ORIGINAL ARTICLE

Inna Belfer Æ Gabriel Phillips Æ Julie Taubman

Heather Hipp Æ Robert H. Lipsky Æ Mary-Anne Enoch

Mitchell B. Max Æ David Goldman

Haplotype architecture of the norepinephrine transportergene SLC6A2 in four populations

Received: 13 January 2004 / Accepted: 13 February 2004 / Published online: 9 April 2004� The Japan Society of Human Genetics and Springer-Verlag 2004

Abstract The norepinephrine transporter (NET) regu-lates levels of monoamine neurotransmitters integral toa variety of behaviors and autonomic functions. TwoSLC6A2 polymorphisms have been used in geneticassociation studies, generating intriguing but nondefin-itive results on traits such as hypertension and mood.One of these SLC6A2 variants is functional but rare.The other is common but not informative over the entire48 kb SLC6A2 region and is insufficient to capture thefunctional diversity potentially contained within anySLC6A2 region. To elucidate SLC6A2 haplotypestructure and define markers sufficient to capturehaplotype diversity within detected haplotype blocks,26 single-nucleotide polymorphisms (SNPs) were geno-typed in 384 individuals evenly divided across FinnishCaucasian, US Caucasian, Plains American Indian, andAfrican American populations. Three conserved blocks,13.6, 12.5, and 25 kb in size and showing little evidencefor historical recombination were observed in all popu-lations. Haplotype diversity in block 1 and numbers ofcommon haplotypes were highest in African Americans,among whom 5–6 optimal markers were sufficient tomaximize diversity of each block. For other populations,2–3 markers/block sufficed, but the optimal markers

differed across populations. The SLC6A2 haplotypemap and 25-marker panel (excluding the monomorphicone) is a comprehensive tool for genetic linkage studieson phenotypes related to NET function.

Keywords Single-nucleotide polymorphism Æ Linkagedisequilibrium Æ Haplotype Æ Norepinephrinetransporter Æ SLC6A2

Introduction

In humans, norepinephrine (NE) is essential to fun-damental cognitive and emotional processes includingattention, learning and memory, perception ofemotions, and perception of pain (Foote et al. 1983;Jasmin et al. 2002). NE is also involved in autonomiccontrol via its actions in the brainstem and as theprimary neurotransmitter at postganglionic sympa-thetic nerve terminals (Hahn et al. 2003). The majorityof brain noradrenergic neurons are concentrated in thelocus coeruleus, a phylogenetically ancient and devel-opmentally precocious structure. These NE neuronsproject to limbic regions critical to cognition andaffect.

NE released at central and peripheral synapses isinactivated through active transport into terminals bythe presynaptically localized norepinephrine transporter(NET) (Iversen 1974). NET recaptures as much as 90%of released NE making it a critical mediator of NEinactivation and presynaptic catecholamine homeostasis(Schomig et al. 1989). Thus, NET plays a role incontrolling the intensity and duration of signal trans-duction (Zahniser et al. 2001). NE interacts with manyother neurotransmitters both in normal cortical regula-tion and in the therapeutic response to psychoactivecompounds, and one critical interacting neurotransmit-ter is dopamine (Jordan et al. 1994). Dopamine is theNE precursor so that levels of both neurotransmittersare regulated by common factors, for example, tyrosine

Parts of this work were presented at the 53rd Annual Meeting ofAmerican Society of Human Genetics, Los Angeles, CA, USA,November 2003.

I. Belfer Æ G. Phillips Æ H. Hipp Æ M. B. MaxPain and Neurosensory Mechanisms Branch,National Institute of Dental and Craniofacial Research,Bethesda, MD, USA

I. Belfer (&) Æ G. Phillips Æ J. Taubman Æ H. Hipp ÆR. H. Lipsky Æ M.-A. Enoch Æ D. GoldmanLaboratory of Neurogenetics,Department of Health and Human Services,National Institute on Alcohol Abuse and Alcoholism,National Institutes of Health, Bethesda, MD20892-1258, USAE-mail: ibelfer@mail.nih.govTel.: +1-301-402-8323Fax: +1-301-443-8579

J Hum Genet (2004) 49:232–245DOI 10.1007/s10038-004-0140-9

Table 1 Primer and probesequences for 5¢ nucleasegenotyping of 26 SLC6A2single-nucleotidepolymorphisms (SNPs)

SNP Primers and probes Sequences

1 Forward primer AAGGTCATCTCATAACCTAGCAGAGAReverse primer TCCCTAAGACGCAACTCTAGCAAllele 1 probe (FAM) CACAGAGCTCCATGTGGAllele 2 probe (VIC) CAGAGCCCCATGTGG

2 Forward primer GGACAACAAAGCACAAAGAGTGTReverse primer GCTTGTGTGGCCAGGATGTAllele 1 probe (FAM) TCAGCTGTTTTAATGAAGAllele 2 probe (VIC) CAGCTGTTTTGATGAAG

3 Forward primer GCTTGTGTGGCCAGGATGTReverse primer CCAGACAAGTGCCAGCTCTTAllele 1 probe (FAM) CAGAGGGAGACTCCTAllele 2 probe (VIC) AGAGGGAGGCTCCT

4 Forward primer AAGGCACTGATACACGTAGTCTCTAReverse primer AAACCCATTCATGAACACTTAGTTCTGAAllele 1 probe (FAM) CTCCCGATCCACTGTAGAllele 2 probe (VIC) CCCGATCCGCTGTAG

5 Forward primer GATCATAGATACACCCACCTCCCTTAReverse primer AGTTCTGAATGCTTTCTTCCATGAAAllele 1 probe (FAM) ATGCTACGAATTATGAllele 2 probe (VIC) AATGCTACAAATTATG

6 Forward primer TCCGACCTCATGAACCTAGCTTReverse primer CTGTCCCCGCTGTTTTGGAllele 1 probe (FAM) CTCTCCCACTTTGGAllele 2 probe (VIC) TCTCTCTCACTTTGGG

