics of human blood pressure 21 Gyapay G et al. A radiation hybrid map of the human variation. Science 1996; 272: 676–680. genome. Hum Mol Genet 1996; 5: 339–346. 3 Hunt SC, Williams RR. Genetic factors in human 22 Flaherty Land Herron B. The new kid on the blockFa hypertension. In: Swales J (ed). Textbook of Hyperten- whole genome mouse radiation hybrid panel. Mamm sion. Blackwell Scientific: Oxford, 1994, pp 519–538. Genome 1998; 9: 417–418. 4 Ward R. Familial aggregation and genetic epidemio- 23 Krushkal J et al. Genome-wide linkage analyses of logy of blood pressure. In: Laragh JH (ed). Hyper- systolic blood pressure using highly discordant sib- tension: Pathophysiology, Diagnosis, and Manage- lings. Circulation 1999; 99: 1407–1410. ment. Raven Press: New York, 1990, pp 81–100. 24 Krushkal J et al. Linkage and association of adrenergic 5 Hunt SC, Williams RR. Genetic factors, family history, and dopamine receptor genes in the distal portion of and blood pressure. In: Izzo J (ed). Hypertension the long arm of chromosome 5 with systolic Primer. American Heart Association: TX, 1993, pp blood pressure variation. Hum Mol Genet 1998; 7: 155–158. 1379–1383. 6 O’Connor DT et al. Heredity and the autonomic 25 Gregor P et al. Molecular characterization of a second nervous system in human hypertension. Curr Hyper- mouse pancreatic polypeptide receptor and its inacti- tens Rep 2000; 2: 16–22. vated human homologue. J Biol Chem 1996; 271: 7 Dao TT et al. Expression of altered alpha2-adrenergic 27 776–27 781. phenotypic traits in normotensive humans at genetic risk of hereditary (essential) hypertension. J Hypertens 1998; 16: 779–792.

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