7 Forward primer GGTTCAGAACACATGGCTTGAGAReverse primer CTGCAGGGAATCAGGAAAGCAllele 1 probe (FAM) CCCACAAGTACCTCCACAllele 2 probe (VIC) CACAAGTGCCTCCAC

8 Forward primer CCTCACCCAGCTCCATCCTReverse primer GGAGACTGTGCTGGAAGTTCGTAllele 1 probe (FAM) CATGCAGGGCTTCAllele 2 probe (VIC) ACATGCAGGACTTC

9 Forward primer CCCAGGTGACCCAGAGCReverse primer CCCAGGTGACCCAGAGCAllele 1 probe (FAM) TTTCCTTCTCGCCCTGTTAllele 2 probe (VIC) CTTTCCTTCTCCCCCTGTT

10 Forward primer AGTTTCCGGTGTCGCTTCAGReverse primer CCAGATGGGAGGCATGGAAllele 1 probe (FAM) CAGGCCCGTGATGAAllele 2 probe (VIC) AGGCCTGTGATGACA

11 Forward primer TGAACACTAGGCCAGCTCCTGTAReverse primer GCAGCTCAGACCAATGGTTTCAllele 1 probe (FAM) AAACAGCAAGGACCAllele 2 probe (VIC) AGAAACATCAAGGACC

12 Forward primer TGCTTCCTGAACTGAGCTAGATCATReverse primer GCCTGGCAGGGTCAGAAGTAllele 1 probe (FAM) TCTCCCCTGCAGTCTAllele 2 probe (VIC) AGTCTCCCTTGCAGTC

13 Forward primer CACTACCAAATTCACCCACTTTCACReverse primer CCCCAACACCCTCATCCAAllele 1 probe (FAM) CAGAGGCCCCAACTAllele 2 probe (VIC) AGGCCCAAACTAT

14 Forward primer AATCAGGCTAAGGTCAAGGTAGTCAReverse primer CCCCCTTAGGGATATGGGAAGATTAllele 1 probe (FAM) TTCAGGTGAGTCCTCAllele 2 probe (VIC) TGTTTCAGCTGAGTCC

15 Forward primer TCACTTTAAATCCATACCTGCTGGTAReverse primer GCCATTGGCTTGAATTTGGTAAllele 1 probe (FAM) ACCACACAAATTATAllele 2 probe (VIC) CCACACAGATTATC

16 Forward primer CTTAGAGATTGCTGTAACTGAAACAAAAAReverse primer AAAGTCTAGATAATAAGAGTTCATTGCACAAAllele 1 probe (FAM) TCCCAAGAAACAGCTAllele 2 probe (VIC) TCCCAAGAGACAGCT

17 Forward primer GAGGCCCTTGGCAGTATCCReverse primer AGTTCCTCATATTTTCCTGCCAAAAllele 1 probe (FAM) CCTGGTCTCTGTTATTAllele 2 probe (VIC) TGGTCTCTGCTATTAG

233

hydroxylase activity. The NET has the ability to trans-port dopamine, and drugs that block the NET increaseextracellular levels of both NE and DA (Tanda et al.1997; Bymaster et al. 2002; Gu et al. 1996). Monoaminetransporters are initial sites of action for several anti-depressant drugs (including several which are relativelyNET selective) as well as psychostimulants includingcocaine and the amphetamines (Pacholczyk et al. 1991;Ritz et al. 1990; Tatsumi et al. 1997; Sacchetti et al.1999). Decreases in NE uptake sites and activityhave been observed in hypertension, diabetes, cardio-myopathy, and heart failure (Esler et al. 1981; Merletet al. 1992; Bohm et al. 1995; Schnell et al. 1996; Backset al. 2001), and insufficient NE clearance may contrib-ute to the progression of these diseases (Bohm et al.1998).

The human NET gene (SLC6A2, hCG2025341) islocated on chromosome 16q12.2 (Bruss et al. 1993) andhas 15 exons spanning �48 kb (Porzgen et al. 1995,1998). The cDNA sequence encodes a 617-amino acidprotein with 12 highly hydrophobic membrane domainsand a high level of amino acid identity to other membersof the Na+/Cl)-dependent monoamine transporterfamily, e.g., HTT(serotonin transporter) and DAT

(dopamine transporter) (Nelson 1998; Hahn and Blakely2002). SLC6A2 has five alternative splice transcripts.Resequencing of SLC6A2identified 13 DNA sequencevariants, among them five low-frequency missense sub-stitutions (Stober et al. 1996). The reported missensesubstitutions Val69Ile, Thr99Ile, Val245Ile, Val449Ile,and Gly478Ser are located in putative transmembranedomains 1, 2, 4, 9, and 10, respectively. The Thr99Ilesubstitution is at the 5th position of a putative leucinezipper in transmembrane domain 2. A rare Ala457Prosubstitution in exon 9 resulting in more than 98% loss offunction has recently been detected (Ivancsits et al.2003). A synonymous substitution also located in exon9, A1287G, has been used in a series of associationstudies to NE-related phenotypes including hyperten-sion and mood disorders (Stober et al. 1996;Leszczynska-Rodziewicz et al. 2002; Samochowiec et al.2002). However, no common functional NET poly-morphisms are known.

The NET markers used so far in linkage studies donot capture the potential information on NET func-tional variation, and the results obtained so far havebeen nondefinitive. A haplotype approach combiningabundant missense polymorphisms with a series of loci

Table 1 (Continued)SNP Primers and probes Sequences

18 Forward primer GGCTCCAGGCCAGAAAGGReverse primer GGCCTCTGCAAGTAGATAGGAGTTCAllele 1 probe (FAM) AGGGCGGCATGAGAllele 2 probe (VIC) AGGGCAGCATGAGA

19 Forward primer GCTCTTCCCTTCTCTGACATCATCReverse primer GCGTGGGTAGTCAGGAAAGGAllele 1 probe (FAM) AGGTGACATAGGAACTAllele 2 probe (VIC) AGGTGACGTAGGAAC

20 Forward primer CTGAAACACAAGAAGAGCGATAGATTReverse primer GGGATTGGCTTCAGGTATAGATAGATAllele 1 probe (FAM) CCAGACATCATTATTTAllele 2 probe (VIC) CAGACAACATTATTTG

21 Forward primer TACAGGTGGCTGAAGCAAAGGReverse primer GGACCCCATCAAGTTTCTGTGAAllele 1 probe (FAM) CAGGGAAGTTACCCAllele 2 probe (VIC) AGGGAAGTCACCCAC

22 Forward primer CACTTTGGCCAGTTTCCTCAACReverse primer AACTGGGCAGTTTACCTATGTGTTGAllele 1 probe (FAM) AAACCCCTGGGTGACAllele 2 probe (VIC) TAAACCTCTGGGTGACAC

23 Forward primer CATGACCCAAACACCTCTCACTAGReverse primer TGTCTCCACTAAAACTCCTGTTGAAGAllele 1 probe (FAM) CACTCCCACACTGGAllele 2 probe (VIC) ACTCCCAAACTGG

24 Forward primer AGCACACTGAGGGCTGTTAAATAACReverse primer CCTGGCTCAGGGTTACACTGAAllele 1 probe (FAM) CTGGGTAAGTCCTGGAllele 2 probe (VIC) TGGGTGAGTCCTGG

25 Forward primer CCAAACAGCCATGCTTAAACCTReverse primer AGCTAATCCAGAGGTGAAACTTTCCAllele 1 probe (FAM) AGTAACGCCTGAGAGGAllele 2 probe (VIC) AACGCTTGAGAGGC

26 Forward primer TCCTACCCATCCTCCAAAACCReverse primer GGTTATCAGGGAAGGCTTCACAAllele 1 probe (FAM) CCAGTCACCCTCACAllele 2 probe (VIC) TCCAGTCAACCTCA

234

chosen for haplotype informativeness offers the poten-tial for detection of effects of any allele of moderateabundance and effect size, regardless of whether the al-lele is presently known or unknown (Gabriel et al. 2002).Concerning linkage disequilibrium (LD), many regionsof the genome have a block-like structure such that allloci within the block region tend to be in strong LD.However, haplotype block boundaries, strength of LD,haplotype diversity, and optimal marker panels to fullycapture haplotype diversity vary across populations.In this study, we report the haplotype structure ofSLC6A2obtained using 26 single-nucleotide polymor-phisms (SNPs) genotyped in four populations: Finnishand American Caucasians, American Indians, andAfrican Americans. We also describe marker panels foreach block, which maximize haplotype informationcontent.

Materials and methods

Participants A total of 384 participants weregenotyped, including 96 individuals from each of fourpopulations: Finns, US Caucasians, African Americans,and Plains American Indians. Informed consent was

obtained according to human research protocols ap-proved by the human research committees of therecruiting institutes, including the National Institute onAlcohol Abuse and Alcoholism, National Institute ofMental Health, Rutgers University, and University ofHelsinki. All participants had been psychiatricallyinterviewed, and none had been diagnosed with a psy-chiatric disorder.

SNP markers The physical position and frequency ofminor alleles (>0.05) from a commercial database(Celera Discovery System, CDS, July, 2003) were usedto select SNPs (including A1287G and Ala457Pro). Atotal of 50 SLC6A2 SNPs were identified in the data-base. 5¢ nuclease assays (vide infra) could be designed for35, and of these, 26 SNP assays detected sequencepolymorphisms and could be genotyped in highlyaccurate fashion. This panel of 26 equally spacedmarkers covered the 48-kb gene plus 4 kb upstream and4 kb downstream.

Genomic DNA Genomic DNA was extracted fromlymphoblastoid cell lines and diluted to a concentrationof 10 ng/ul; 1-ul aliquots were dried in 384-well plates.

Table 2 Locations and allelic frequencies of 26 SLC6A2 SNPs in 96 individuals from each of four populationsa. # Single-nucleotidepolymorphism (SNP) marker with location shown in Fig. 1. Orientation is #26–#1 (5¢–3¢)

# SNP ID(CDS)

SNP ID(NCBI)

Variation Position(CDS)

Location Allelic frequency (for allele 2)

US Caucasians FinnishCaucasians

PlainsIndians

AfricanAmericans

1 hCV1232486 rs258099 T>C 42997914 3¢ intergenic 0.67 0.71 0.68 0.172 hCV1232479 rs171798 A>G 43000649 3¢ intergenic 0.82 0.78 0.88 0.463 hCV1232477 rs7188230 A>G 43001386 3¢ intergenic 0.81 0.76 0.97 0.434 hCV26354929 no rs A>G 43003652 3¢ intergenic 0.16 0.27 0.02 0.345 hCV11291484 no rs G>A 43004999 3¢ intergenic 0.86 0.83 0.98 0.716 hCV1232474 rs15534 C>T 43008627 3¢ UTR 0.13 0.17 0.02 0.367 hCV26354922 rs8049681 A>G 43009804 Intron 14 0.13 0.16 0.02 0.388 hCV1232472 rs1800887 G>A 43011565 Intron 11 0.83 0.73 0.97 0.479 Ala457Pro no rs G>C 43013237 Exon 9 1.00 1.00 1.00 1.0010 hCV3020068 rs5569 C>T 43013319 Exon 9 0.27 0.38 0.44 0.1111 hCV1232470 rs5568 G>T 43015030 Intron 7 0.62 0.66 0.77 0.8612 hCV1232469 rs1861647 C>T 43016748 Intron 6 0.31 0.40 0.45 0.1213 hCV3020083 rs47958 C>A 43018692 Intron 6 0.47 0.42 0.43 0.3114 hCV1232465 no rs G>C 43020661 Intron 5 0.68 0.60 0.56 0.8915 hCV3020076 rs1345429 A>G 43022288 Intron 5 0.52 0.47 0.54 0.4016 hCV1232458 rs40616 A>G 43023961 Intron 5 0.51 0.56 0.55 0.6317 hCV11291524 no rs T>C 43025783 Intron 4 0.70 0.63 0.56 0.8718 hCV3020077 rs1875544 G>A 43033100 Intron 4 0.25 0.34 0.37 0.0919 hCV1232444 rs187714 A>G 43038757 Intron 4 0.29 0.42 0.46 0.2520 hCV7719239 rs933555 T>A 43040802 Intron 3 0.73 0.59 0.55 0.7421 hCV1232442 rs41154 T>C 43042550 Intron 2 0.28 0.42 0.47 0.2322 hCV1232440 rs6499771 C>T 43044584 Intron 2 0.82 0.82 0.84 0.8423 hCV1232433 rs36028 C>A 43048097 Intron 2 0.21 0.15 0.08 0.1824 hCV1232429 no rs A>G 43050568 Intron 2 0.87 0.85 0.86 0.9525 hCV1232426 rs36031 C>T 43052435 Intron 2 0.71 0.59 0.53 0.6726 hCV1232422 rs4783899 C>A 43058059 5¢ intergenic 0.34 0.46 0.48 0.37

aPhysical locations are from the Celera Discovery system [CDS] database, July 2003. NCBI IDs are from the National Center forBiotechnology Information database, November 2003

235

Polymerase chain reaction (PCR) amplification Geno-typing was performed by the 5¢ nuclease method (Shiet al. 1999) using fluorogenic allele-specific probes. Oli-gonucleotide primer and probe sets were designed basedon gene sequence from the CDS, July 2003. Primers anddetection probes for each locus are listed in Table 1.

In each reaction well, 2.5 ll of PCR Master Mix(Applied Biosystems, CA, USA) containing AmpliTaqGold DNA Polymerase, dNTPs, gold buffer, and MgCl2

was mixed with 900 nmol of each forward and reverseprimer and 100 nmol of each reporter and quencherprobe. DNA was allowed to stand at 50�C for 2 min andat 95�C for 10 min, amplified by 40 cycles at 95�C for15 s and 60�C for 1 min, and then held at 4�C. PCR wascarried out with a GeneAmp PCR system 9700 (AppliedBiosystems).

Allele-specific signals were distinguished bymeasuringendpoint 6-FAM or VIC fluorescence intensities at 508and 560 nm, respectively, and genotypes were generatedusing Sequence Detection System Software Version 1.7(Applied Biosystems). Genotyping error rate was directlydetermined by regenotyping 25% of the samples,randomly chosen, for each locus. The overall error ratewas <0.005. Genotype completion rate was 0.98.

Fig. 1 Location of single-nucleotide polymorphisms (SNPs) geno-typed in the human SLC6A2 gene. Coding exons are shown as solidblocks; 5¢ and 3¢ UTRs are indicated by unfilled rectangles. Physicallocations are from the Celera Discovery System [CDS] database,July 2003

Table 3 Pairwise linkagedisequilibrium (D¢) among eightsingle-nucleotidepolymorphisms (SNPs) inhaplotype block 1 across fourpopulations

U.S. Caucasians marker 1 2 3 4 5 6 7 81 1.00 0.66 0.66 0.65 0.60 0.60 0.662 0.71 0.71 0.70 0.66 0.66 0.713 1.00 1.00 1.00 1.00 1.004 0.86 1.00 1.00 1.005 1.00 1.00 0.866 1.00 1.007 1.008Finnish Caucasians marker 1 2 3 4 5 6 7 81 1.00 0.64 0.55 0.27 0.30 0.30 0.542 0.79 0.79 0.34 0.37 0.37 0.823 1.00 0.82 0.86 0.86 1.004 0.95 1.00 1.00 1.005 1.00 1.00 0.956 1.00 1.007 1.008Plains Indians marker 1 2 3 4 5 6 7 81 1.00 0.40 0.25 0.00 0.00 0.00 0.402 0.32 1.00 1.00 1.00 1.00 0.323 0.74 1.00 1.00 1.00 1.004 1.00 1.00 1.00 0.745 1.00 1.00 1.006 1.00 1.007 1.008African Americans marker 1 2 3 4 5 6 7 81 1.00 1.00 1.00 0.88 1.00 1.00 1.002 0.81 0.92 0.83 0.93 0.93 0.813 1.00 0.82 1.00 0.93 0.754 0.88 0.97 0.97 1.005 0.87 0.87 0.876 0.97 0.977 1.008

236

Haplotype analysis Haplotype frequencies were esti-mated using a Bayesian approach implemented withPHASE (Stephens et al. 2001). These frequencies closelyagreed with results from a maximum likelihood methodimplemented via an expectation–maximization (EM)algorithm (Long et al. 1995).

Results and discussion

Of a total of 26 SLC6A2SNPs, 25 were polymorphic inall four populations. Dramatic interpopulation differ-ences in allele frequencies were observed for most of theSNPs. Ala457Pro, the previously reported in vitrofunctional variant, was monomorphic in the 384 indi-viduals representing the four populations. Allele fre-quencies of SLC6A2 SNPs and their locations in thegene are shown in Table 2. The majority of the markersare located in the intronic sequence of the gene, onesynonymous substitution (A1287G) is located in exon 9,one marker is located in the 3¢ UTR region, five are inthe 3¢ region, and one is in the 5¢ region (Fig. 1). Allgenotype frequencies conformed to Hardy–Weinbergequilibrium.

Within SLC6A2, three conserved LD blocks (1,13.6 kb; 2, 12.5 kb; 3, 25 kb) were observed in all fourpopulations. Definition of haplotype blocks and blockboundaries is an inexact science. Some disruptions of D¢(a measure of LD) occurring within blocks are clearlyattributable to low allele frequencies that lead to in-creased variance in estimation of D¢. We discounted lowD¢ values, which appeared to originate from this cause.In the SLC6A2 haplotype block regions, D¢ was gener-ally >0.85 from one end of the region to the other. D¢averaged was 0.83, 0.94 and 0.94 in blocks 1, 2, and 3,and perhaps more importantly, the median D¢ valuewithin haplotype blocks was 0.97, 0.97, and 1.00 forblocks 1, 2, and 3, meaning that most of the SNP lociwere in very high LD. We note that in the situation thathaplotype block boundaries are drawn too widely, anincreased number of haplotypes will be observed for theblock and an increased number of markers will berequired to capture this diversity. SLC6A2 haplotypeblock boundaries could be drawn somewhat differentlythan we have done, and it can also be observed thatthere is some variation from population to population.For example, there was some disruption of LD withinblock 1 in both Finns and Plains Indians. However, the

Table 4 Pairwise linkage disequilibrium (D¢) among eight single-nucleotide polymorphisms (SNPs) in haplotype block 2 across fourpopulations

U.S. Caucasians marker 10 11 12 13 14 15 16 1710 0.83 0.94 0.91 0.88 0.92 0.91 0.9411 0.90 0.94 0.90 0.93 0.94 0.8912 1.00 1.00 0.97 0.89 1.0013 1.00 1.00 1.00 1.0014 0.96 0.88 0.9415 0.95 0.9616 0.8817Finnish Caucasians marker 10 11 12 13 14 15 16 1710 0.57 1.00 1.00 1.00 1.00 0.97 1.0011 0.60 0.72 0.61 0.70 0.71 0.5812 1.00 1.00 1.00 0.97 1.0013 1.00 1.00 1.00 1.0014 0.97 0.97 1.0015 0.98 1.0016 0.9717Plains Indians marker 10 11 12 13 14 15 16 1710 0.94 0.98 0.97 0.95 0.95 0.94 0.9811 0.94 1.00 1.00 1.00 1.00 1.0012 0.97 0.98 0.95 0.94 1.0013 0.97 1.00 0.98 1.0014 0.95 0.94 1.0015 0.97 0.9816 0.9717African Americans marker 10 11 12 13 14 15 16 1710 1.00 0.95 1.00 0.95 1.00 0.75 0.9511 1.00 0.88 1.00 1.00 1.00 1.0012 1.00 1.00 1.00 0.75 1.0013 1.00 0.94 0.92 0.8614 1.00 0.75 1.0015 0.93 0.8916 0.7617

237

marker panels we genotyped were sufficient to capturediversity in the blocks in the four populations we stud-ied, as described below.

Pairwise LD values within each haplotype block aresummarized in Tables 3, 4, and 5; all pairwise LD valuesamong 25 SNPs across four populations are representedin Tables 6, 7, 8, and 9. Haplotype frequencies for thethree blocks in four populations are shown in Table 10.For each population and haplotype block, 3–6 common(‡0.05) haplotypes accounted for most of the total: 85–96% of Caucasian and Plains Indian haplotypes and 75–89% of African American haplotypes. The number ofcommon (‡0.05) haplotypes were block 1: 4, 5, 3, 6;block 2: 5, 5, 4, 5, and block 3: 4, 5, 5, 6 for U.S. andFinnish Caucasians, Plains Indians, and African Amer-icans, respectively.

For each haplotype block, a panel of markers suf-ficient to maximize genetic information content wasavailable. Excluding Ala457Pro, the number of SNPsavailable for the three haplotype blocks were 8, 8, and

9 for blocks 1, 2, and 3, respectively. Knowing this, thevalue of additional SNP markers for haplotype diver-sity (informativeness) can then be evaluated. We beganwith the haplotypes derived from all available markersin the block and successively subtracted markers, firstsubtracting markers that resulted in no change what-ever in haplotype diversity and then subtractingmarkers which changed diversity in the most minimalfashion and so on until we were left with a single,independent, highest heterozygosity SNP marker. Thefigures graphically depict a reversal of this process,showing that diversity can be maximized using asmaller group of tag SNPs selected from a larger panel.After each marker addition using the pathway deter-mined by the subtraction analysis, haplotype frequen-cies, and diplotype heterozygosity, the chosen measureof diversity were recalculated. At some point for eachhaplotype block and each population, the addition of anew SNP marker did not appreciably increase diversity,as shown in Fig. 2.

Table 5 Pairwise linkage disequilibrium (D¢) among nine single-nucleotide polymorphisms (SNPs) in haplotype block 3 across fourpopulations

U.S. Caucasians marker 18 19 20 21 22 23 24 25 2618 0.97 0.97 0.97 1.00 0.89 1.00 0.97 0.9619 1.00 1.00 1.00 1.00 1.00 1.00 0.9720 1.00 1.00 1.00 1.00 1.00 1.0021 1.00 1.00 1.00 1.00 1.0022 0.85 1.00 1.00 0.4523 1.00 1.00 0.7524 1.00 1.0025 1.0026Finnish Caucasians marker 18 19 20 21 22 23 24 25 2618 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.0019 1.00 1.00 1.00 1.00 1.00 1.00 1.0020 1.00 1.00 1.00 1.00 1.00 1.0021 1.00 1.00 1.00 1.00 1.0022 1.00 1.00 1.00 0.8623 1.00 1.00 0.7624 1.00 1.0025 1.0026Plains Indians marker 18 19 20 21 22 23 24 25 2618 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.9419 1.00 1.00 1.00 1.00 1.00 1.00 0.9520 1.00 1.00 1.00 1.00 1.00 0.9521 1.00 1.00 1.00 1.00 0.9522 1.00 1.00 1.00 1.0023 1.00 1.00 1.0024 1.00 1.0025 0.9526African Americans marker 18 19 20 21 22 23 24 25 2618 1.00 1.00 1.00 0.90 0.97 0.24 1.00 0.8719 0.86 0.87 0.96 0.96 0.75 0.95 0.6420 1.00 0.73 0.97 0.75 0.88 0.6121 0.72 0.97 0.75 0.91 0.6322 0.96 0.94 0.73 0.6223 0.96 0.86 0.5824 0.82 0.7925 0.3426

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Table

6Pairwiselinkagedisequilibrium

(D¢)among25SLC6A2single-nucleotidepolymorphisms(SNPs)

inU.S.Caucasians.

Marker

#9,Ala457Pro,wasmonomorphic

andthus

excluded 1

23

45

67

810

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

11.00

0.64

0.64

0.62

0.56

0.56

0.64

0.44

0.68

0.41

0.10

0.46

0.15

0.10

0.35

0.02

0.04

0.04

0.04

0.28

0.14

0.56

0.04

0.05

20.69

0.69

0.67

0.62

0.62

0.69

0.40

0.72

0.34

0.58

0.35

0.13

0.58

0.27

0.05

0.14

0.14

0.14

0.06

0.02

0.52

0.14

0.06

31.00

1.00

1.00

1.00

1.00

0.29

0.70

0.34

0.55

0.31

0.59

0.55

0.27

0.08

0.15

0.15

0.15

0.07

0.01

0.03

0.15

0.05

40.84

1.00

1.00

1.00

0.29

0.99

0.34

0.74

0.32

0.76

0.74

0.27

0.04

0.10

0.10

0.10

0.10

0.13

0.03

0.10

0.09

51.00

1.00

0.84

0.82

0.64

0.83

0.45

0.83

0.50

0.45

0.81

0.25

0.28

0.28

0.28

0.10

0.02

0.01

0.28

0.02

61.00

1.00

0.79

0.99

0.81

0.68

0.80

0.71

0.68

0.79

0.13

0.17

0.17

0.17

0.14

0.19

0.04

0.17

0.10

71.00

0.79

0.99

0.81

0.68

0.80

0.71

0.68

0.79

0.13

0.17

0.17

0.17

0.14

0.19

0.04

0.17

0.10

80.29

0.99

0.34

0.74

0.32

0.76

0.74

0.27

0.04

0.10

0.10

0.10

0.10

0.13

0.03

0.10

0.09

10

0.81

0.93

0.91

0.87

0.92

0.91

0.96

0.12

0.09

0.09

0.09

0.06

0.15

0.14

0.09

0.04

11

0.88

0.92

0.87

0.91

0.92

0.86

0.30

0.24

0.24

0.24

0.34

0.16

0.12

0.24

0.35

12

1.00

1.00

1.00

0.99

1.00

0.12

0.08

0.08

0.08

0.11

0.19

0.24

0.08

0.02

13

1.00

1.00

1.00

1.00

0.02

0.02

0.02

0.02

0.36

0.13

0.18

0.02

0.11

14

1.00

0.99

0.93

0.12

0.09

0.09

0.09

0.13

0.20

0.25

0.09

0.03

15

0.99

1.00

0.02

0.00

0.00

0.00

0.39

0.19

0.26

0.00

0.10

16

1.00

0.02

0.01

0.01

0.01

0.36

0.13

0.18

0.01

0.11

17

0.13

0.09

0.09

0.09

0.07

0.20

0.16

0.09

0.05

18

0.96

0.96

0.96

0.98

0.85

0.98

0.96

0.95

19

1.00

1.00

0.99

0.99

0.98

1.00

0.99

20

1.00

0.99

0.99

0.98

1.00

0.99

21

0.99

0.99

0.98

1.00

0.99

22

0.95

1.00

0.99

0.44

23

0.97

0.99

0.70

24

0.98

0.98

25

0.99

26

239

Table

7Pairwiselinkagedisequilibrium

(D¢)

among25SLC6A2single-nucleotidepolymorphisms(SNPs)

inPlainsIndians.

Marker

#9,Ala457Pro,wasmonomorphic

andthus

excluded 1

23

45

67

810

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

10.99

0.40

0.49

0.49

0.49

0.49

0.42

0.96

1.00

0.92

0.41

0.92

0.84

0.38

0.92

0.16

0.44

0.42

0.44

0.93

0.52

0.93

0.44

0.39

20.32

0.63

0.63

0.63

0.63

0.59

0.98

0.99

0.98

0.65

0.98

0.76

0.77

0.98

0.02

0.07

0.16

0.07

0.81

0.63

0.80

0.07

0.11

31.00

0.82

1.00

0.93

0.75

0.98

0.95

0.98

0.98

0.98

0.98

0.98

0.98

0.01

0.22

0.21

0.22

0.21

0.09

0.21

0.22

0.26

41.00

1.00

1.00

1.00

0.98

0.95

0.98

0.98

0.98

0.99

0.98

0.98

0.01

0.22

0.21

0.22

0.21

0.09

0.21

0.22

0.26

51.00

1.00

1.00

0.98

0.95

0.98

0.98

0.98

0.99

0.98

0.98

0.01

0.22

0.21

0.22

0.21

0.09

0.21

0.22

0.26

61.00

1.00

0.98

0.95

0.98

0.98

0.98

0.99

0.98

0.98

0.01

0.22

0.21

0.22

0.21

0.09

0.21

0.22

0.26

71.00

0.98

0.95

0.98

0.98

0.98

0.99

0.98

0.98

0.01

0.22

0.21

0.22

0.21

0.09

0.21

0.22

0.26

80.70

0.96

0.70

0.72

0.70

0.77

0.72

0.70

0.26

0.42

0.41

0.42

0.24

0.05

0.25

0.42

0.44

10

1.00

1.00

1.00

1.00

0.97

0.97

1.00

0.47

0.50

0.50

0.50

0.59

0.52

0.57

0.50

0.41

11

0.93

1.00

1.00

1.00

1.00

1.00

0.30

0.21

0.22

0.21

0.21

0.49

0.18

0.21

0.19

12

0.97

1.00

0.95

0.94

1.00

0.46

0.49

0.48

0.49

0.58

0.53

0.56

0.49

0.40

13

1.00

1.00

0.97

1.00

0.37

0.42

0.42

0.42

0.57

0.56

0.56

0.42

0.38

14

0.97

0.97

1.00

0.46

0.48

0.48

0.48

0.58

0.53

0.56

0.48

0.39

15

0.97

0.97

0.43

0.47

0.46

0.47

0.64

0.46

0.63

0.46

0.38

16

0.97

0.37

0.42

0.42

0.42

0.49

0.74

0.47

0.42

0.38

17

0.46

0.48

0.48

0.48

0.58

0.53

0.56

0.48

0.39

18

1.00

1.00

1.00

0.96

0.98

1.00

1.00

0.93

19

1.00

1.00

0.97

0.98

1.00

1.00

0.95

20

1.00

0.97

0.98

1.00

1.00

0.95

21

0.97

0.98

1.00

1.00

0.95

22

0.98

1.00

0.97

0.97

23

0.98

0.98

0.98

24

1.00

1.00

25

0.95

26

240

Table

8Pairwiselinkagedisequilibrium

(D¢)among25SLC6A2SNPsin

FinnishCaucasians.Marker

#9,Ala457Pro,wasmonomorphic

andthusexcluded

12

34

56

78

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

11.00

0.62

0.53

0.27

0.29

0.29

0.52

0.10

0.93

0.02

0.25

0.01

0.09

0.22

0.12

0.01

0.01

0.02

0.01

0.81

0.57

0.80

0.01

0.00

20.78

0.77

0.35

0.37

0.37

0.80

0.15

0.91

0.25

0.87

0.29

0.58

0.81

0.14

0.35

0.45

0.44

0.45

0.75

0.60

0.73

0.45

0.39

31.00

0.82

0.86

0.86

1.00

0.15

0.79

0.22

0.84

0.22

0.85

0.78

0.14

0.40

0.34

0.33

0.34

0.74

0.59

0.73

0.34

0.29

40.95

1.00

1.00

1.00

0.10

0.81

0.20

0.85

0.19

0.86

0.80

0.09

0.42

0.38

0.37

0.38

0.53

0.61

0.50

0.38

0.33

51.00

1.00

0.95

0.28

0.70

0.33

0.80

0.34

0.82

0.81

0.29

0.29

0.18

0.17

0.18

0.28

0.12

0.23

0.18

0.10

61.00

1.00

0.35

0.69

0.40

0.80

0.40

0.81

0.80

0.36

0.37

0.24

0.23

0.24

0.25

0.12

0.20

0.24

0.15

71.00

0.35

0.69

0.40

0.80

0.40

0.81

0.80

0.36

0.37

0.24

0.23

0.24

0.25

0.12

0.20

0.24

0.15

80.07

0.79

0.16

0.80

0.15

0.82

0.76

0.06

0.44

0.37

0.36

0.37

0.55

0.60

0.52

0.37

0.29

10

0.52

1.00

1.00

1.00

1.00

0.96

1.00

0.10

0.24

0.26

0.24

0.23

0.31

0.27

0.24

0.26

11

0.55

0.68

0.56

0.67

0.68

0.53

0.09

0.20

0.20

0.20

0.41

0.82

0.47

0.20

0.25

12

1.00

1.00

1.00

0.96

1.00

0.07

0.22

0.23

0.22

0.24

0.28

0.28

0.22

0.24

13

1.00

1.00

1.00

1.00

0.25

0.24

0.25

0.24

0.24

0.80

0.30

0.24

0.19

14

0.96

0.96

1.00

0.08

0.23

0.24

0.23

0.25

0.28

0.29

0.23

0.25

15

0.97

1.00

0.24

0.23

0.24

0.23

0.20

0.60

0.27

0.23

0.18

16

0.96

0.24

0.23

0.24

0.23

0.23

0.72

0.29

0.23

0.18

17

0.11

0.25

0.27

0.25

0.20

0.31

0.23

0.25

0.27

18

0.99

0.96

0.99

0.98

0.98

0.99

0.99

0.99

19

1.00

1.00

0.99

0.99

0.99

1.00

1.00

20

1.00

0.99

0.99

0.99

1.00

1.00

21

0.99

0.99

0.99

1.00

1.00

22

0.97

1.00

0.99

0.85

23

0.97

0.99

0.74

24

0.99

0.99

25

1.00

26

241

Table

9Pairwiselinkagedisequilibrium

(D¢)among25SLC6A2single-nucleotidepolymorphisms(SNPs)

inAfricanAmericans.Marker

#9,Ala457Pro,wasmonomorphic

andthus

excluded 1

23

45

67

810

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

10.90

0.91

0.91

0.90

0.93

0.93

0.88

0.24

0.67

0.24

0.29

0.24

0.30

0.31

0.24

0.19

0.03

0.02

0.03

0.31

0.04

0.17

0.11

0.10

20.76

0.88

0.82

0.90

0.86

0.72

0.35

0.72

0.32

0.38

0.32

0.17

0.19

0.32

0.25

0.03

0.02

0.02

0.03

0.08

0.16

0.03

0.04

30.95

0.80

0.96

0.91

0.71

0.80

0.73

0.71

0.38

0.71

0.20

0.23

0.71

0.29

0.03

0.02

0.04

0.12

0.05

0.15

0.02

0.01

40.83

0.95

0.95

0.96

0.75

0.79

0.59

0.36

0.59

0.33

0.35

0.59

0.07

0.11

0.14

0.17

0.31

0.10

0.16

0.09

0.05

50.82

0.82

0.86

0.77

0.73

0.59

0.17

0.59

0.31

0.24

0.59

0.07

0.13

0.09

0.11

0.25

0.02

0.12

0.07

0.02

60.96

0.94

0.82

0.72

0.68

0.38

0.68

0.35

0.38

0.68

0.07

0.09

0.13

0.16

0.18

0.08

0.17

0.06

0.07

70.97

0.79

0.84

0.65

0.38

0.65

0.36

0.38

0.65

0.14

0.07

0.10

0.13

0.12

0.08

0.18

0.04

0.06

80.49

0.84

0.45

0.24

0.45

0.10

0.07

0.45

0.37

0.03

0.02

0.04

0.03

0.04

0.25

0.02

0.01

10

0.66

0.93

0.88

0.93

0.92

0.64

0.93

0.35

0.08

0.08

0.09

0.09

0.14

0.14

0.04

0.15

11

0.66

0.97

0.66

0.96

0.96

0.66

0.19

0.09

0.21

0.10

0.48

0.21

0.13

0.04

0.08

12

0.88

0.99

0.92

0.64

0.99

0.35

0.08

0.08

0.09

0.09

0.14

0.12

0.04

0.15

13

0.88

0.91

0.92

0.88

0.08

0.05

0.02

0.10

0.21

0.10

0.41

0.04

0.17

14

0.92

0.64

0.99

0.35

0.08

0.08

0.09

0.09

0.14

0.12

0.04

0.15

15

0.90

0.92

0.07

0.32

0.09

0.17

0.03

0.13

0.21

0.18

0.23

16

0.64

0.07

0.23

0.03

0.12

0.03

0.16

0.23

0.16

0.23

17

0.35

0.08

0.08

0.09

0.09

0.14

0.12

0.04

0.15

18

0.96

0.96

0.97

0.65

0.69

0.15

0.95

0.81

19

0.86

0.91

0.90

0.91

0.63

0.94

0.57

20

0.98

0.75

0.91

0.63

0.87

0.57

21

0.74

0.90

0.60

0.93

0.62

22

0.83

0.96

0.77

0.61

23

0.50

0.84

0.60

24

0.76

0.78

25

0.29

26

242

The required number of tag SNPs varies according tothe haplotype diversity of the region (and population) andthe information content of the markers available. Hap-lotype diversity in block 1 was greatest in African Amer-icans, and tomaximize it, more markers were needed (5–6markers). For the other populations, 2–3markers sufficedfor block 1, but the optimal markers differed acrosspopulations. Thus, each SNP had different informationcontent in different populations. For association/linkagestudies, different tag SNPs couldbe used in different targetpopulations. Alternatively, the entire panel of 25 SNPscould be applied to reliably capture haplotype diversityacross populations. As illustrated in Fig. 2, genotypinglarger panels of markers yields a steadily diminishing re-turn, but another purpose of this approach is to capturemore information on certain rarer haplotypes. The focusof haplotype-based genetic association studies has beenthe detection of effects of moderately abundant loci, be-cause haplotypes and functional alleles of low frequencyare not well represented in small datasets. However,power increases in larger datasets that may be availablefor certain noradrenergic related phenotypes, for exam-ple, diseases such as hypertension and mood disorders,which are readily diagnosed and which afflict very largesegments of populations.

For SLC6A2, the 25-locus SNP panel defines athree-block LD structure across the entire generegion and would be sufficient to capture the signalof any moderately abundant SNP. For example,A1287G is the marker most extensively used in NETlinkage studies, and this SNP is located in block 2, forwhich the SLC6A2 SNP panel includes another sevenmarkers in addition to A1287G. When A1287G is ex-cluded, 99% of the information content of block 2 is stillcaptured.

In conclusion, the SLC6A2haplotype map and mar-ker panel are a comprehensive tool for genetic linkagestudies on phenotypes related to noradrenergic function.This map is a surrogate for moderately abundant effec-tive alleles, which may be unknown or unrecognized asfunctional.

Acknowledgements We are grateful to Dr. Alec Roy and Dr. MattiVirkkunen for subsets of their population datasets, to LonginaAkhtar for assistance with cell culture, and Ilona Lorincz fortechnical assistance.

Supported by NIDCR Intramural Grant Z01, NIAAA Intra-mural Grant Z01 AA000301 (National Institutes of Health, Beth-esda, MD, USA), and the Comprehensive Neuroscience ProgramGrant USUHS G192BR-C4 (Henry Jackson Foundation, Rock-ville, MD, USA).

Table 10 Frequencies ofhaplotypes constructed from 25single-nucleotidepolymorphisms (SNPs)a.(1=allele 1, 2=allele 2)

a Haplotypes were constructedusing PHASE as described in‘‘Materials and methods’’ andfrom 192 chromosomes repre-senting each population

U.S. Caucasians Finnish Caucasians Plains Indians African Americans

Common haplotypes (block 1)22212112 0.64 0.62 0.65 0.1612212112 0.11 0.08 0.21 0.211121221 0.1 0.08 0 0.2611212112 0.05 0 0.1 011122111 0 0.1 0 022121221 0 0.06 0 011112111 0 0 0 0.111122221 0 0 0 0.0612112112 0 0 0 0.05Common haplotypes (block 2)11122212 0.35 0.29 0.2 0.1222211121 0.27 0.31 0.43 0.0812112122 0.14 0.12 0 0.4912122212 0.1 0.14 0.23 0.1512112222 0.05 0 0.08 021211121 0 0.07 0 012112212 0 0 0 0.05Common haplotypes (block 3)112121221 0.31 0.25 0.28 0.29221221212 0.24 0.33 0.35 0.07112122221 0.17 0.14 0.08 0.04112111121 0.13 0.16 0.15 0.04121221212 0.04 0.08 0.08 0.11112111221 0 0 0 0.08112122222 0 0 0 0.13112121211 0 0 0 0.07

243

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